Use of immunologic reactions for identification and serologic diagnosis

Modern tests for diagnosis of infectious diseases immunofluorescens, CPR. ELISA, gene diagnosis. Using of immunologic reactions in diagnosis of viral infections.

Allergy. Types of hypersensitivity

 

 

Agglutinins and the Agglutination Reaction

Agglutinins are antibodies capable of clumping the corresponding microbes by producing visible conglomerates. The addition of the corresponding immune sera to a suspension of microbes provokes clumping of microbial cells in the form of flakes or granules. This phenomenon is known as agglutination. The agglutination reaction takes place on mixing erythrocytes, yeasts and other cells with the corresponding immune sera.

It was described by A. Charrin, G. Roger (1889), V. Isaiev and V. Ivanov (1894) and was investigated in detail by M. Gruber and H. Durham in 1896. In the same year F. Widal reported that the serum of typhoid patients was capable of specifically clumping (agglutinating)

typhoid bacteria. Later it was established that in a whole series of infectious diseases antibodies (agglutinins) are produced in the blood of patients, which are capable of clumping the corresponding causative agents of infectious diseases.

The agglutination reaction, like the flocculation and precipitin reactions, is under the control of the physicochemical conformities of the interrelations of colloidal systems. The antibody (agglutinin) and antigen (agglutinogen) take part in the agglutination reaction. Their interaction takes place in definite quantitative proportions, and in the presence of an electrolyte (0.85 per cent NaCI solution). In mechanism and outer manifestation the agglutination reaction is similar to the precipitin reaction. Both reactions are accompanied by the production of visible precipitates of antigen with the difference that in the agglutination reaction microbial bodies serve as the antigen, in the precipitin reaction the antigen is the product of the breakdown of microbial bodies, very minute particles of dissolved antigens requiring a large amount of antibodies for complete interaction.

 

 

The agglutination reaction is characterized by specificity, but group agglutination can be found, that is, the clumping of closely related microbes though in weaker serum dilutions.

The antigenic structure of bacteria is quite varied. In one and the same bacterial strain there may be group, species, and  type antigens. Similar bacteria are composed of various antigenic groups, and during immunization of animals the corresponding agglutinins are produced in the blood. This can be represented in the table shown on the next page.

As may be seen from this table, the serum received against microbe  A agglutinates microbe A readily, since agglutinins a₁ b₁ c₁ completely correspond to the agglutinogens a b c. This serum agglutinates microbe B (to a lesser degree) due to the homologous b₁ c₁-agglutinins and bc-agglutinogens, and also microbe C (to an even lesser extent) due to the common character of c₁-agglutinin and c-agglutinogen. These interrelations are found between the serum against microbe B and microbes B, C and A, etc.

The variety of antigens in microbial cells is a regular process and reflects the law of homologous series of in intraspecies and interspecies variability of bacteria (see section on variability of bacteria).

 

 

Thus, upon immunization of the animal with one species of microbe agglutinins may occur not only to this species, but to other related bacterial species which have general group antigens.

For revealing specific agglutinins in sera of animals immunized by a complex of antigens of the bacterial cell the method of adsorption of agglutinins is employed (Castellani’s exhaustion reaction). By adding certain species of bacteria to the serum of an immunized animal, in which there are several agglutinins, those which clump only organisms of this species are removed, after which the serum freed from these agglutinins is checked for the presence of other agglutinins by adding other species of bacteria.

The method of agglutinin adsorption is used to study the antigenic structure of bacteria which are used for preparing agglutinating and therapeutic sera, vaccines, and diagnostic preparations. Agglutinating sera obtained by this method are called monoceptor sera. They make it possible to determine more precisely the species and type specificity of the causative agents of salmonellosis and dysentery. In motile microbes there are flagellar (H) and somatic (0) antigens. During immunization of animals with motile bacteria H-agglutinins and 0-agglutinins are correspondingly produced. Flagellar agglutinins cause a more rapid clumping of microbes in the form of loose flakes, while somatic agglutinins produce comparatively slowly conglomerates of bacteria in the form of fine granules. H-agglutination is otherwise known as large flaky and 0-agglutination as fine granular agglutination.

Bacteria containing the Vi-antigen are only weakly or eveot agglutinated by 0-sera, but agglutinate well with Vi-sera. This shows that 0- and Vi-antigens as well as 0- and Vi-antibodies have a different structure.

 

 

 

Slide agglutination test

 

The reaction of agglutination may take place as a result of the action of non-specific factors (without the presence of an agglutinating serum), the main colloidal solutions of dyes and acids. Such non-specific reactions may also take place in the presence of an isotonic solution alone m microbes which were exposed to considerable changes as a result of long storage, and also in R-forms of bacteria. The extent of manifestation of the specific agglutination reaction depends on the salt concentration (electrolyte), serum concentration, density of bacterial suspension, pH, influence of temperature, shaking and mixing, etc.

The agglutination reaction is widely employed in the practice of serological diagnosis of enteric fever, paratyphoids A and B {Widal’s reaction), brucellosis (Wright’s reaction), typhus fever  reaction with Rickettsia prowazeki), tularaemia, leptospirosis and other diseases, in which with the help of known microbes (diagnosticums) the corresponding agglutinins are determined in patients’ sera. For determining Vi-antibodies in carriers of enteric fever salmonellae Vi-agglutination has had wide application in laboratory diagnosis. The agglutination reaction is used for the identification of isolated microbes in patients and sick animals with the application of previously known agglutinating sera.

To obtain a quick response accelerated agglutination reactions are used as tentative methods in some cases Nobel’s reaction for detecting typhus and enteric fever, Huddleson’s reaction for brucellosis, Minkevich’s reaction for typhus fever and tularaemia and the agglutination reaction with luminescent sera for revealing causative agents of intestinal infections, anthrax, etc.

In surgical practice of blood transfusion the reaction of isohaemagglutination has had wide application with the help of which blood groups may be determined. For this purpose it is necessary to have two haemolytic sera (β and γ) obtained from people with A and B blood groups.  One or two drops of each of these sera are put separately on a slide or china dish, and one small drop of the blood under test is added. The blood and serum are carefully mixed and, according to the reaction of isohaemagglutination, the blood group is established.

The agglutination reaction may also  take place without the participation of antibodies. Some plants (leguminous) were found to contain haemagglutinins which agglutinate human erythrocytes of definite blood groups; phytohaemagglutinins have been revealed in saline solutions of the fruits and seeds of definite types of plants; they are used in haematological studies.

To obtain agglutinating animal sera (rabbit, etc.), the animals are immunized with a suspension of freshly isolated bacteria of a certain species or type according to a certain schedule, taking into account the dose and the intervals between vaccinations. At the end of immunization blood is taken from the animals and the serum obtained is inactivated, conserved and titrated. The titre of the agglutinating serum is known as the smallest amount or the greatest dilution which causes a clearly marked agglutination reaction. On the labels of ampoules of manufactured sera the titres are written as fractions indicating the maximum dilution (1 : 3200, 1 : 6400, 1 : 12800, 1 : 25600, etc.) at which they cause agglutination of the corresponding antigen (agglutinogen). Agglutinating sera are produced as non-adsorbing and adsorbing, multivalent, species and type specific.

 

Serological examination.

All immunological tests are based on specific antibody-antigen interaction. These tests are called serological since to make them one should use antibody-containing sera.

Serological tests are employed in the following cases: (a) to deter­mine an unknown antigen (bacterium, virus, toxin) with the help of a known antibody; (b) to identify an unknown antibody (in blood serum) with the help of a known antigen. Hence, one component (ingredient) in serological tests should always be a known entity.

The main serological tests include tests of agglutination, precipi­tation, lysis, neutralization, and their various modifications.

Agglutination Test. The term agglutination means clumping of microorganisms upon their exposure to specific antibodies in the presence of electrolyte. The presumptive and standard agglutination tests (AT) are widely utilized in the diagnosis of numerous infectious diseases.

To perform agglutination tests, one needs three components: (1) antigen (agglutinogen); (2) antibody (agglutinin); (3) electro­lyte (isotonic sodium chloride solution).

Standard agglutination test is employed for determining the serogroup and serovar of microorganisms and is performed according to the scheme presented in Table 1. All ingredients are dispersed into test tubes in a definite sequence. Serum is diluted in simple nu­merical ratios such as 1:100, 1:200, 1:400, etc.

Into each tube with diluted serum, transfer 1-2 drops of the antigen (1-2 milliard microorganisms per ml), shake vigorously, and place into a 37 °C incubator for 2 hrs; then make a preliminary estimation of the test results, beginning with the controls (serum and antigen). The absence of agglutination in the control tubes and the presence of suspended flocculi in the tested tubes point to a positive test. Al­low the test tubes to stand at room temperature for 18-20 hrs and then make the final assessment of the results. Intensity of the reac­tion is denoted with pluses. In complete agglutination (++++), the liquid is completely transparent, while on the bottom of the test tube there is a floccular sediment of agglutinated microorgan­isms. The lesser the number of agglutinated microorganisms, the more turbid is the fluid and the smaller is the floccular pellet on the bottom (+++, ++, +).

A negative test (–), there is no sedi­ment, the suspension remains uniformly turbid, showing no differ­ence from the content of the test tube with the antigen control.

External manifestations of the agglutination test depend on the type of antigen and the size of cells. In bacteria, the interaction be­tween somatic antigens (0-antigens) and specific antibodies is slow and a fine granular sediment forms in 18-20 hrs. Small grains of the agglutinate do pot break upon shaking. Such agglutination is ob­served in bacteria of tularaemia, Brucella, etc. The presence of the flagellar H-antigen (Salmonella of typhoid, paratyphoid, food tox-infections) induces a rapid develop­ment of  agglutination. Readily breakable large loose flocculi form in 2-4 hrs  (Fig. 1).

Table 1

Schematic Representation of the Agglutination Reaction

 

 

 

 

 

 

 

 

 

                                

Figure 1. Agglutination test

 

Agglutination of living Leptospira is studied in wet-mount preparations with lateral illumination. Aggluti­nated Leptospira appear as lumines­cent “spiders” against a dark back­ground.

The use of the AT in the serological diagnosis of infectious dis­eases, such as typhoid and paraty­phoids (Widal’s reaction), epidemic typhus (Weigl’s reaction), brucellosis (Wright‘s and Huddleson’s reaction), tularaemia and other diseases, is based on determining antibodies (agglutinins) in the patient’s serum.

To perform the test, take 3-5 ml of blood from the jugular vein, fin­ger or earlobe in au adult or 1 ml from the heel in small children. Separate the serum from blood and dilute it. with isotonic saline in a ratio blood may contain normal  antibodies which are capable of inducing agglutination reaction in small dilutions.

As to the antigen that is utilized in this test, diagnosticums (sus­pensions of known killed and occasionally living microorganisms) are employed for this purpose. Diagnosticums of killed microorgan­isms are fairly stable, retaining their properties for several years and present no risk of contamination.

The procedure of the AT with the patient’s blood and evaluation of the results do not differ from those used in the standard AT aimed at determining the species of microorganisms.

Indirect agglutination (haemagglutination) (IHA) test. Occasion­ally, antigens employed for the agglutination reaction are so highly dispersed that an agglutinogen-agglutinin complex evades detec­tion by the naked eye. To make this reaction readily visible, meth­ods of adsorption of such antigens on larger particles with their subsequent agglutination by specific antibodies have been designed. Adsorbents employed for this purpose include various bacteria, particles of talc, dermal, collodium, kaolin, carmine, latex, etc. This reaction has beeamed indirect (or passive) agglutination test.

Red blood cells display the highest adsorptive capacity. The test conducted with the help of erythrocytes is called indirect, or pas­sive, haemagglutination (IHA or PHA). Sheep, horse, rabbit, chicken, mouse, human, and other red blood cells can be used for this test. These are prepared in advance by treating them with formalin or glutaraldehyde. The adsorptive capacity of erythrocytes augments following their treatment with tannic or chromium chloride solu­tions.

Antigens usually used in the IHA test are polysaccharide anti­gens of microorganisms, extracts of bacterial vaccines, antigens of viruses and Rickettsia, as well as other protein substances.

Erythrocytes sensitized with antigens are called erythrocytic di­agnosticums. Most commonly used in preparing erythrocytic diagnosticums are sheep red blood cells possessing high adsorptive activity.

Procedure. Blood taken from the jugular vein of an adult sheep is placed into a glass jar with beads, defibrinated by shaking for 10-15 min, and filtered through a cotton-gauze filter. Following 10-minute centrifugation at 2000 X g, red blood cells are washed 3-4 times in isotonic saline solution, and the sediment is resuspended in the same solution. Then, a five-fold volume of 4 per cent formalin (pH 7.0) is added to the jar and erythrocytes are left to stand at 4 °C for 3-4 days. The erythrocytes are precipitated once again and the procedure is repeated with a fresh solution of formalin. After this the red blood cells are washed with a 20-fold volume of physiolog­ical saline and adjusted to 20 per cent concentration. The fixed red blood cells are kept at 4 °C.

The suitability of erythrocytes is checked by the following crite­ria: (1) no haemolysis should be observed after freezing and thawing of 5 per cent erythrocyte suspension in distilled water; (2) mixing of 0.1 ml of 0.2 per cent suspension of erythrocytes with isotonic NaCI solution brings about no spontaneous agglutination.

To sensitize red blood cells, to eight volumes of distilled water add one volume of antigen, one volume of 50 per cent suspension of for­malin-treated erythrocytes, and one volume of 0.1-0.2 per cent solu­tion of chromium chloride and tannin in dilution 1:20000-1:2000000.

Allow the mixture to stand for 10-15 min at room temperature; then add an equal amount of isotonic saline and centrifuge it for 20 min at 2000 X g. The sediment of sensitized erythrocytes is washed two-three times with a 20-fold volume of physiological saline solution, then resuspended to 5 per cent concentration in the stabi­lizing solution consisting of equal volumes of 30 per cent solution of sucrose and human donor blood.

As a control, use formalin-treated red blood cells sensitized by another antigen or formalin-treated non-sensitized red blood cells.

It is convenient to set up an indirect haemagglutination test on micropanels of the Takata apparatus using a microtitrator for dilut­ing the material. The sera to be assayed are heated for 30 min at 56 °C to remove non-specific haemagglutinins, then two-fold dilutions on a stabilizing solution are prepared, and each dilution (by one drop) is placed into two wells of the U-shaped Takata microtitrator. To each well in the first row one drop of 1 per cent suspension of red blood cells sensitized with antigen is added, to the second row, the same amount of control erythrocytes. The plates are thoroughly shaken and put in a 37 °C incubator for 30-40 min.

The results of the test are assessed by the presence of haemaggluti­nation. It is considered positive if the titre of haemagglutination with the erythrocytes assayed exceeds by at least four times the titre of haemagglutination with the control erythrocytes. The sensi­tized red blood cells should be invariably checked for the absence of spontaneous agglutination.

 

 

 

Reaction with incomplete antibodies. Interrelations between protective (defensive) antigens and the corresponding antibodies are of a completely different nature. During this interactioo typical immunological reactions are observed (neutralization, precipitation and complement–fixation). It has been suggested that protective antigens provoke the formation of incomplete or blocking antibodies capable of rendering harmless the aggressins of anthrax bacilli, capsular proteins of the causative agents of plague, tularaemia and of other bacteria.

Incomplete (monovalent) or blocking antibodies are fixed by the antigens, but do not cause their agglomeration. In contrast to ordinary (complete) antibodies they proved to be more stable to heat, pressure, and chemicals, and quite easily penetrate through the placenta. They include rhesus-agglutinins, non-precipitating thermolabile antibodies and reagins of allergic patients, and of patients with systemic lupus,  infectious polyarthritis, and  collagenosis. Incomplete hetero-, iso-, and autoantibodies may cause drug leuko- and thrombocytopenia.

Incomplete agglutinins and haemagglutinins have been demonstrated in immunization of animals with the capsular antigen of the causative agent of plague. They were found in the sera of patients suffering from dysentery, typhus, and brucellosis in titres 4-32 times those of complete antibodies; in the sera of animals immunized with the cholera vaccine their titre was 4-8 times that of complete antibodies.

Of most interest are agglutinins against the rhesus-antigens of erythrocytes of children suffering from haemolytic disease which is the result of the presence of a rhesus-factor in the erythrocytes inherited from the father. After penetrating into the blood of the mother the rhesus-factor provokes the production of rhesus-agglutinins which later enter the blood of the foetus through the placenta and cause agglutination of erythrocytes. Haemolytic disease is due to the incompatibility of the rhesus-factor in the blood of the mother and the foetus.

The rhesus-factor is capable of causing the production of two types of agglutinins: (1) complete (bivalent) agglutinins which in a saline and colloidal medium may cause the agglutination reaction of erythrocytes containing a particular antigen, and (2) incomplete (monovalent) agglutinins inhibiting agglutination, which do not cause the agglutination reaction in a saline solution.

For detecting incomplete antibodies special methods are employed. The Coombs’ test is used, in particular to detect incomplete agglutinins in rhesus-negative mothers. To determine the fixation of agglutinins by the patients’ erythrocytes, antiglobulin serum is added, which, in a saline solution, is capable of causing marked agglutination of erythrocytes sensitized by incomplete agglutinins. A molecule of antiglobulin binds two molecules of incomplete agglutinins fixed to two different erythrocytes, due to which the agglutination reaction takes place.

 

Coombs’ test represented schematically (1–direct; 2– indirect)

 

The direct reaction (Fig. 1, left) demonstrates the presence in the patient’s blood of antibodies bound with the erythrocytes by means of antiglobulin: in the indirect reaction (Fig. 1, right) free incomplete antibodies are revealed by adding to the serum normal erythrocytes, bacteria or rickettsia and then antigamma globulin.

 

 

 

 

Reversed indirect haemagglutination (RIHA) test is used for detecting bacterial and viral antigens in the  materials to be exam­ined as well as for the rapid diagnosis of a number of infections.

In contrast to IHA. erythrocytes in this test are sensitized not by antigens but by antibodies whose agglutination occurs upon ad­dition of the antigen.

Erythrocytes are first fixed with formalin or glutaraldehyde and bound to gamma-globulin which is isolated from immune sera and purified from other serum proteins. Binding of gamma-globulin with the erythrocyte surface is mediated by chromium chloride. For this purpose, to 8 volumes of distilled water add 1 volume of immunoglobulins obtained from immune serum, 1 volume of 50 per cent sus­pension of formalin-treated red blood .cells, and 1 volume of 0.1-0.2 per cent solution of chromium chloride. Allow the mixture to stand at room temperature for 10-15 min, thetreat the erythro­cytes as in the passive haemagglutination test.

This test is commonly used to identify the causative agents in post-mortem material taken from the organs of man and animals, for example, from the brain, spleen, liver, and lungs. Prepare 10 per cent suspension of the above organs with isotonic sodium chlo­ride solution, centrifuge it at 10 000 X g for 30-60 min, and use the  supernatant as an antigen.

Procedure. Prepare two-fold dilutions of the material to be studied (antigen) with a stabilizing solution. Place one drop of each dilution of the antigen into 3 neighbouring wells of the micropanel (the reac­tion requires 3 parallel rows of wells). Into each well of the first row add 1 drop of the stabilizing solution, into wells of the second row, 1 drop of homologous immune serum in a 1:10 dilution, those of the third row, 1 drop of heterologous immune serum. The second and third rows serve as controls of reaction specificity. Let the mix­ture stand at room temperature for 20 min.

To all wells add one drop of 1 per cent suspension of sensitized erythrocytes (erythrocyte antibody diagnosticum) and shake the plates well. Read the results of the reaction in 30-40 min. In the presence of the specific antigen, haemagglutination is observed in the first and third rows (with heterologous serum) and is absent in the second row -where the antigen is preliminarily neutralized by homo­logous serum.

To ensure the accuracy of the test, the sensitized red blood cells are checked for spontaneous agglutination.

Reversed indirect haemagglutination inhibition (RIHAI) test makes it possible to detect the presence of antibodies in human and animal sera.

Procedure. Dilute sera by ten-fold with isotonic sodium chloride solution, heat for 20 min at 65 °C to destroy nonspecific inhibitors, then prepare two-fold serum dilutions with stabilizing solution and the working dose of the antigen containing four agglutinating units. Introduce by one drop of each dilution into the wells of the micro-panel and add to each of them one drop of the antigen whose dilution corresponds to the working dose. Allow the mixture to stand for 20 min at room temperature, then place one drop of the erythrocyte antibody diagnosticum into each well, and shake them thoroughly. Read the results of the reaction after 1.5-2 hour-incubation at room temperature. The titre of the serum is its greatest dilution which completely inhibits haemagglutination with four agglutinating units of the antigen.

The test is validated by checking whether sensitized erythrocytes may undergo spontaneous agglutination in the presence of: (a) stabilizing solution; (b) normal antigen (from material free of the virus); (c) the serum tested. Among advantages of the test one can cite its universality and the possibility to use it for finding various antigens.

Assessment of the haemagglutination results. Estimation of the results of IHA, BIHA and RIHAI tests is relied on the degree of erythrocyte agglutination; (++++)> complete agglutination; (+++), almost complete agglutination; (++), partial agglutina­tion; (+), traces of agglutination; (–), no agglutination.

The test is considered positive, if agglutination is complete (++++) or almost complete (+++)i the diagnosticum does not induce spontaneous agglutination in the presence of each compo­nent required for the reaction, and the control test of the specificity of the antigen or antibody is positive.

Recent years have seen wide application of serological reactions which make use of antibodies or antigens labelled in some way. The label may be different but should meet the basic requirement: it should be easily detected by means of definite reactions or under the microscope. Apart from retaining the specificity of immunological reactions, serological reactions with labels make it possible to rapidly obtain the results and are usually distinguished by high sensi­tivity. It is not unnatural, therefore, that they have found wide­spread application for the rapid diagnosis of viral and bacterial in­fections.

Latex agglutination test: latex beads coated with specific antibody are agglutinated in the presence of the homologous bacteria or antigen. This test is used to determine the presence of the  H. influenzae, N meningitidis, several species of streptococci, and the yeast Cryptococcus neoformans.

 

The following types of labels are employed most frequently: (1) fluorochromes capable to fluoresce in ultraviolet rays or the blue-violet area of the spectrum of visible light, for example, fluoresce-inisothiocyanate (FITC) which is utilized for performing immunofluorescent reactions; (2) ferritin, a protein containing up to 23 per cent of iron, which is readily visible by electron microscopy and thus is well suitable as a label in immunoelectron microscopy; (3) en­zymes which induce the breakdown of the substrate with the formation of stained products when they get in contact with the substrate; they are used for performing immunoenzymic reactions; (4) radio­active labels utilized in highly sensitive radioimmunoassays.

The above serological tests vary in their sensitivity and diagnostic value. Some of them (e.g., radioimmunoassay) require the employ­ment of sophisticated recording equipment, as well as special mea­sures of protection from radiation.

Immunofluorescence (IF) test relies on the utilization of FITC or other fluorochromes which are chemically conjugated with anti­bodies. The antibodies labelled by FITC (as part of immunofluorescent sera) retain the immunological specificity and interact with strictly delinite antigens. Antibody-labelled antigen complexes are readily recognized by intense yellow-green fluorescence during exam­ination with the luminescent microscope.

There are several variants of immunofluorcscence (or Coons’ reaction).

1. Direct immunofluorescence envisages the employment of immunofluorescent sera against each antigen tested.

2. Indirect immunofluorescence is based on the use of two sera. First, unlabelled antibodies against the antigen to be assayed are utilized. At the second stage, the formed antigen-antibody complex is treated with FITC-labelled serum containing antibodies against gamma-globulins of that species of animals from whom the antisera used at the first stage of the reaction were obtained (anti-species se­rum).

For example, if the serum employed at the first stage of the reac­tion has been obtained by rabbit immunization, at the second stage one employs labelled anti-species rabbit serum obtained by immuniz­ing with rabbit gamma-globulins of donkeys or some other ani­mals. In this case antiglobulin-labelled antibodies coat the antigen tested with a second layer (the first one has been formed by unlabelled antibodies which, in turn, serve as antigens for antiglobulin (anti-species) serum). As a result, the antigen becomes visible under the luminescent microscope.

Immunofluorescence may be used for studying various antigens: cultures of bacteria, fungi, Protozoa, preparations from patients’ material; infected cultures of cells, tissue sections, etc. The mate­rial to be examined is placed on a glass slide and fixed (most often in acetone for 10 min at room temperature) after which it is dried for 20 min at 37 °C. The subsequent treatment of preparations de­pends on the variant of immunofluoroscence utilized.

 

Figure. 2 Fluorescein labeled antibody test methods Note that on must have fluorcscein-labeled antibody that is specific rur the organist in question in order to use the direct method, however, the indirect method can be adapted to any organism using fluorcscein labeled ant human gamma globulin as the only labeled antiserum.

 

 

In direct immunofluorescence the preparation is stained with spe­cific labelled serum in a humid chamber for 30 min at 25 °C, after which it is washed with buffer saline (pH 7.2) for 10 min at 25 °C.

In indirect immunofluorescence the preparation is first treated with unlabelled specific serum in a humid chamber for 30 min at 25 °C and then washed with buffer solution (as in the case of direct immunofluorescence). After that the preparation is stained with anti-species labelled serum in a humid chamber for 30 min at 25 ”C and washed with buffer salt solution.

 

 

 

Ready preparations are dried with filter paper and examined un­der a luminescent microscope, first, with a dry objective (40 x), then, after having placed on the preparation a drop of non-fluorescent oil, with the immersion objective (90 x). Attention is paid not only to the presence of green or green-yellow luminescence, but also to its intensity and the arrangement of luminescence in the cell examined.

To rule out false positive results, a number of control techniques is available. Of particular significance among them is a control with a heterologous antigen (for example, with bacterial culture which does not correspond to the serum tested from an antigcnic stand­point). In studying infected cultures of cells, a control with normal noninfected culture should be used (to exclude autofluorescence and non-specific binding of labelled antibodies with the cell surface). To inhibit auto fluorescence of the preparation, one can use bovine al­bumin labelled with sulpharodamine.

While retaining the pecificity of immunological tests, the mmunofluorescence reaction is distinguished by its simplicity and rapidi­ty. Indirect immunofluorescence may be employed not only for in­vestigating antigens but also for determining the number of antibod­ies in immune serum. On the other hand, the IF test cannot be considered as a highly sensitive reaction. Furthermore, non-specific adsorption of labelled antibodies on the preparation with the appear­ance of false positive results is also possible.

Fluorescein-labeles antibody. Fluorescein-labeled antibodies are used in a procedure for rapidly determining the presence of specific antigens or antibodies to a known antigen. As illustrated in Figure2, the indirect method can be used to detect the presence of antibodies to any bacterium. All that is required is known bacteria, the patient’s serum, and some fluorescein-labeled antihuman y-globulin.

The direct method, also illustrated in Figure 2, can be used to confirm a tentative identification of an isolated organism. In this case, however, the microbiologist is limited by the availability of fluorescein-labeled specific antibody to the bacterium in question. The commercial availability of many different monoclonal antibodies has facilitated the rapid identification of many bacteria and viruses. Such antibodies can be selected to provide a much higher specificity than can be obtained with a heterologous population of antibodies. Monoclonal antibodies can be linked to fluorescent dyes and used to detect microorganisms in tissues and infected cell cultures.

Direct method: 1 Antigen. 2.Cover with fluorescein-labeled group A streptococcus antiserum and incubate 3.Wash away the excess fluoroscein-labeled antiserum and examine.

Indirect method: 1.Antigen. 2.Cover with unlebeled serum from patient with active syphilis and incubate 3. Wash away the excess unlabeled patient’s antiserum. 4. Cover with fluoroscein-labeled anti-human gammaglobulin antiserum and incubate. 5.Wash away the excess  fluoroscein-labeled antiserum and examine.

 

 

Precipitins and the Precipitin Reaction. Precipitins are antibodies which bring about the formation of a minute deposit (precipitate) upon interaction with a specific antigen. The precipitin reaction is a specific interaction of the antigen (precipitinogen) and antibody (precipitin) in the presence of an electrolyte (0.85 per cent NaCI solution) with the formation of a deposit or precipitate. In 1897 R. Kraus roved that the transparent filtrate of a broth culture of plague bacteria became turbid when mixed with an antiplague serum with the subsequent falling-out of flakes to the bottom of the test tube. An analogous phenomenon was observed upon the interaction of cholera and typhus culture filtrates with the corresponding sera. R. Kraus named this a precipitin reaction, and the antibody producing it –  precipitin. After the injection of eel serum protein into rabbits in 1899 F. Chistovich in E. Metchnikoffs laboratory established that antibodies (precipitins) occur in their blood which,  upon interaction with eel serum protein, produce a precipitate. The precipitin reaction is most clearly obtained when the transparent filtrate (antigen solution) is layered on the transparent precipitating serum. A greyish-white ring appears comparatively rapidly at the junction of the reacting components.

In mechanism this reaction is similar to the flocculation reaction and the physicochemical interrelations of highly dispersed colloids form the basis for it.

The precipitin reaction is specific and sensitive. It allows the detection of antigen  precipitinogen) in a dilution up to 1 : 1000000 and 1 : 10000000.

Precipitinogens during parenteral injection provoke the formation of specific precipitins in the body and combine with them. Proteins of animal, plant and microbe origin: blood, serum, extracts from different organs and tissues, foodstuffs of a proteiature (meat, fish, milk), filtrates of microbial cultures or affected tissues, may all be used as precipitinogens.

Precipitinogens of the causative agents of anthrax, plague and tularaemia are thermoresistant. Some precipitinogens withstand heat up to 120-180 °С.

The precipitin reaction is used in the diagnosis of anthrax, tularaemia, etc., in the typing and studying of antigen structure of certain groups of bacteria. In forensic medicine with the aid of the precipitin reaction the origin of blood spots and sperm is determined, in sanitary examination an admixture of milk of one species of animal to another is revealed, the addition of artificial honey to natural, the falsification of meat, fish and flour goods, etc., and in biology the genetic links between related species of animals, plants and micro-organisms are established.

The precipitin reaction is most widespread in the diagnosis of anthrax (Ascoli’s test) for detecting the antigen of anthrax bacilli in extracts from the organs of animals, skin, wool, hair and also for the control of the manufactured goods: fur jackets, fur collars, shaving brushes and other goods. This reaction is known as the thermoprecipitin reaction, since the extract which undergoes investigation is preliminarily boiled, then filtered to obtain a transparent solution and layered on the precipitating anthrax serum. The thermoprecipitin reaction {ring precipitation} is widely used for the diagnosis of plague and tularaemia during the investigation of extracts prepared from the internal organs (spleen, liver) of the cadavers of wild rodents.

In special laboratories there is a set of precipitating species-specific sera which are obtained by long-term immunization of animals with the corresponding antigens (precipitinogens). At the end of the immunization course blood is taken from animals (rabbits, donkeys, sheep, goats), a serum is obtained and the strength of its action is determined

The titre of the precipitating serum is known as the maximum dilution of antigen (precipitinogen) in which a clearly expressed precipitin reaction is obtained. Whole precipitating serum is taken because the dispersion of the precipitating serum is less in comparison to that of the precipitinogen. For this reason in order to obtain optimal quantitative proportions of particles of acting components the antigen and not the serum is diluted.

In order to differentiate antibodies against different antigens, the method of the diffusion precipitin reaction with antiserum mixed with gelatin or agar is used. After the antigen solutions are layered easily discernible precipitation zones occur within this agar, each specific pair of antigen-antibody complex having its own zone. This method was later improved. In wells made in agar in Petri dishes solutions of antigens and antibodies are poured which diffuse into the gel, and after coming in contact with one another form lines of precipitation. The merging of the ends of precipitating lines provides evidence for the similarity of antigens of comparative systems.  At present there are several modifications.

The precipitin reaction can be carried out on paper. If the mixture of antigens is isolated by the method of paper electrophoresis and then the strips of paper are treated with immune serum, then a precipitate is produced in definite places, the localization of which determines the nature of antigen if the origin of the antibody is known, and vice versa. The precipitin reaction became the basis of the method of immunoelectrophoresis proposed by P. Grabar and K. Williams. At first, the antigen is separated in the electric field, after which it is developed by antiserum poured in a groove running parallel to the line along which the antigens moved during electrophoresis. Each antigen gives an individual band with the antibody. From the number and arrangement of the bands one can determine the presence of these or other antigens in the solution under test. With the help of immunoelectrophoresis antigen fractions which were unknown earlier have been revealed in various complexes. It allows to detect pathological deviations in the sera of patients. The immunoradiographic method is very effective.

 

 

 

 

 

Modifications of the Precipitin Reaction. Several modifications of the classic precipitin reaction have been developed. Such changes are designed either to increase the sensitivity of the reaction or to identify specific Ag-Ab reactions occurring in a system containing multiple Ags and Abs.

Double diffusion (Ouchterlony technique). When soluble Ag and soluble Ab are placed in separate small wells punched  into agar that has solidified on a slide or glass plate, the Ag and the Ab will diffuse through the agar. The holes are located only a few millimeters apart, and Ab and Ag will interact to form a line of precipitate in the area in which they are in optimal proportions. Because different Ags diffuse at different rates, and because different Ags can require different concentrations of Ab for optimal precipitation, the position of the precipitin band usually varies for each Ag. In specimens containing several soluble Ags, multiple precipitin lines are observed, each occurring between the wells at a position that depends on the concentration of that particular Ag and its Ab (Fig.1). Thus, this simple diffusion method can be used to detect the presence of one or more Ags or Abs in a clinical specimen. In addition, if set up differently, this gel method can be used to detect similarities or differences in Ags. For example, Figure 2 shows the use of various Abs to distinguish between human IgM and IgG, and to detect specific epitopes on human IgG

 

 

 

 

Radial immunodiffusion. Radial immunodiffusion is used to measure the amount of a specific Ag  present in a sample and can be used for many Ags. The most widely used diagnostic application of this procedure is to measure the amount of a specific Ig class present in a patient’s serum. The assay is carried out. by incorporating monospecific antiserum (antiserum containing only Ab to the Ag being assayed for) into melted agar and allowing the agar to solidify on a glass plate in a thin layer. Holes then are punched into the agar, and different dilutions of the Ag are placed into the various holes. As the Ag diffuses from the hole, a ring of precipitate will form at that position where Ag and Ab are in optimal proportions (Fig. 3). The more concentrated the Ag solution, the farther it must diffuse to be in optimal proportion with the constant Ab concentration in the agar gel. Thus, the diameter of the precipitin ring is a quantitative measure of Ag concentration. Using known concentrations of the Ag in question, a standard curve can be prepared by plotting the diameter of the precipitin ring versus Ag concentration.

Once a standard plot is obtained, the diameter of the precipitin ring formed with the unknown Ag can be measured to calculate its concentration.

Nephelometry is supplanting radial immunodiffusion as a method of measuring various Ig classes present in a patient’s serum. As discussed earlier, when Abs to various  classes of Igs are mixed with serum, Ag-Ab complexes form and create a precipitate in a previously clear solution. Tubes containing known concentrations of Abs to Igs are incubated with different volumes of a patient’s serum. The precipitation reaction, seen as a cloudiness, is measured in an instrument called a nephelometer. This instrument interprets the Ag-Ab precipitates as increased light scattering compared to a control tube containing no precipitate. As with the radial immunodiffusion procedure, iephelometry, standard curves are performed using Ig class standards of known concentrations and Abs to the various Ig classes.

Through a certain concentration range, it is possible to generate a straight line plot when Ig concentration is plotted as a function of the amount of light scattering indicated by the nephelometer. This technique is being used increasingly in hospital  laboratories and clinics as a quantitative measure of serum Ig class concentration in patient blood.

 

FIGURE 3 Radial immuno-diffusion This test is based on a precipitate that is formed as antigen diffuses into a semisolid medium that contains antibody Because the amount of antibody in the agar bed is constant, the diameter of the precipitin ring formed is a function of the amount of antigen applied A. Ann IgA is incorporated into the agar and small wells are punched out, into which patient serum or a standard antigen solution is placed The top row consists of standard amounts of known IgA The wells in the bottom row receive sera from four different patients B. A curve is drawn by plotting the diameter of the precipitin rings formed by the standard IgA solutions against the logarithm of the concentration of the standard IgA The concentration of IgA in each patient’s serum then is read from this standard curve after measuring the diameter of the ring formed by the patient’s serum in the bottom row.

Precipitation Test. The term precipitation test (PT) refers to sedimentation from the solution of the antigen (precipitinogen) upon its exposure to immune serum (precipitin) and electrolyte. Using the precipitation testю Using the precipitation test (PT), one can demonstrate the antigen in such tiny amounts which can­not be detected by chemical techniques.

Conduction of the PT requires liquid and transparent antigens representing ultramicroscopic particles of colloid solution of pro­tein, polysaccharides, etc. Antigens are represented by extracts from microorganisms, organs, and tissues, products of breaking down of microorganism cells (lysates, filtrates). Resistance of precipitinogens toward a high temperature is used for  for obtaining antigens from the causative agents of anthrax, plague (the boiling technique).Precipitating sera are prepared in a batch manner by hyperimmunization of animals (rabbits) with bacterial suspension,  filtrates of broth cultures, autolysates, salt extracts of microorganisms, serum proteins, etc.

The titre of the precipitating serum, in contrast to the titre of other diagnostic sera, is determined by the maximum dilution of the antigen which is precipitated by a given serum. This is explained by the fact that the antigen participating in the precipitation reac­tion has an infinitesimal magnitude and that in a volumetric unit of the serum there are much more antigens than antibodies. Commercial­ly available precipitating sera have the titre of no less than 1:100 000.

Procedure. In a narrow test tube (0.5 cm diameter) pour 0.3-0.5 ml of undiluted precipitating serum. With a Pasteur pipette slow­ly layer on the wall (the tube is held-in a tilted position) the same volume of antigen. Then carefully, in order to avoid mixing of the fluids, set up the tube in a vertical position. Provided the layering of the antigen on the serum has been correct, one can see a distinct borderline between the two layers of liquid. The PT should always be coupled with control testing of the serum and antigen.

Precipitation test:  1 – in a test tube;  2—in gel; 3 –immunoelectrophoresis (order of picture: left–right–down )         

 

The precipitation test is widely employed in the laboratory prac­tice for the diagnosis of infectious diseases of the bacterial (anthrax, plague, tularaemia) and viral (natural small pox, acute respiratory infection) nature.

The results of the test are read in 5-10 min, 1-2 hrs or 20-24 hrs, depending on the type of an antigen and antibodies. If the reaction is positive, a precipitate in the form of a white ring forms on the borderline between the serum and the extract tested.

 

 

In forensic medicine the precipitation test is employed for classi­fying the species of the protein (blood stains, sperm).

The PT may demonstrate not only species but also group specificity of the protein. Thus, the degree of homogeneity of various spe­cies of animals and plants has been determined with its help.

The use of the precipitation test for sanitary and hygienic control of foods luns makes it possible to uncover adulteration of meat, fish, and flour products, as well as admixtures in milk. etc.

Disadvantages of the PT are instability of the precipitate (the ring) which disappears even upon the slightest shaking and impossibility to establish the number of various antigens participating in the formation of the precipitate.

The precipitation reaction in gel is free of these disadvantages. Test of precipitation in gel (PG) is based on the interaction of homologous antibodies and antigens in an agar gel and the formation of visible bands of precipitation. As a result of counter-diffusion into gel, the antibodies and antigen form immune complexes (aggregates) visualized in the form of opalescent (white) bands (Fig.4).

When several antigens diffusing irrespective of each other are present, the number of bands corresponds to the number of antigens. Serologically homogeneous antigens form precipitation bands which merge with each other, whereas bands of heterogeneous antigens cross each other. This property permits determination of the homo­geneity of the antigenic structure of various objects tested.

Components in the precipitation reaction in gel are agar gel, anti­gen, and antibodies. For the purpose of quality control of the precip­itation test in gel, the test system comprised of known homologous antibodies and antigens is utilized.

The antigen used in a precipitation test should be concentrated, while the sera (from patients or immunized animals) should be of a high titre.

Procedure. To prepare gel, use 0.8-1 per cent solution of Difco’s agar or agarose on isotonic sodium chloride solution, which is lay­ered on clean slides 1-2 mm thick.  In a solidified agar cut the well? and remove the agar from them with a Pasteur pipette. Into one series of wells place serum, into the other, antigens and put the slides into a humid chamber for several days. In reading the results of the reaction, compare the localization and nature of precipitation lines in the test and control wells. To measure the levels of the antigen and antibodies, study their multiple dilutions.

The precipitation test in gel is widely employed in the diagnosis of diseases caused by viruses, Rickettsia. and bacteria producing exotoxins. It has become of great practical significance with regard to determining the toxigenicity of Corynebacteria of diphtheria.

Immunoelectrophoresis (IEP) test allows analysis and identifica­tion of individual antigens in a multi-component system (Fig. 1, 3). The IEP test is based on the  electrophoretic division of antigens in the gel with their subsequent precipitation by antibodies of the im­mune serum. To perform immunoelectrophoresis, glass plates with. an agar layer are used. First, the antigens placed in the centre of a plate are divided in the electrical field. Then, immune serum is added to an agar slit running parallel to the line of the antigen division. As a result of their mutual diffusion, the antigens and anti­bodies form the arches of precipitation at the place where they meet.

Counterimmunoelectrophoresis (CIEP) test is based on counter-diffusion in the electrical field of antigens and antibodies and the appearance of a visible precipitate inside a transparent gel. In agar or agarose gel cut the wells 2-3 mm in diameter so that the distance between wells for the serum and antigen is 5-6 mm. The wells are arranged in pairs (one for the antigen, the other for the serum) or in triple series (one for the antigen, the second for the serum tested, the third for the control serum). Wells for the serum are positioned closer to the anode and those for the antigen closer to the cathode. The reaction is set up with several dilutions of the antigen and lasts 90 min. The results of the test are read immediately upon the cessa­tion of electrophoresis by noting the number and localization of pre­cipitation lines and comparing them with the control system.

 

Lysins and the Lysis Reaction. Lysins are specific antibodies which cause the dissolution of bacteria, plant and animal cells.

Under the influence of antibodies and a substance contained iormal serum, complement, the dissolution of microbial cells (bacteriolysis) takes place, or bactericidal action accompanied by destruction of microbes without any noticeable morphological changes occurs.

In 1884 V. Gromann established the bactericidal action of normal serum on the microbes of anthrax. V. Isaiev and R. Pfeiffer revealed antibodies (bacteriolysins) dissolving bacteria in the blood of immune animals. The Isaiev-Pfeiffer phenomenon may be reproduced in guinea pigs, actively or passively immunized against cholera. When a culture of cholera vibrios is injected into the peritoneal cavity of an immunized guinea pig, they lose their motility fairly rapidly, swell, become spherical, then granular and then finally completely disappear and dissolve. The same phenomenon is observed during simultaneous injection of live cholera vibrios and anticholera serum into a guinea pig. E. Metchnikoff and J. Bordet established that bacteriolysis may be observed outside the body by adding fresh immune serum to a bacterial suspension. In later investigations it was established that bacteriolysis depends not only on the antibody which appears under the influence of immunization, but also on the thermolabile substance (complement) found in all kinds of fresh serum, and which is disintegrated by heating at 56 C for half an hour. Thus, bacterial lysis takes place with the participation of two components: a specific antibody contained in the immune serum and a non-specific substance of any normal or immune serum — complement. The antibody which together with complement causes bacterial lysis ond system). Both systems are placed separately in a thermostat for half an hour. Then they are mixed, poured into one test tube, and the mixture is placed again in a thermostat for 30-60 minutes If the complement-fixation reaction proves to be positive, that is, if complement is bound to the antigen-antibody complex of the first system, then the second system (erythrocytes + haemolytic serum) does not change as no complement is left for it. Within a day the erythrocytes settle to the bottom, and the supernatant liquid is transparent and colourless. During a negative reaction the complement will not combine with the complex of the first system, while the liquid becomes pink (lacquered) without any precipitate of erythrocytes.

The complement-fixation reaction has a high specificity and a marked sensitivity.

According to the mechanism of action this reaction is the most complex m comparison to reactions of agglutination and precipitation and proceeds m two phases. In the first phase precipitation occurs between the antigen and antibody (mutual adsorption), and in the second, fixation of the complement by the antibody-antigen complex takes place.

Complement participates in all immunological reactions, while in some reactions the presence of complement is obligatory (lysis, complement-fixation), in others it is non-obligatory (neutralization of toxin by antitoxin, precipitation, agglutination and opsonization).

The complement-fixation reaction is used in the diagnosis of glanders, syphilis (Wassermann reaction), etc. In recent years it has been used successfully in discerning typhus fever, Q fever and other rickettsioses and many virus diseases. Modifications of the complement fixation reaction have been devised for determining antibodies as well as antigens in the blood of patients. Preparation and titration of the ingredients and the method of the test are described in more detail in a manual.

 

 

Lysis Test. The term lysis reaction refers to dissolvement of the antigen conjugated with antibodies in the presence of a complement. Depending on the nature of antigens participating in the lysis reaction, it may be called spirochaetolysis, vibrionolysis, bacteriolysis, haemolysis, etc. Antibodies involved in the corresponding reactions are called spirochaetolysins, vibrionolysins, haemolysins, etc. Lysins exert their action only in the presence of a complement.

Most microorganisms with the exception of the cholera vibrio and Treponema are resistant to the lytic action of antibodies. Hence,. the lysis test has failed to find the wide-scale use in laboratory practice.

In carrying out the lysis reaction (Table 1), the immune serum is  heated for 30 min at 56 °C to inactivate the complement present in it. Suspension of microorganisms is prepared from their 24-hour culture (1 milliard per 1 ml). To the first test tube with 0.5 ml of the tested serum diluted 1:10 (or 1:20,1:40, etc.) 0.1 ml of the prepared suspension of microorganisms and 0.1 ml of undiluted complement are added. To the second tube serving as a control of complement in-activation in the immune serum no complement is added. In the third tube serving as a control of the complement for the absence of lysins the immune serum is replaced with isotonic sodium chloride solution.

Place the tubes into a 37 °C incubator for 2 hrs, then streak with the loop from each test tube onto Petri dishes with solid nutrient medium. Incubate the inoculated cultures at 37 °C for 24 hrs and acount the colonies.

If the serum to be tested contains lysins, the number of colonies on a nutrient medium inoculated with the material to be assayed will be many times lower than in dishes containing the material from the control tubes.

The haemolysis reaction is used as an indicator system in the complement-fixation test.

 

 

The complement-fixation (CF) test belongs to complex serological reactions. It requires five ingredients to be performed; namely, an antigen, an antibody and complement (the first system), sheep red blood cells and haemolytic serum (the second system). Specific interaction of the antigen and antibody is attended by adsorption (binding) of the complement. Since the process of complement binding cannot be visualized, Bordet and Gangou have proposed to employ a haemolytic system (sheep erythrocytes plus haemolytic serum) as an indicator which shows whether the complement is fixed by the antigen-antibody complex. If the antigen and antibody correspond to each other, the complement is bound by this complex and no haemolysis takes place. If the antibody does not correspond to the antigen, the complex fails to be formed and free complement combines with the other system causing haemolysis.

CF, as other serological tests, may be used for identifying specific antibodies by a known antigen as well as for determining an antigen by known antibodies. The performance of the CF test calls for special preparation. The glassware (test tubes, pipettes, vials) are thoroughly washed and care is takeot to use them for other purposes. All ingredients of the reaction are prepared and titrated prior to the main test.

1. Before the test, the serum (either obtained from a patient or the diagnostic one) is heated on a water bath at 56 C for 30 min in order to inactivate its inherent complement.

Some sera, particularly those from immunized animals, possess anticomplement properties, i.e., they can bind complement in the absence of a homologous antigen. This property of some sera is eliminated by treating them with carbon dioxide, heating at 57-58 0C, single freezing at —20 0C or —70 0C, and removing the sediment by centrifugation, addition to the scrum of a complement in a 1:10 ratio, and its heating after 18-20-hour incubation in the cold. To prevent the anti-complement activity of sera, they are kept in the lyophilized or frozen form at low temperatures.

2. Cultures of variable killed microorganisms, their lysates, bacterial components, of abnormal and normal organs, and tissue lipids, as well as viruses and virus-containing materials may be used as antigens for CF. Many antigens from microorganisms are available commercially.

3. The anti-complement activity of antigens is eliminated by such methods as thermolysis (multiple freezing and thawing), treatment with lipid solvents (ether, chloroform, acetone), and alcohols (methanol, ethanol).

Guinea pig serum collected immediately before a reaction is used as a complement; dry complement may also be employed. To obtain the basic solution for subsequent titration, the complement is diluted 1:10 with isotonic saline.

4. Sheep erythrocytes are employed as a 3 per cent suspension in isotonic sodium chloride solution. A blood sample (100-150 ml) is drawn from the jugular vein, put, into a sterile jar with glass beads, defibrinated by shaking for 10-15 min, and filtered through 3-4 layers of sterile gauze to remove fibrin. Erythrocytes are washed three times with isotonic saline by adding it to the erythrocytic sediment until the initial volume of blood is achieved. Red blood cells may be stored for 5-6 days at 4-6 “C. Their shelf life is increased if they are preserved by using either formalin (0.1 ml of undiluted formalin per 80 ml of defibrinated blood) or some other technique.

5. Haemolytic serum for complement-fixation is obtained in the following manner. Rabbits are immunized by injecting into the ear vein 50 per cent suspension of washed sheep erythrocytes (by 1-ml portions 4-6 times every other day). Seven days after the last injection the serum tested is obtained. If the serum titre is equal to or over 1:1200, bloodletting is performed. The serum is heated for 30min at 56 0C. To prevent bacterial growth, a conservant (merthiolate 1:10000 or 1 per cent boric acid) is added to haemolytic serum.

6. A haemolytic system consists of haemolytic serum (taken in a triple titre)and 3 per cent suspension of sheep erythrocytes, mixed in equal volumes. To sensitize the erythrocytes by haemolysins, incubate the mixture at 37 °C for 30 min.

Titration of haemolytic serum. Serum is titrated by mixing 0.5 ml of the serum (in dilution 1:600; 1:1200; 1:1600; 1:3200, etc.) with 0.5 ml of 3 per cent suspension of red blood cells and 0.5 ml of fresh complement in dilution 1:10 (Table 2). The volume of the mixture for reaction in the control tubes is adjusted to 1.5 ml by adding 0.5 ml of isotonic sodium chloride solution. The results of the reaction are read after 1-hour incubation at 37 °C. The titre of haemolytic serum is defined as its maximum dilution causing complete haemolysis. The serum is stored in the lyophilized form.

Complement titration. Before the test, the basic solution of complement (1:10) is dispensed into a series of test tubes in quantities varying from 0.05 to 0.5 ml, and then isotonic sodium chloride solution is added to each tube, bringing the volume of the fluid to 1.5 ml. The test tubes are incubated at 37 °C for 45 min, then the haemolytic system is added to them and they are reincubated for 30 min, after which the complement titre is measured (Table 3).

To perform the test, one takes the working dose of complement (contained in an 0.5-ml volume) exceeding the titre by 20-30 per cent.

Table 2

Schematic Representation of Haemolytic Serum Titration

 

 

Table 3

Schematic Representation of Complement Titration

 

 

Antigen titration. Antigens employed in complement fixation may adsorb a certain amount of complement, i.e., may have inherent anti-complement properties. For this reason antigens are titrated in the presence of the working dose of complement before the lest. Commercially available specific antigens have less marked anti-complement properties. Their titre is determined less frequently (for example, once a month in view of its possible reduction during storing). In this case various amounts of the basic dilution specified in the instruction are used.To determine the titre of the antigen, it is dispensed into a number of test tubes in decreasing amounts varying from 0.5 to 0.05 ml, bringing the volume to 1 ml by adding sodium chloride solution. Then, 0.5 ml of the working dose of complement is added to each tube and they are placed in 37 °C incubator for 1 h. After that 1-ml portions of the haemolytic system are added to all tubes which are incubated for 1 h once again, and then the results of the reaction are read.

The titre of the antigen is its lowest amount sufficient to bring about complete haemolysis. For complement fixation the working dose of the antigen constituting about half to two-thirds of the titre is utilized. Antigens in whose presence the complement titre decreases by over 30 per cent are not suitable for the test.

Basic complement-fixation test. The total volume of ingredients involved in the reaction is 2.5 ml, the volume of the working dose of each of them is 0.5 ml. The diagram of the complement-fixation reaction shows (Table 4) that in the first test tube one introduces serum in the appropriate dilution, antigen and complement; into the second, serum in the appropriate dilution, complement and isotonic sodium chloride solution (serum control); into the third one, antigen, complement and isotonic saline (antigen control).

Table 4

Schematic Representation of the Basic CF Test

 

Simultaneously, a haemolytic system is prepared by mixing 2-ml portions of haemolytic serum in a triple titre (in relation to the one denoted in the instruction) and’ 3 per cent suspension of sheep erythrocytes (with regard to the initial volume of blood). The test tubes are incubated at 37 ‘C for 1 h, then 1 ml of the haemolytic system (the second system) is added to each of the first three tubes (the first system). After thoroughly mixing the ingredients the test tubes are reincubated at 37 °C for 1 h. The results of the reaction are read both preliminarily after removal of the tubes from the incubator and, finally, after they have stood for 15-18 hrs in a refrigerator or at room temperature.

In the final reading of the results the intensity of the reaction is expressed in pluses: (++++), a markedly positive reaction characterized by complete inhibition of haemolysis (the fluid in the tube is colourless, all red blood cells have settled on the bottom); (+++, ++), positive reaction manifested by the intensification of the liquid colour due to haemolysis and by a diminished number of red blood cells in the residue; (+), mildly positive reaction (the fluid is intensely colourful and there is only a small amount of erythrocytes collected on the bottom of the tube). If the reaction is negative (—), there is a complete haemolysis, and the fluid in the tube is intensely pink (varnish blood).

 

 

 

 

 

 

 

A number of complement-fixation modifications have been proposed, which are distinguished by elevated sensitivity and lesser volume of the ingredients used. Thus, the volume of ingredients for the CF test in the cold is 1 ml. For the drop CF test one takes 1 drop of serum + 1 drop of antigen +1 drop of complement + 2 drops of haemolytic system.

Despite its complexity, complement fixation is a sensitive and specific test, being used for these reasons for the diagnosis of many infectious diseases. Using the CF reaction, one can detect complement-binding antibodies in the blood serum obtained from patients with syphilis (Wassermann’s reaction), glanders, chronic gonorrhoea, rickettsiosis, viral diseases, etc.

Complement-binding antibodies make their appearance in the first days of the infection, yet their titre is relatively low. As a rule, antibodies reach the highest titre on the 7 th-10th-14 th day of the disease. Therefore, the most reliable are data obtained as a result of examining paired sera withdrawn at the onset of the disease and during convalescence.

 

Immunologic diagnosis in virology

According to the nature of the material to be tested and the proce­dures utilized, the methods for diagnosing viral infections may be categorized into rapid, viroscopic, virological, and serological (Table).

Table

 Methods of the Diagnosis of Viral Infections

 

Most of the relevant diagnostic techniques rely on the interaction between virus antigens and homologous antibodies in a fluid medium (complement-fixation (CF) test, haemagglutination inhibition (HAI) test, indirect haemagglutination (IHA) test, reversed indirect haem­agglutination (RIHA) test, reversed indirect hemagglutination inhibition (RIHAI) test, radioimmunoassay (RIA), or in gel (the test of precipitation in gel (PG), radial hemolysis (RH) test, immunoelectrophoresis (IEP) test, or during fixation of any ingre­dient in a solid medium (enzyme-linked immunosorbent assay (ELISA), radioimmunoassay (RIA), haemadsorption on a solid-medium (HadsSM) test, immunofluorescence (IF) test, haemadsorp­tion (Hads) test, and haemadsorption inhibition (Hadsl) test). In order to improve test sensitivity, antigens or antibodies are adsorbed on erythrocytes (IHA, RIHA, RIHAI, HadsSM, RH) or linked to enzymes (ELISA), isotopes (RIA, PG), and fluorochromes (IF); an alternative principle is erythrocyte lysis induced by the antigen-antibody interaction in the presence of complement (CF, RH).

The appropriate test procedures are described in detail in chapters dealing with serological diagnosis and with virus detection and identification in cell cultures. This chapter is devoted to the specific features of these tests and modifications which are used in the diagno­sis of viral infections.

Haemagglutination inhibition is based on blocking viral haemagglutinin by antibodies. The test is performed on plexiglass plates and interpreted as positive if erythrocytes fail to agglutinate on adding them to mixture of the  virus and specific serum. In order to remove or destroy non-specific haemagglutinaton inhibitors, test sera are pretreated with potassium periodate, kaolin, bentonite, acetone, or other agents. Then, the  sera are diluted two-fold in isotonic podi­um chloride solution, and every dilution is supplemented with an equal amount of virus-containing fluid which has four haemagglutinating units. The  mixture is incubated for 30-60 min at a tempera­ture optimal for a given virus (0°, 4°. 20°, 37 °C), and an equal volume of 0.5-1 per cent erythrocyte suspension is added. The mixture is reincubated for 30-45 min, and the results of the test are read. The  serum titre is defined as the  greatest serum dilution at which haemaglutination is inhibited.

 

Microhaemagglutination inhibition test using Takata’s micro-panel and loop is also widely employed.

Haemadsorption inhibition test is used for identifying haemadsorbing viruses and determining serum antibody titres. Specific serum (0.2 ml) diluted 1:5 is placed in test tubes with a culture of virus-infected tissue and following its incubation for 30-60 min. 0.2 ml of 0.5 per cent erythrocyte suspension is added. Nonimmune serum from the same animal species and erythrocytes are instilled in the control test tubes. The tubes are incubated for 20-30 min at a temperature which is optimal for the haemadsorption of the virus to be isolated. A conclusion about a species of the virus is based oil the  absence of erythrocyte adsorption in the test tubes in the presence of typical haemadsorption in the  control test tubes.

Neutralization test for an infective and cytopathic effect of viruses is performed in virus-sensitive live systems. A virus-containing specimen is serially diluted, and specific serum, diluted to a titre indicated on the ampoule label, is added. The mixture is incubated for 30-60 min at 37 °C and is used to infect tissue culture, chicken embryos, or laboratory animals. A sensitive system inoculated with the virus treated iormal serum serves as control.

Neutralization test is considered positive if the cell culture displays no CPE, chicken embryos show no changes, and the animals live without exhibiting any signs of disease. The findings obtained are used to determine a neutralization index which is a ratio of the virus titre in the control (where CPE is observed) to the test titre. The test is considered negative if the neutralization index is below 10, am­biguous if it varies from 11 to 49, and positive with an index of 50 or higher (significant virus-antiserum correlation).

The most sensitive version of the N test is inhibition of virus plaque formation by virus-specific antiserum (virus plaque reduction test). For this test, a virus-containing specimen (50-100 plaque-forming units) is supplemented with antiserum (diluted to a speci­fied titre), and, after 30-60-min incubation in a heating block, the mixture is applied onto monolayers of sensitive cell cultures. Match­ing of the virus to the employed antiserum is expressed in reduced plaque formation as compared with control. The N test helps to ascertain the  virus species and type (variant).

Colour test (colorimetric neutralization test). Cell activity in the nutrient medium results in  accumulation of acid products, which induces a corresponding change in the  pH (making the medium or­ange-coloured). Inoculation of the  cell culture with cytopathogenic viruses (enteroviruses, reoviruses, etc.) leads to inhibition of cell metabolism. As a result, the pH of the medium undergoes no change and the medium remains red.

0.25-ml portions of the working virus dilution (100-1000 CPE₅₀) and the respective serum dilution are pipetted into the  test tubes. Let the mixture stand for 30-60 min at room temperature, and, after adding 0.25 ml of the cell suspension into each test tube, stopper them with rubber plugs, or pour sterile vaseline oil into them. The mixture is incubated at 37 °C for 6-8 days. The results are inter­preted colorimetrically: pH equal to or above 7.4 (red-coloured me­dium) indicates virus reproduction, whereas pH of 7.2 or less (orange-coloured medium) suggests virus neutralization by antibodies.

Enzyme-linked immunosorbent assay (ELISA) or the immunoenzymic test relies on the capacity of the enzyme antibody label to break down the substrate with the formation of stained products. Antibodies linked to the enzyme regain their ability to conjugate with antigens. The number of formed enzyme-antigen-antibody complexes corresponds to the intensity of substrate staining.

Peroxidase and alkaline phosphatase are commonly utilized as enzymes while 5-aminosalicylic acid, orthophenylendiamine, and other substances are used as the substrate for peroxidase.

 

 

Currently, a solid phase modification of ELISA is most often employed in microbiology. The essence of this variant consists in the fact that at first antigens (or antibodies) are sorbed on a solid material and only after that the remaining ingredients of the serological reaction are added. Plastic plates, beads, films or tubes made of various synthetic inert materials (polystyrene, methacrylate, etc.) are usually used as a solid phase carrier of antibodies or anti­gens. Being adsorbed on the surface of such materials, antibodies or antigens, even in a dry state, retain their immunological specificity and ability to participate in serological reactions for a long time.

There are numerous methodological variants of immunoenzymic detection of antigens; in most cases the antigen is caught by anti­bodies bound to the solid phase. Following incubation with the ma­terial, the antigen tested attaches to the antibody and thus to the solid phase. Then the “linked” antigen is demonstrated by means of enzyme-labelled antibodies against this antigen, the direct variant of ELISA- In an indirect variant anti-species (antiglobulin) enzyme-labelled sera are used. The amount of enzyme linked to the solid phase is equal to the amount of the antigen. Activity of the enzyme is determined quantitatively by the intensity of post-incubation staining with the appropriate substrate. This analysis can be made by means of an automatic device, with the results being registered by a special spectrophotometer.

ELISA is distinguished by a fairly high sensitivity and rapidity of obtaining the results (within 2 hours). Improvement in the sensitiv­ity of the solid phase ELISA modification requires the use of anti­bodies with a high degree of specificity. Despite their relatively low-affinity, monoclonal antibodies appear promising in this regard. Hence, the  development of methods for obtaining highly affinitive monoclonal antibodies is one of the top priorities facing modern microbiologists.

The  following buffer solutions are necessary to perform ELISA.

1. Coupling buffer: 0.05 M of sodium-carbonate-bicarbonate buffer (CBB) (pH 9.5-9.7) for sorption of the antigen or antibodies on a solid carrier. The composition of the buffer is as follows: 1.18 g of Na₂CO₃, 3.47 g of NaHCO₃ and 200 mg of NaNO₃. The volume of the  buffer is adjusted to 1 L with distilled water,

2. Incubation buffer: phosphate-salt solution (pH 7.3-7.5) which is used for diluting the components introduced into the reaction after sorption of the first component on the carrier. The composition of the  buffer is as follows:

17.9 g of Na₂HPO₄“H₂O; 0.8 g of NaH₂PO₄“HaO; 42.5 g of NaCI; 2.5 ml of Twin-20. The volume of the buffer is brought to 5 L with distilled water and the buffer is stored at 20-25 °C.

3. Washing buffer: isotonic sodium chloride solution containing 0.05 per cent. of Twin-20. Phosphate-salt solution with 0.05 per cent of Twin-20 may also be utilized as a washing buffer. Orthophenylendiamine or 5-aminosalicylic acid serves as a substrate for peroxidase.

Orthophenylendiamine is prepared ex tempore in the following manner:

10 mg of orthophenylendiamine, 6.1 ml of 0.1 M of citric acid, 6.4 ml of  0.2 M of Na₂HPO₄“12H₂O (for complete solution the mixture is heated on a water bath), 12.5 ml of distilled water, 0.35 ml of 3 per cent H₂O₂.

Dilute 80 mg of 5-aminosalicylic acid in 100 ml of distilled water, adjust the pH of the solution to 6.0 ex tempore with the help of 1 M NaOH. Prior to using, add 1 ml of 0.05 per cent H₂O₂ to each 9 ml of the solution.

To perform ELISA, one should have polystyrene plates with flat-bottom wells and automatic pipettes. To quantitate the results, the spectrophotometer (a registrator of extinction at a 492 nm wave length) is used.

Procedure. The first stage of ELISA is sorption of the corresponding dilution of antibodies or antigen (in concentration of 10-20u.g/ml)on carbonate-bicarbon­ate buffer in a 0.2-ml portion on a solid phase for 1-2 hrs at 37 °C and 10-12 hrs at 4 °C (sensitization). Then, the wells are washed (to remove the antibody or antigen which has not been sorbed on the carrier) with tap water and washing buffer containing 0.05 per cent Twin-20 for 5 min (twice) at room temperature. After that place into each well (solid phase) 0.2 ml of 1 per cent solution of bo­vine serum albumin in CBB and incubate for 1 hr at 37 °C to ensure covering of those sites of the well surface, which have remained free after sensitization, sorption of the first component of the reaction on the solid carrier- Wash the well to remove the unbound bovine serum albumin and introduce the material to be tested (antigen or antibodies) (in 0.2 ml aliquots) diluted with a phosphate-salt solution (pH 7,2) containing 0.05 per cent Twin-20. Each dilution of the material is pipetted into two wells and placed in a 37 °C incubator for 1-3 hrs. Wash off the antigens or antibodies which have not reacted in the immune test and introduce 0.2-ml portions of conjugated antibodies against the test antigen or antibodies in a working dilution on a phosphate-salt solution containing 0.05 per cent Twin-20. Then, incubate the mixture at 37 °C for two hours. The unbound conjugate is washed off with buffer three times for 10 min.

Put 0.1 ml of substrate (chromogen) solution into the well and allow it to stand for 30 min in the dark at mom temperature. In the process of incubation in the presence of peroxidase orthophenylendiamine is stained yellow and aminosalicylic acid, brown.

To stop the reaction of substrate splitting, add 0.1 ml of 1 N H₂SO₄ (or 1 M NaOH) into the well.

Control of the reaction: the test antigen or antibodies are replaced with a ho­mologous component of the reaction.

Control of the conjugate:  0.2 ml of 1 per cent bovine serum albumin per CBB + 0.2 ml of conjugated antibodies in the working dilution.

The results of the reaction are read either visually or instrurnentally. In the first case, one looks for the  greatest dilution of the material tested in which the staining is more intense than in the  control (by bovine serum albumin). In read­ing the results of the  test with the  help of a spectrophotometer, a positive dilu­tion is the greatest dilution of the material tested at which the level of extinc­tion exceeds by at least two times the level of extinction of the corresponding dilution of the  heterologous component of the reaction.

To obtain antibodies, conjugated with the enzyme, one needs highly active precipitating sera against the antigen or against animal or human globulins from which the gamma-globulin fraction is isolated by precipitation with poly­ethylene glycol, ammonium sulphate, and by means of the rivanol-alcohol technique. Immunoglobulins are conjugated by the enzyme with the help of glutaraldehyde. Non-conjugated enzyme is removed by dialysis or chromatography on Sefadex. To prevent bacterial growth, merthiolate in a volume of up to 0.01 per cent of the mixture is added to the conjugates and the latter are kept at 4 °C or in the frozen state.

Radioimniunoassay (RIA). The antigen or antibodies for RIA are labelled with radioactive isotopes, most commonly with ¹²⁵I. RIA is very sensitive and allows the detection of 1-2 ng of the sub­stance tested, or even less. Special radiometric equipment is ne­cessary to perform this assay.

Variable RIA modifications are available, with the solid phase variant being the one most frequently utilized in practice. As in the case of solid phase ELISA, antibodies (antigen) are sorbed on a solid phase carrier [on the surface of plates with wells, beads, and films from polystyrene or other polymer synthetic ma­terials). Adsorbed (immobilized) antigens and antibodies preserve their capacity to participate in serological reactions for a long time.

Figure 1 presents the diagrams of conducting RIA by three methods, viz., competitive, reverse, and indirect.

In the competitive method of RIA antibodies specific in relation to the antigen tested are sorbed on the surface of polystyrene wells. Then, the antigen-containing material to he assayed is placed into the wells and after a definite period of time sufficient for the spe­cific interaction of the antigen with immobilized antibodies to take place, the purified antigen labelled with a radioactive isotope is added. With regard to antigenic specificity, it should correspond to the antibodies immobilized on the surface of wells.

If the material to be examined contains the antigen correspond­ing to immobilized antibodies, some of the active centres of the latter are blocked. In this case the labelled antigen placed into the -wells will conjugate with immobilized antibodies to a lesser degree (as compared to the  control), the difference being expressed in varying levels of radioactivity in the liquid part of the reacting mixture (Fig. 1, a}.

In performing reversed RIA purified unlabelled antigen homologous to the antigen tested is sorbed on the surface of the wells. The antigen-containing material is conjugated in a separate test tube with labelled antibodies specific with regard to the antigen immobilized on the surface of the wells. If the material studied contains the an­tigen capable of interacting with labelled antibodies, the active cen­tres of the latter are blocked either partially or completely. In this case following the introduction of this mixture into the wells with the sorbed antigen, the labelled antibodies will be fixed on their surface in lower amounts (as compared with the control), which can be judged by the degree of radioactivity of the well contents (Fig. 1, b).

 

 

Figure 1. Formation of an immune complex during the determination of an unknown antigen with the help of competitive (a), reversed (b), and indirect (c) radioimmunoassays: 1— anti-virus antibodies; 2 — labelled viruses; 3 — test viruses; 4 — labelled antibodies; 5— known virus; 6 — unknown human serum studied for antibodies; 7 — ¹²⁵I-labelled antibodies against human globulin

 

 

The conduction of solid-phase RIA appears most convenient when an indirect method with anti-species labelled antibodies (the method of double antibodies) is used .

 

 

Indirect RIA may be employed for detecting both antibodies (serological diagnosis) and unknown antigens. In both cases an anti-species labelled serum containing the antibodies against gamma globulins is used. To carry out the serological diagnosis by indirect RIA. the antigen is sorbed on the well surface and then the patient’s diluted serum is added. If it contains the corresponding antibodies, the antigen-antibody complex is formed on the well surface. Upon the subsequent introduction into the wells of the anti-species radio-labelled serum, the antibodies present in it are adsorbed on the formed antigen-antibody complex, with human antibodies (gamma globulins) playing the role of an antigen in the given case. The greater the number of antibodies in the patient’s serum, the larger the level of the radioac­tive label linked to the well surface. Measurement of radioactivity in the liquid phase of the well contents gives evidence about the number of antibodies in the patient’s serum (Fig. 1, c).

Complement-fixation test is used in virology for the retrospective diagnosis of numerous viral infections by demonstrating specific antibodies in paired human sera and for the evaluation of various clinical specimens for virus-specific antigens.

Virological application of the complement-fixation test is peculiar in that it is performed in the cold (for 12 hours, at 4 °C) and that an additional control with the so-called normal antigen is used (antigens from cells which are known to have reproduced the virus). This anti­gen is used in the same dilution as the viral one. A working comple­ment dilution is prepared ex tempore. A microcomplement-fixation test is also available (Table 2)

Immunofluorescence test, both direct and indirect, is used to demonstrate viruses in clinical specimens, inoculated cell cultures, and in animals (Table 3).

Radial haemolysis test involves haemolysis of antigen-sensitized erythrocytes by virus-specific antibodies in the presence of comple­ment in agarose gel. The test is routinely used in the serological diagnosis of influenza, other respiratory infections, rubella, parotitis and arbovirus (togavirus) infections.

Agarose (30 mg) is melted in 2.5 ml of phosphate buffer (pH 7.2), cooled to 42 °C, and mixed with 0.3 ml of sensitized erythrocytes and 0.1 ml of complement. One drop of boric acid is added, the mixture is carefully stirred and spread onto panels or slides with a warm 5-ml pipette. The thickness of the resultant layer should not ex­ceed 2 mm. Three-four minutes after agarose solidincatioa the panel is covered with a lid, inverted, and allowed to stand for 30 min at room temperature. Wells are punched in the solidified agarose and filled with test or control serum. The panel is covered with a lid and placed upturned into a moist chamber (Petpi dishes with a mois­tened piece of cotton wool) at 37 °C for 16-18 hrs.

Table 3

Diagnosis of Viral Infections by Means of the IF Test

 

The results of the test are evaluated by the size of hemolysis areas round the serum-filled wells. The controls should present no evidence of hemolysis.

For this test, sheep erythrocytes are washed with phosphate buffer (pH 7.2) and 0.3 ml of 10 per cent suspension is prepared, with a pH optimally adjusted for a given virus (e.g., 6.2-6.4 for tick-borne encephalitis virus). A 0.1-ml por­tion of undiluted antigen is added to erythrocytes, thoroughly mixed, and left to stand at room temperature for 10 min. Sensitized erythrocytes are precipitated by centrifuging for 10 min at 1000 X g, the pellet is washed with phosphate buffer (pH 7.2), and resuspended in 0.3 ml of borate-phosphate buffer (pH 6.2-6.4).

Test of haemadsorption on solid medium is a modification of en­zyme-linked immunosorbent assay and an indirect haemagglutination test. Because of its high sensitivity, it can be used as a rapid diag­nostic test in viral infections.

The procedure of the test is as follows. Wells of disposable poly­styrene panels are treated with immune globulin (immune serum), and suspension of antigen-containing material is placed in them. Thirty-sixty minutes later the wells are repeatedly washed with buffer, suspension of erythrocytes with adsorbed specific immunoglobulin is added, and haemagglutination is evaluated in 30-60 min.

If a specific antigen is present in the material, it is bound by the serum adsorbed on well surface, and, in turn, binds immunoglobulins on the erythrocyte surface. This results in erythrocyte agglutination (haemagglutination).

The test in the above modification is used for identifying antigens of rotaviruses and other enteroviruses in faeces.

Reversed indirect haemagglutination (RIHA) test is used for detecting bacterial and viral antigens in the  materials to be exam­ined as well as for the rapid diagnosis of a number of infections.

In contrast to IHA. erythrocytes in this test are sensitized not by antigens but by antibodies whose agglutination occurs upon ad­dition of the antigen.

Erythrocytes are first fixed with formalin or glutaraldehyde and bound to gamma-globulin which is isolated from immune sera and purified from other serum proteins. Binding of gamma-globulin with the erythrocyte surface is mediated by chromium chloride. For this purpose, to 8 volumes of distilled water add 1 volume of immunoglobulins obtained from immune serum, 1 volume of 50 per cent sus­pension of formalin-treated red blood .cells, and 1 volume of 0.1-0.2 per cent solution of chromium chloride. Allow the mixture to stand at room temperature for 10-15 min, thetreat the erythro­cytes as in the passive haemagglutination test.

The specificity of the  antibody diagnosticum is checked in the reac­tion of passive haemagglutination inhibition, using a homologous antigen. The reaction should be inhibited by a homologous antigen by at least 16 times and remain unaffected by a heterologous one. Examine the diagnosticum for spontaneous haemagglutination as well.

This test is commonly used to identify the causative agents in post-mortem material taken from the organs of man and animals, for example, from the brain, spleen, liver, and lungs. Prepare 10 per cent suspension of the above organs with isotonic sodium chlo­ride solution, centrifuge it at 10 000 X g for 30-60 min, and use the  supernatant as an antigen.

Procedure. Prepare two-fold dilutions of the material to be studied (antigen) with a stabilizing solution. Place one drop of each dilution of the antigen into 3 neighbouring wells of the micropanel (the reac­tion requires 3 parallel rows of wells). Into each well of the first row add 1 drop of the stabilizing solution, into wells of the second row, 1 drop of homologous immune serum in a 1:10 dilution, those of the third row, 1 drop of heterologous immune serum. The second and third rows serve as controls of reaction specificity. Let the mix­ture stand at room temperature for 20 min.

To all wells add one drop of 1 per cent suspension of sensitized erythrocytes (erythrocyte antibody diagnosticum) and shake the plates well. Read the results of the reaction in 30-40 min. In the presence of the specific antigen, haemagglutination is observed in the first and third rows (with heterologous serum) and is absent in the second row -where the antigen is preliminarily neutralized by homo­logous serum.

To ensure the accuracy of the test, the sensitized red blood cells are checked for spontaneous agglutination.

Reversed indirect haemagglutination inhibition (RIHAI) test makes it possible to detect the presence of antibodies in human and animal sera.

Procedure. Dilute sera by ten-fold with isotonic sodium chloride solution, heat for 20 min at 65 °C to destroy nonspecific inhibitors, then prepare two-fold serum dilutions with stabilizing solution and the working dose of the antigen containing four agglutinating units. Introduce by one drop of each dilution into the wells of the micro-panel and add to each of them one drop of the antigen whose dilution corresponds to the working dose. Allow the mixture to stand for 20 min at room temperature, then place one drop of the erythrocyte antibody diagnosticum into each well, and shake them thoroughly. Read the results of the reaction after 1.5-2 hour-incubation at room temperature. The titre of the serum is its greatest dilution which completely inhibits haemagglutination with four agglutinating units of the antigen.

The test is validated by checking whether sensitized erythrocytes may undergo spontaneous agglutination in the presence of: (a) stabilizing solution; (b) normal antigen (from material free of the virus); (c) the serum tested. Among advantages of the test one can cite its universality and the possibility to use it for finding various antigens.

Assessment of the haemagglutination results. Estimation of the results of IHA, RIHA and RIHAI tests is relied on the degree of erythrocyte agglutination; (++++), complete agglutination; (+++), almost complete agglutination; (++). partial agglutina­tion; (+)» traces of agglutination; (—), no agglutination.

The test is considered positive, if agglutination is complete (++++) or almost complete (+++)i the diagnosticum does not induce spontaneous agglutination in the presence of each compo­nent required for the reaction, and the control test of the specificity of the antigen or antibody is positive.

 

Schematic Representation of Hemagglutination inhibition test for identification of influenza virus

 

Schematic Representation of Hemagglutination inhibition test for serological diagnosis of influenza

 

 

Schematic Representation of Neutralization test for serological diagnosis of poliomielitis

 

 

Immunopathology

C. Pirquet gave the name allergy (Gk. allos other, ergein to work) to the altered reactivity of the body under the effect of pathogenic microbes, toxins, medicines and other substances.

Allergy is an altered reactivity of the body which manifests itself in the disturbance of the usual course of general or local reactions, often during repeated entrance into the body of substances known asallergens. These reactions may be increased in comparison to the standard, that is, strengthened and speeded up (hyperergia), or lowered, that is, weakened and slowed down (hypoergia), or these reactions may be completely absent (anergia), for example, during absolute immunity.

In 1839 F. Magendie obtained an increased sensitivity in rabbits which perished from the third injection of egg white, which had been completely harmless during the first injection. Analogous phenomena were described in 1894 by S. Flexner. In his experiments animals died from a second injection of dog serum. In 1888 C. Richet and J. Hericourt noticed that during repeated injections of eel serum into dogs the latter did not acquire resistance to it but, on the contrary, became quite sensitive and perished.

In 1902 C. Richet and P. Portier established an increased sensitivity in dogs to repeated (within 11-12 days after the first injection) administrations of extracts from the tentacles of Actinia. This state of increased sensitivity to repeated injections of protein was named anaphylaxis by C. Richet.

In 1902 T. Smith easily reproduced anaphylactic shock in guinea pigs by the injection of horse serum.

Allergic reactions are subdivided into two groups: (1) immediate and (2) delayed reactions, although it is difficult to draw a strict distinction between them.

Allergic reactions of immediate action are associated with B-lymphocytes and antibodies circulating in the blood, allergic reactions of delayed action with T-lymphocytes and cell antibodies.

Based on the mechanisms of these reactions, Gell and Coombs divided hypersensitivities into four types (Table 7). Although the four types are distinct, notice that hypersensitivity to any given antigen may occur because of any one or more of these four mechanisms.

 

TABLE 7.

 

Classifications of Immunologically Mediated Tissue Injury

 

 

Immediate Allergic Reactions. The group of immediate allergic reactions includes anaphylacticshock, serum sickness, hay fever, acute rheumatism, rheumatoid arthritis, Arthus’ phenomenon, and other pathological conditions. These reactions are manifested soon after the antigen is introduced. The antigen-antibody reaction in the tissues and fluid tissue media takes part in their development. Immediate allergic reactions are transmitted from one animal to another passively, i. e. by the administration of immune serum of sensitized animals. In most cases immediate-type allergy may be relieved by desensitization.

Anaphylaxis (Gk. ana away from, back from, phylaxis protection). Anaphylaxis is a form of altered reactivity, a state of the organism’s increased sensitivity induced by repeated injection of foreign proteins (sera, antibiotics, etc.).

 

The first dose of the antigen (protein) causing increased sensitivity is known as the sensitizing dose (L. sentire to feel). The second dose, from die injection of which anaphylactic shock develops, is known as the reacting dose. The sensitizing dose is injected into animals subcutaneously, intraperitoneally, intravenously, or by the intracardiac route; and the reacting dose is injected intravenously or by the intracardiac route and in a larger amount than the sensitizing dose.

The state of increased sensitivity in animals does not develop immediately, but after a certain incubation period (8-21 days).

The clinical picture of anaphylactic shock is various in different species of animals. The best example of anaphylactic shock can be reproduced in guinea pigs. If a guinea pig is preliminarily sensitized by a subcutaneous injection of 0.01 ml of horse serum, and then within 8-21 days 0.1-0.5 ml of the same serum is injected directly into the heart, then anaphylactic shock develops quite rapidly. Within 1-2 minutes the guinea pig becomes restless, the fur stands on end, the guinea pig begins to rub its nose with its paws, involuntary defecation and urination, sneezing, acute dyspnoea, tonic and clonic spasms are observed and breathing becomes slower and difficult. Within 5-10 minutes the animal dies from asphyxia, with a fall in temperature, decrease of complement and impairment of blood clotting. Autopsy reveals emphysema of the lungs due to a spasm of the bronchial muscles, inability of the blood toclot, and hyperaemia and haemorrhages in the mucous membrane of the stomach, intestine and other organs.

In dogs anaphylaxis is accompanied by a spasm of the hepatic veins causing the phenomena of stasis in the liver and an insufficient blood supply to the heart. The animal dies from collapse. In rabbits anaphylaxis is characterized by a spasm of the end arteries of the pulmonary circulation, a blockade of the pulmonary circulation, a drop in blood pressure in the systemic circulation, slowing down of cardiac activity and a sharp dilatation of the right ventricle. Death results from respiratory arrest and drop in the blood pressure.

In man all three types of anaphylaxis are observed. However, the type pertinent to guinea pigs occurs predominantly. Spasm of smooth muscles is an important factor in the mechanism of the development of anaphylaxis.

Anaphylactic shock in humans arises from repeated injections of immune sera during treatment of patients with various infectious diseases (diphtheria, tetanus, anthrax, anaerobic infections), and of antibiotics (penicillin, etc.). It is characterized by a series of symptoms: dyspnoea, quick pulse, cold limbs, temperature rise, spasms, oedemas, pains in the joints, body rash, and affection of the central nervous system, sympathetic and parasympathetic systems, etc. In some cases anaphylactic shock terminates in death.

Individuals who develop anaphylactic hypersensitivities produce IgE antibodies as a result of exposure to a specific allergen (figure).

 

 

 

Figure.  In allergic individuals, exposure to an antigen (allergen) that reacts with IgE leads to degranulation of mast cells. Degranulation is triggered when the allergen is a cross-linkage between the two adjacent IgE molecules on the surface of a mast cell. The release of histamine causes vasodilation. During systemic anaphylaxis, such as occurs following allergic reactions to drugs and bee stings, blood pressure drops due to vasodilation and breathing and heart irregularities develop. If untreated the persons as breathing and heart beating cease. Epinephrine is administered to restore respiratory and heart function.

 

These IgE antibodies bind via their Fc regions to the surfaces of mast cells and basophils. The surface of a mast cell can be covvered with as many as 500,000 IgE receptors. Mast cells and basophils also contain granules in their cytoplasm. When an allergen reacts with several IgE molecules on a sensitized mast or basophil cell, the cell degranulates, that is, it releases the contents of its granules into the surrounding blood or tissues. The release of the contents of basophil or mast cell granules establishes the basis for several physiological responses. One of the most important substances released is histamine, which causes blood vessels to dilate (vasodilation) and become more permeable. Blood flow is increased and the escape of fluid and cells from the blood vessels is also increased This results in tissue swelling and redness.

In addition, histamine causes increased secretion of mucus, contraction of smooth muscle, and constriction of bronchial air passageways. Other substances that are newlysynthesized and released when mast cells and basophils are stimulated by antigen include prostaglandins and leukotrienes. Some leukotrienes are called slow-reacting substances of anaphylaxis (SRS-A),These molecules also contribute to vasodilation and smooth muscle contraction. Leukotrienes and prostaglandins also stimulate nerve endings to cause pain and itching.

Allergic processes in general, and anaphylaxis in particular, are accompanied by disturbed tissue respiration and constitute quite a complex process developing in the sensitized body.

 The phenomena of allergy and anaphylaxis are inherent in comparatively highly organized organisms capable of reactivity. They were preceded by simple forms of parasitism, and septic and toxic infections.

In the mechanism of anaphylaxis a definite role is played by the reaction of the antigen and antibody in tissues attended with the production of serotonin, heparin, bradykinin, etc. The union of the antigen with the antibody fixed in the cells stimulates the cells, and a pathological process develops in the tissues, as a result of which the smooth muscles contract. The presence of M and G immunoglobulins in the blood reduces hypersensitivity because the cytophilic E immunoglobulins adsorbed on the cells cannot bind with the antigen.

Local manifestation of anaphylaxis. Repeated subcutaneous injections of horse serum to rabbits at six day intervals cause an infiltration and necrosis of tissues (Arthus phenomenon) after the 5th-7th injection. Local anaphylaxis develops after the injection of other antigens (bacteria, toxins, antibiotics, etc.).

Passive anaphylaxis. An increased sensitivity may be reproduced iormal guinea pigs passively, that is, by injecting immune serum of sensitized animals. The state of sensitization does not appear immediately, but within 24 hours after subcutaneous injection, within 12 hours after intraperitoneal injection and within 4 hours after intravenous injection. Increased sensitivity is retained in guinea pigs for 3 to 8 weeks.

Hyposensitization (anti-anaphylaxis). If the reacting dose does not provoke anaphylactic shock then the animal loses its increased sensitivity to this antigen, desensitizes on the 2nd-3rd week, and then becomes sensitive again, sometimes to an even greater extent. Desensitization ensues also after anaphylactic shock. A. Bezredka suggested a specific, simple and quite an effective method of desensitization by a fractional injection of the antigen (serum). When the antigen (serum, albumin) is introduced repeatedly in small doses, fixation of the antibodies circulating in the blood occurs with each introduction because of the colloid and chemical character of the immune reaction, and the formation of high concentrations of histamine and other toxic substances that induce anaphylactic shock is prevented.

To prevent anaphylactic shock, an intracutaneous test is made previously by injecting 0.1 ml of      1 : 100 diluted serum into the flexor surface of the forearm. If the reaction is negative (a papule no larger than 0.9 cm in diameter surrounded by a limited area of hyperaemia), 0.1-0.5 ml of undiluted serum is injected subcutaneously in 20-30 minutes and the whole dose is injected 30 to 60 minutes later. If the intracutaneous test proves positive (a papule of more than 1 cm in diameter and a large zone of hyperaemia), the 1 : 100 diluted serum is injected subcutaneously in doses of 0.5, 1.0, 2.0, and 5.0 ml at intervals of 20 minutes and then 0.1-0.2 ml of indiluted serum is injected at intervals of 30 minutes.

Anaphylactic shock may be averted by non-specific substances, e. g. the injection of a reacting dose of serum under ether anaesthesia, or by the effect of chloralhydrate and alcohol. Dimedrol (Allergan, Benadryl, diphenhydramine), Diprasin (promethazine hydrochloride), atropine, ether, chloroform, urethane, Novocain (Procaine), bile acid salts, saponin, hirudin, sodium hyposulphite, calcium chloride, etc., have desensitizing properties. Penicillinase is administered in penicillin shock.

Immediate reactions also include atopy (Gk. atopia strangeness) which is natural hypersensitivity occurring sponta-neously in persons susceptible to allergy. Unlike anaphylaxis, it develops only among humans. The group of atopies includes allergic rhinitis, hay fever, bronchial asthma, Quincke’s disease (angioneurotic oedema), urticaria, eczema of the newbom, intolerance (idiosyncrasy) to egg white, the meat of crawfish, strawberries, and drugs (iodine, chloroform, antibiotics, etc.).

In atopy the skin and mucous membranes have an increased capacity for adsorbing from the blood special antibodies to haptens; these antibodies are called reagins and are found in class E gamma globulins. Hereditary predisposition is important in the pathogenesis of atopic reactions. The medical histories of half of the patients are marked by similar diseases of the parents. Atopic reactions are unamenable to hyposensitization.

The activity of allergens is determined by their structure and the position of the determinant groups in their molecules. The allergens are of bacterial and fungal origin, protein-polysaccharide-lipid complexes. Different allergens have antigenic determinants in common (polyvalent character of allergic reactions). Sensitization to an allergen is attended with the production of antibodies.

Reagins are thermolabile, they are incapable of complement fixation and do not pass through the placenta. Reagins are demonstrated by immunodiffusion by means of the radioallergosorbent test, etc.

The production of blocking antibodies has an inhibiting effect on reagin synthesis.

Allergens are subdivided into household and epidermal (the dust of feather quilts and pillows, skin epidermis, dandruff of dogs, cats, and horses, etc.), occupational (library dust, dust of wool and cotton, certain dyes, soaps, varnishes, wood pulp, explosives and synthetic substances, etc.), plant (the pollen of plants during pollination of meadow grasses, garden and potted plants), food (eggs, strawberries, shellfish, citrus fruits, coffee, chocolate, and other foods), drug (codeine, acetylsalicylic acid, sulphanilamides, penicillin and other antibiotics).

antibody-dependent cytotoxic hypersensitivity. Antibody-dependent cytotoxic hypersensitivity reactions (type II hypersensitivity) occur by a different mechanism than anaphylactic hypersensitivity (fig.). In type II hypersensitivity reactions, an antigen on the surface of the cell combines with an antibody. This stimulates phagocytic attack or initiates the sequence of the complement pathway that results in cell lysis and death.

Antibody-dependent cytotoxic response occurs after transfusions with incompatible blood types. Blood serum contains antibody to any antigens that do not occur in the plasma membranes of the redblood cells of that individual. A person with type A blood has antigen A on red blood cell surfaces and circulating anti-B antibody. If that person were given a transfusion with type B blood that has antigen B on blood cell surfaces and anti-A antibody in the serum , the circulating antibodies in the recipient would react with the surface antigens of the donor cells. Formation of antigen-antibody complexes on the surface of the red blood cells would activate the complement system, resulting in the lysis of the donated cells. It is therefore essential that blood transfusions be made with compatible blood types.

 

 

 

Figure. In a type II hypersensitivity reaction, antigen on a cell, such as on a red blood surface, combines with antibody.

 

A transfusion reaction can occur when matching antigens and antibodies are present in the blood at the same time. This is a type II hypersensitivity reaction against a foreign antigen. Foreign erythrocytes in the transfused blood are agglutinated (clumped), complement is activated, and red blood cells undergo hemolysis (rupture). The patient’s symptoms include fever, low blood pressure, back and chest pains, nausea, and vomiting.

Persons with type 0 blood are sometimes called universal donors and individuals with type ABblood are sometimes called universal recipients. The reason for this is that type 0 red blood cells lack both A and B antigens on their surfaces. They lack the antigens generally associated with transfusion in- compatibility. Regardless of the circulating antibodies in the recipient, the donated blood cells do nothave antigens with which to react. The anti-A and anti-B antibodies in the donated blood are rapidly diluted when introduced into the larger volume of blood in the recipient. Similarly, persons with type AB blood do not have circulating antibodies against either A or B antigens. They lack antibodies that react with the A and B antigens on blood cells that are introduced regardless of cell type. However, the concepts of the universal donor and the universal recipient refer only to the major A and B antigens. Thereare various other antigens on blood cell surfaces, including the Rh antigen, that can cause incompatibility reactions. Therefore, except in emergencies, transfusions are given only after adequate analysis of cellantigens and only with matching blood types.

Rh incompatibility between mother and fetus is another example of type II hypersensitivity (figure).

 Approximately 85% of the human population has an antigen on their red blood cells known as the Rh factor. These people are Rh positive. Antibodies that react with Rh antigens are not found in A serum of Rh-negative people. Exposure to the Rh antigen will sensitize Rh-negative individuals to produce anti-Rh antibodies. Rh incompatibility occurs when the father is Rh positive, the mother is Rh negative, and the fetus is Rh positive. In this case, the mother develops Rh antibodies in response to exposure to the Rh antigens of the fetus. Generally, the mother is only exposed to fetal Rh antigens at the time of birth. She does not develop an immune response until after the birth of the first child. In subsequent pregnancies, however, the anti-Rh antibodies (lgG) circulating through the mother’s body can cross the placenta and attack the cells of a subsequent Rh-positive fetus.

This causes anemia. During development of the fetus, fetal blood is purified because molecules pass from the fetus across the placenta and then through the mother’s liver. After birth, the fetal blood is no longer purified by the maternal circulatory system and the infant develops jaundice. Thisdisease is called hemolytic disease of the newborn (previously called erythroblastosis fetalis). It is characterized by an enlarged liver and spleen. If untreated, the mortality rate is about 10%.

 

Hemolytic disease of the newborn can be treated by removal of the fetal Rh-positive blood. Blood is replaced by transfusion with Rh-negative blood Cells in the new blood will not be attacked by the anti-Rh antibodies that crossed the placenta and now are circulating within the newborn At a later time,when the anti-Rh antibodies passively acquired (passive natural immunity) from the mother have been diluted and eliminated, these transfused cells are replaced by Rh-positive cells produced by the infant Cell destruction does not occur at this time because the maternal antibodies have been eliminated from the infant’s circulatory system.

 

Figure. Before birth the products formed by the attack of anti-Rh antibodies passacross the placenta and are detoxified by the maternal liver After birth the liver of the newborn is unable to detoxify these products, which attack the nerves as well as red blood cells, causing disease

 

To prevent hemolytic disease of the newborn, passive artificial immunization of the Rh-negative mother with Rhogam (anti-Rh antibodies) is done a the time of birth. Anti-Rh antibodies react with the fetal Rh-positive cells that enter the mother at the time of birth through traumatized tissue The reaction of anti-Rh antibodies with Rh-positive cells limits the development of an immune response m the mother Anti-Rh antibodies bind to the Rh antigens that may have been mtroduced from the baby to the mother at the time of birth.

Their recognition by the immune system of the mother is prevented. Thus, artificial passive immunization is used to prevent the development of active natural immunity. This treatment is repeated at eachv birth when the baby is Rh positive and the mother is Rh negative As a result of this treatment, a serious antibody-dependent cytotoxic hypersensitivity reaction can be prevented.

 

immune complex-mediated hypersensitivity. Immune complex-mediated hypersensiti-vity (type III hypersensitivity) reactions occur when the formation of antibody-antigen complexes triggers the onset of an inflammatory response. Such an inflammatory response is part of the normal immune response. If there are large excesses of antigen,  the antigen-antibody-complement complexes may circulate and become deposited in various tissues. Inflammatory reactions from such deposition of immune complexes can cause localized physiological damage to kidneys, joints, and skin. These reactions are called type III hypersensitivity or Arthus immune-complex reactions.

In the Arthus reaction, the site becomes infiltrated with neutrophils.  This leads to extensive injury to the walls of the local blood vessels because lysosomal enzymes and vasoactive substances (substances that cause dilation of blood vessels) are released from these cells. An Arthus reaction sometimes occurs m the lungs because of repeated exposure to antigens on the surfaces of inhaled particulate matter. Symptoms generally include cough, fever, and difficulty m breathing. These symptoms typically develop over a period of 4 to 6 hours. The attack usually subsides within a few days after the removal of the source of the antigen. Persons in particular occupations have a high risk of developing this condition. For example, farmers often develop this reaction because of repeated exposure to the airborne spores of actinomycetes growing on hay. Sugar cane workers, mushroom growers, cheesemakers, and pigeon fanciers are also prone to this condition because of exposure to airborne antigens associated with their activities.

Serum Sickness. Serum sickness develops within 8-12 days after a single primary introduction usually of large doses of serum (from 10 ml and more). Bezredka’s method does not avert serum sickness. In some cases in sensitized people serum sickness ensues quite rapidly after the injection of serum, and then it resembles anaphylactic shock. Serum sickness is characterized by the appearance of a rash resembling urticaria and is accompanied by a severe itch, elevated temperature, oedema, pains in the joints, swelling of the lymph nodes, disturbances of the cardiovascular activity, and a change in the white blood count (first leukocytosis, then leukopenia and relative lymphocytosis).

 

Figure. Type 3 hypersensitivity reactions result in inflammation and deposition of immune complexes m blood vessel walls. The immune response that forms antigen-antibody-complement complex triggers degranulation of mast cells and basophils. This causes increased vascular permeability Enzymes released m the process lead to tissue damage and platelet aggregation. The platelets that aggregate block oxygen transfer and cause further damage.

 

The patient recovers within a few days. The mechanism of serum sickness as that of anaphylactic shock is based on the interaction of the antigen and antibody. Serum sickness is prevented by the use of matured therapeutic sera, sera preliminarily heated at 56° C for 0.5-1 hour, or sera purified from ballast protein fractions. The above mentioned methods of treating sera lower their toxic action. Treatment of serum sickness is carried out with Dimedrol, Diprasin and other antihistaminics.

This condition of glomerulonephritis can also be brought about by persistent infections (fig.). As a result of such infections, antigen-antibody complexes are formed that are deposited within the glomeruli of the kidneys Immune complexes formed by antibody reactions with antigens produced by Streptococcus pyogenes (the causative agent of strep throat), hepatitis B virus (the cause of serum  hepatitis), Plasmodium species (protozoa that cause malaria), and Schistosoma spp. (helminthic worms that cause schistosomiasis) may lead to this condition. The persistence of these infections provides a continuing supply of antigen to react with circulating antibodies produced by the infected individual. The immune complex that forms accumulates m the kidneys Eventually nephritis due to complex-mediated hypersensitivity results.

                                                                

 

 

Figure. Multiple reactions triggered by antigen-antibody complexes occur as a result of serum sickness As a result of immune complex deposition, PMNs degranulate and release destructive enzymes, platelets clot, basophils degranulate, and some cells (mesangial cells) proliferate, blocking the glomerulus These reactions cause kidney damage and the disease glomerulonephritis.

 

cell-mediated (delayed) HYPERSENSitiVITY. Cell-mediated or delayed hypersensitivity (type IV hypersensitivity) reactions involve sensitized T_DTH  lymphocytes. This type of hypersensitivity is not associated with antibodies circulating in the blood and therefore cannot be transmitted passively by means of serum of a sensitized organism (Fig.). As the name implies, these reactions occur only after a prolonged delay after exposure to the antigen Such reactions often reach maximal intensity 24 to 72 hours after initial exposure. Delayed  hypersensitivity reactions occur as allergies to various microorganisms and chemicals. Contact dermatitis results from exposure of the skin to various chemicals. Skin rashes are typical of delayed hypersensitivity reactions.

 

                                          

Figure. Delayed (cell-mediated) hypersensitivity. The macrophage ingests the antigen, processes it, and presents an epitope on its surface in association with class II MHC protein. The helper T (Th-1) cell is activated and produces gamma interferon, which activates macrophages. These two types of cells medi­ate delayed hypersensitivity

 

The state of delayed hypersensitivity may be transmitted to another organism, though not by serum but adoptively, by injection of lymphoid cells of a sensitized organism. Hypersensitivity of the delayed type is therefore a cellular form of immunity induced by sensitized lymphocytes.

The sensitized lymphocytes carry on their surface receptors which are antideterminants, specific to the given antigen. They bind with the foreign antigen by means of these receptors and destroy it with their enzymes and by producing special humoral factors, lymphokinins, which act as auxiliary vehicles of cellular immunity. Some types of lymphokinins may mobilize non-immune lymphocytes and include them in the reactions of cellular immunity.

Delayed allergic reactions are encountered in infectious diseases (tuberculosis, leprosy brucellosis, and others), diseases caused by allergens of plant origin, in transplantations, tumours, and in ageing.

 

 

                                                                              Contact dermatitis

 

Intracellular bacterial parasites, such as Mycobacterium tuberculosis and Listeria monocytogenes,  elicit a delayed-type hypersensitivity response. In these situations, activated tdth cells recognize foreign antigens on the surfaces of infected cells and activate macrophages Activated macrophages then eliminate the bacteria. Some bacteria may evade being killed or degraded by the macrophages In such cases, additional macrophages proliferate and aggregate, leading to the formation of a characteristic nodular or granular mass called a granuloma in the tissues surrounding the original infection. The presence of granulomas may be indicative of a chronic cell-mediated response to a microorganism. By surrounding the microorganism, they attempt to prevent its spread to other parts of the body.

Koch’s Phenomenon.When a tuberculous guinea pig is injected sub-cutaneously with a suspension of tubercle bacilli, there is a massive inflammatory reaction at the injection site that tends to wall off the injected material and often leads to necrosis; this is called Koch’s phenomenon. This reaction does not require living tubercle bacilli but occurs similarly with tuberculoprotein (PPD). These soluble preparations produce local inflamma­tory reactions, particularly edema, infiltration with lymphoid cells and macrophages, hemorrhage, and marked enlargement of the regional lymph nodes; and focal reactions, consisting of hemorrhagic inflamma­tion and dense cellular infiltration within existing tu­berculous lesions. Because focal reactions may “stir up” tuberculous activity, administration of excessive doses of tuberculoprotein to hypersensitive individuals during skin tests must be avoided.

The immune response to invasion by fungal cells is very similar to that of bacteria. In addition, many pathogenic fungi can survive inside macrophage and elicit a delayed-type hypersensitivity response. Thus chrome infections with Histoplasma capsulatum, Coccidioides immitis, and Sporothrix schenckii can lead to granulomatous (granuloma formation) and in flammatory responses. Some parasitic infections may invoke a delayed type hypersensitivity response. Chronic parasitic infections can lead to granuloma formation and inflammation in the tissues.

Delayed reactions are reactions of the organism to an antigen, which are followed by a second stage marked by antibody production and, accordingly, the development of immediate-type reaction. It is possible that both reactions develop parallel to and independent of each other.

Infectious allergy. Infectious allergy develops during tuberculosis, leprosy, glanders, brucellosis, tularaemia, actinomycosis, syphilis, dermatomycosis and toxoplasmosis, etc. It can be retained long (for many years) even after recovery. Due to the specificity of allergic reactions they are widely used for diagnosis of infectious diseases. For this purpose the corresponding allergens are employed which are injected subcutaneously or cutaneously. At the site of injection of the allergen in patients with, for example, tularaemia and brucellosis within 12-48 hours reddening is produced, with swelling and pain. A disadvantage of the diagnostic value of allergic tests is that they may be positive in those inoculated against the diseases or those who had them many years ago.

The pathogenesis of allergy consists of a whole number of damages to the body, e. g. lowering of the dispersity in the humoral medium and occlusion of the capillaries, excitation of the nerve endings and cells of the smooth muscles. Under the effect of the antibody-allergen complex fixed on the surface of the cells a sharp change in metabolic processes and disturbances in life activity of the cells occur. As a result of blocking by the antibody-allergen complex the cells do not receive the necessary substances and as a result excrete an excess of acetylcholine (nerve cells), serotonin (blood cells), heparin (liver cells), and histamine (connective tissue), etc. In small concentrations these substances are necessary for the normal life activity of the body, while in excess they cause dilatation of the capillaries and increase vascular permeability, and thus intensify the allergic reaction.

The capillary membranes and smooth muscles are the main sites for manifesting the allergic-anaphylactic reaction. The primary reaction is marked by arterial stasis due to contraction of the arterioles. During asthma the symptoms of disturbances in water metabolism and in the function of the cerebral cortex are observed. During eczema a primary neurovascular process is developed in the skin exhibiting an allergic reaction.

Energy production is preceded by a definite time interval characterized by the accumulation of a sufficient concentration of antibodies, the interaction of which with the allergens is accompanied by the formation of high molecular aggregates consisting of antibodies and allergens. Antibodies which are produced in response to the penetration of allergens into the cells of the macro-organism may enter the blood or lymph or may be fixed on the surface of cell walls.

Depending on where the antibody-allergen complex is formed and localized a definite form of allergic reaction develops. If the allergen is found in the skin then urticaria appears, whereas if the allergen unites with the antibody in the upper respiratory tract then an allergic rhinitis arises. The formation of an antibody-allergen complex in the eye conjunctiva is accompanied by conjunctivitis, and during localization of the antibody-allergen complex in the mucous membrane of the bronchi an allergic reaction manifests itself as asthma, while the entrance of a certain amount of the antibody-allergen complex into the blood leads to anaphylactic shock.

Of great significance in the development of allergic disease is the condition of the body, the allergic constitution, the degree of excitation of the vegetative nervous system, permeability of the capillaries, and peculiarities of metabolism. In the etiology of allergy it is impossible to exclude the role of heredity. If the parents are exposed to allergic reactions they transmit to their children the predisposition to the manifestation of not any one definite form of allergy, but to allergy in general. This predisposition to allergic diseases is transmitted through the blood of the donor suffering from allergic disease.

The frequency of allergy depends on age and intensity of the synthesis of proteins in general, and on the production of antibodies in particular. In the newborn or breast-fed children due to a weak development of the nervous and other systems the phenomena of allergy are encountered comparatively rarely and proceed less intensively. At an age of 18 months and up to the period of puberty allergy is observed considerably more frequently and proceeds more severely. In adults the predisposition to allergic diseases is lowered, and in old age the exposure to allergy becomes slight.

The relation of allergy to immunity. There are three points of view on this problem. The first, the classical one, identified immunity with allergy; the second .opposed immunity to allergy but attributed an auxiliary defence role in the infectious process to the latter; the advocates of the third point of view considered allergy to be a condition which was harmful, rather than beneficial, to the organism.

Experiments have proved that it is possible to separate allergy from immunity in tuberculosis. It has been established that active immunity does not suffer in hyposensitized animals in whom allergy was relieved in this manner. Immunity may develop, consequently, without the simultaneous development of an allergic state.

In some cases allergic reactions are attended with tissue necrosis if the dose of the antigen and the level of sensitivity are quite high. Cavities form in the lungs in tuberculosis. Destructive changes develop in the bone, vascular and nervous systems in patients with syphilis. Streptococcal diseases lead to rheumatic lesions of the heart. The allergic factor aggravates considerably the course of many severe chronic infectious diseases (brucellosis, glanders, actinomycosis, fungus diseases, etc.).

 

Despite the obvious harmful consequences of allergy, however, one cannot deny that it has defence action due to the increased efficacy of tissue immunity. It is general knowledge that latent infection with Mycobacterium tuberculosis makes the organism more resistant to tuberculosis. Modem active immunization against tuberculosis is based on this principle; it increases body reactivity, leads to the development of productive inflammation, the formation of granulomas, and localization of the causative agent on a restricted area, and prevents generalization of the process. The defence role of an allergic reaction is directed not only against pathogenic micro-organisms, but also against exotoxins due to intensive binding by the cells of the inflamed tissue.

Taking into account the high proportion of allergic diseases, the necessity arose to elaborate on the current scientific level such important problems as the division of allergens into districts (the determination of their geographical map) and the diagnosis, treatment, and prevention of pathological processes caused by them.

Among the numerous measures of allergy management and prevention, the following are very important: removal of contact with the allergens (food, drug, household, occupational and other allergens); sometimes, being guided by the geographical map of allergens, it is advisable to change the patient’s place of residence. Hormonal preparations (ACTH and others) inhibiting the process -of antibody production yield quite good results.

To reduce the patient’s sensitivity and susceptibility to the allergens, those to which he is hypersensitive are injected subcutaneously. As the result of the injection monovalent (incomplete) antibodies appear which bind the allergens and in that way prevent the formation of bivalent antibodies capable of combining with the allergens and forming antibody-allergen complexes. The method of desensitization is claimed to be effective in allergy caused by household, occupational, and plant allergens and rarely effective in food and drug allergy. In diseases marked by the production by the cells of large amounts of histamine and other substances it is advisable to prescribe antihistaminics:

Dimedrol (diphenhydramine), Pipolphen (promethasine) and Suprastin (chloropyramine). These drugs, however, do not always prevent the development of severe allergic conditions, sometimes causing their augmentation due to impairment of the protective barrier.

 

ALLERGY DIAGNOSTIC TESTS. Many infectious diseases are associated with the development of the body’s elevated sensitivity toward the causative agents and products of their metabolism. Allergy tests used for the diagnosis of bacterial, viral, and protozoal infections, as well as mycosis and helminthiasis, rely exactly on this phenomenon. Allergy tests are quite specific but not infrequently they can be observed in vaccinated individuals and in those with a history of the disease in question.

All allergy tests are divided into two groups, namely, in vivo and in vitro tests.

The first group (in vivo) consists of cutaneous tests made di­rectly on the patient and revealing allergy of immediate (in 20 min) or delayed (in 24-48 hrs) type.

Allergy in vitro tests are based on investigating the sensitization processes in the test tube rather than in the patient’s body. They are employed when for some reason cutaneous tests cannot be performed or when they yield ambiguous results.

To make allergy tests, one utilizes allergens, i.e., diagnostic preparations determining the status of the body’s specific sensitization. Infective allergens employed in the diagnosis of contagious diseases represent purified nitrates of broth cultures, less commonly suspensions of killed microorganisms or antigens isolated from them.

The following standard allergens available commercially are used in practical microbiology: tuberculin PPD (purified protein derivative), dry, purified protein substance of tuberculosis mycobacteria; anthraxin, proteiucleopolysaccharide complex of the anthrax corpuscular antigen; pestin, protein polysaccharide com­plex of corpuscles of the causative agent of plague; brucellin and mallein, filtrates of broth cultures of the causative agents of brucellosis and glanders; tularin, killed suspension of tularaemia bacteria in 3 per cent glycerol.

Cutaneous Tests. Infective allergens are most often administered either intracutaneously or epidermally by rubbing them into scarified sites of the skin, less commonly they are injected subcutaneously.

In the intracutaneous method 0.1 ml of an allergen is injected into the middle portion of the anterior surface of the forearm, using a special thieedle. In 28-48 hrs (hypersensitivity of a delayed type) the results of the reaction are assessed by measuring the size of the erythema and infiltrate formed at the site of injection.

Non-infective allergens (pollen, household dust, foodstuffs, medi­cinal and chemical preparations) are administered epicutaneously by means of scarification, intracutaneously, and by applications onto the intact skin. The results are read within 20 min (hypersensi­tivity of the immediate type) from the size of the swelling and erythe­ma (i.e., the test is considered positive if the swelling is 20 mm in diameter and oedema and itching are present).

Cutaneous tests are widely used for detecting individuals infected with mycobacteria of tuberculosis (Mantoux’s test) and the causa­tive agents of brucellosis (Burnet’s test), leprosy (Mitsuda’s test), tularaemia. glanders, actinomycosis, dermatomycosis, toxoplasmosis, some forms of helminthiasis, etc.

Since allergens introduced into the body induce additional sensitization and in some cases may even cause allergic complications, one should adhere to very strict rules pertinent to selecting indi­viduals for allergic diagnostic tests.

In Vitro Tests. These methods of investigation are safe for the patient, highly sensitive, and allow to carry out a quantitative assessment of the body’s allergization. To date, a number of tests have become avail­able in -which reactions with T- and B-lymphocytes, tissue basophils, neutrophil granulocytes, etc. are employed for this purpose. These tests include inhibition of leucocyte migration and lymphocyte blast transformation, specific rosette formation, the parameter of neutrophil granulocyte damage, Shelley’s basophil test, reaction of tissue basophil degranulation. Still another test involves determi­nation of IgE in blood serum.

The specific rosette-formation test. Rosettes are formations devel­oping in vitro upon adherence of red blood cells to the surface of immunocompetent cells. Rosette formation may be spontaneous since human T-lymphocytes contain receptors to sheep red blood cells. The rate of spontaneous rosette formation iormal people is 52-53 per cent and can be used as an indicator of the functional status of T-lymphocytes. This phenomenon is also reproduced when sensitized lymphocytes are mixed with suspension of tanned erythrocytes on which the corresponding allergens are fixed.

Procedure. Lymphocytes are isolated from blood with the help of special column or by differential centrifugation in a gradient of density, washed off with Hanks’ solution and counted in 1 ml in Goryaev’s chamber, and then stained with trypan blue to de­termine their viability. To tan the red blood cells, to 0.1 ml of washed erythrocytes add 5 ml of tannic acid in 1:20 000 dilution and allow it to stand for 10 min with the subsequent washing in phosphate buffer (pH 7.2) and suspension in 5 ml of buffer. The allergen is fixed on tanned erythrocytes following mixing 1 ml of suspended red blood cells with 4 ml of phosphate buffer (pH 6.4} and 1 ml of allergen solution. The mixture is allowed to stand at 20 °C for 30 min, washed off with Hanks’ solution containing normal human serum (1:10), and then the red blood cells are resuspended in 2 ml of Hanks’ solution.

Lymphocytes isolated from the patient’s blood are mixed with sensitized erythrocytes in a 1 to 4 ratio (4 min of erythrocytes per 1 min of lymphocytes). The following control is utilized for the reac­tion: (1) mixture of allergen-treated erythrocytes with non-sensitized lymphocytes; (2) non-treated erythrocytes with lymphocytes of the patient examined. The test and control tubes are incubated at 37 °C for 2 hrs, and the results of the reaction are read. A total of 1000 lymphocytes are counted and the proportion of “rosettes” is determined. The term “rosette” refers to a lymphocyte to which no less than three red blood cells have become attached. To assess the degree of the body’s sensitization by one or another allergen, compare the results obtained in the test versus control tubes.

The tissue basophil degranulation test is based on the fact that tissue basophils of the rat sensitized by cytophil antibodies present in the patient’s blood serum undergo degranulation under the im­pact of allergen.

Procedure. On glass slides treated in advance with 0.3 per cent solution of neutral red place 0.05 ml of the patient’s blood serum, 0.05 ml of allergen and 0.05 ml of suspension of tissue basopliils obtained by washing of the abdominal cavity of a sacrificed rat with Tyrode’s solution without glucose. Cover the mixture with a cover slip whose edges are ringed with petrolatum. Incubate the prepa­rations at. 37 “C for 10-15 min and then examine under the micro­scope. Control of the degranulation reaction is: (1) suspension of tissue basophiis and allergen: (2) suspension of cells and the test se­rum. Degranulation in the control should be under 10 per cent. Count 100 tissue basophils, both normal and degranulated. Degranulation is expressed in a weaker staining of granules, appearance of vacuoles, irregularity of the edges of cells, their swelling, and rup­tures.

The results of the reaction are calculated in percentage by the formula

x = N₁ – N₂,

where N₁, is the  degranulation rate in the test: N₂ is the degranula­tion rate in the control.

The test is considered weakly positive when degranulation is 10-20 per cent; positive when it is 20-30 per cent; and markedly positive when it is 30 per cent or more.

Shelley’s basophil test. This reaction relies-on the fact that human or rabbit basophil granulocytes undergo degranulation in the  pres­ence of the patient’s serum and allergen to which a given patient is sensitive.

Procedure. Leucocytes are obtained from heparinized blood by its centrifugation at 2000 X g for 3 min. On a glass slide with a dye (0.3 per cent solution of neutral red in alcohol) mix in equal vol­umes rabbit leucocytes, the patient’s serum and allergen, cover it with a cover-slip sealing the edges with paraffin to prevent drying. Simultaneously set up three controls: (1) rabbit leucocytes + 2 equal volumes of isotonic .sodium chloride solution: (2) rabbit leucocytes + patients’ serum + isotonic sodium chloride solution; and (3) rabbit leucocytes + allergen + isotonic saline.

Let the preparations stand at. room temperature for 1 h and be examined under the oil-immersion objective. Count 20-25 basophil granulocytes determining the number of unaltered and altered cells. The test is considered positive when degranulation has affected at least one-third of basophil granulocytes.

 

The lymphocyte blast transformation (BT) test.

This reaction is based on the ability of normal peripheral lymphocytes to transform into non-differentiated germinal cells of the blast type following their in vitro cultivation in the presence of specific allergens and non-specific stimulators, mitogens (phytohaemagglutinin or PHA, concanavalin A, lipopolysaccharides, and other substances).

Procedure. A 3-5-ml aliquot of blood from the vein is transferred into a test tube with 1 ml of heparin diluted as 1:100 with Eagle’s medium without antibiotics. To the tube add 10 per cent solution of gelatin in the amount equal to one-tenth of the volume of the withdrawn blood (0.3-0.5 ml). Place the tube into a 37 “C incubator for 20-40 min. Then, aspirate the supernatant liquid plasma and visually count the number of cells in Goryaev’s chamber. Optimal for the reaction is the concentration of 1 -10⁶ cells per ml. If the number of cells is greater, the suspension is diluted to the necessary concentration with Eagle’s medium; if it is too small, the suspen­sion is centrifuged and the residue is suspended in the necessary amount of the medium. Then, add to the suspension antibiotics (peni­cillin and streptomycin) diluted in Eagle’s medium in a volume with which the final concentration of antibiotics constitutes 10 000 V per 100 ml of the medium. The suspension obtained is dispensed in special vials or test tubes of 2-ml capacity. One of the vials is con­trol, into the others specific or non-specific mitogens are added. The test tubes are incubated at 37 °C. The results of the test are read in 48-72 hrs. For this purpose, the contents of the vials is dispensed into centrifuge tubes and centrifuged’. The supernatant fluid is de­canted and the sediment is used to prepare smears which are stained by one of the techniques usually employed for blood cells and then examined microscopically- Two hundred lymphocytes, including the number of unaltered cells, cells which have undergone blast transformation, and intermediate cells, are counted.

To determine the rate of blast transformation, the sum of blasts and intermediate forms is determined per 100 cells. A similar calcula­tion is made in the control as well.

It has been proven that under normal conditions 40 to 90 per cent of lymphocytes can transform into blasts. Decreased blast trans­formation in the presence of allergens or PHA points to some ab­normality.

The leucocyte migration inhibition (LMI) test.

This test relies on suppres­sion of monocyte and leucocyte migration under the impact of me­diators produced by sensitized lymphocytes in the presence of a specific allergen.

Procedure. Into two or several places on a glass slide (depending on the number of the antigens tested) place four drops of the blood to he studied. Then, two drops of medium 199 are added to each of the large drops and one drop of the same medium and one drop of the antigen tested are added to the remaining large drops. The drops are well mixed and the mixture is transferred to glass capil­laries 7 mm long and 0.6-0.7 mm wide. The capillaries are soldered at one end and centrifuged at 1000 X g for 5 min. As a result, blood cells precipitate in the capillary as a homogeneous column occupying one-third of the capillary. After that the second end of the capillary is soldered and the capillary is placed into a Petri dish at a 10-15° angle. The dishes with the capillaries are incubated at 37 °C for 24 hrs,

The results of the reaction are assessed microscopically by measur­ing the height of the column of cell migration (in mm) from the sur­face of the cell sediment to the nutrient medium in the test versus control capillaries. Then, the coefficient or rate of cell migration inhibition is calculated by the following formula:

 

Height of the control column—height of the test column x 100 %

Height of the test column

 

The reaction is considered positive if the rate of cell migration inhibition is 30 per cent or more.

The test of neutrophil granulocyte damage (NGD).

Neutrophil granulocytes in the sensitized body show an elevated sensitivity to the respective allergen. It is on this phenomenon that assessment of the body’s sensitization in tuberculosis and other allergic condi­tions is based.

Procedure. Place 0.02 ml of the allergen (several types of diagnostic allergens for the test, of neutrophil granulo­cyte damage are available commercially), which is suspected to be responsible for allergization, based on 5 per cent sodium citrate into the test tube and 0,02 ml of 5 per cent sodium citrate into the con­trol one. Introduce 0.08 ml of the patient’s blood into each tube and put them in a 37 °C incubator for 2 hrs. After that the smears are prepared, fixed, and stained with haematoxylin. In assessing the results of the reaction, count 100 neutrophil granulocytes both in the test and control tubes, including damaged, destroyed, and deformed cells.

The rate of neutrophil granulocyte damage (RNGD) is calculated by the formula

           N – N₁

RNGD=  —————-,

           100

where N is the number of damaged neutrophil granulocytes in the test tube, N₁ is the number of damaged neutrophil granulocytes in the control tube, 100 is the number of cells.

Iormal individuals this parameter does not exceed 0.02-0.04.

 

AUTOIMMUNITY. In some individuals the immune response fails to recognize self-antigens. In such cases the immune system attacks one’s own body, a condition known as autoimmunity. The inability to recognize self-antigens results in reactions that kill some of one’s own cells. There are a number of autoimmune diseases that result from the failure of the immune response to recognize self antigens. Such autoimmune diseases often result in the progressive degeneration of tissues.

 

 

 

 

 

 

 

Some autoimmune diseases affect single sites within the body. Graves disease, for example, is an autoimmune disease that affects the thyroid. In Graves disease the body produces an antibody that reacts with the receptor for thyroid stimulating hormone. In contrast, some autoimmune diseases affect sites throughout the body. In systemic lupus erythematosus, numerous autoantibodies are produced that react with self-antigens They attack blood cells and cells at multiple body sites Antigen-antibody complexes circulate and settle in the glomeruli of the kidney In cases of myasthenia gravis, antibodies react with nerve-muscle junctions In autoimmune haemolytic anaemia, antibodies react with red blood cells, causing anaemia. Immunosuppressive substances are available to prevent the self-destruction of body tissues by the body’s own immune response.

Various other disease conditions reflect the failure of the immune system to recognize self-antigens. These self-antigens are similar to antigens associated with pathogenic microorganisms. For example, rheumatic fever is an autoimmune disease that results following an infection with group A streptococci (Streptococcus pyogenes) (FIG.). Some antibodies made in response to group A streptococcal antigens can also react with myosin of the heart muscle tissue. After a strep throat, therefore, antibodies made against the group A streptococci cross react with myosin in some individuals, causing tissue damage to the heart. These individuals develop rheumatic fever. Damaged heart valves may cause heart failure years later. The immune complexes between antibody and myosin or related antigens may also cause arthritis and kidney failure.

 

FIG. An autoimmune response can occur following an infection with Streptococcus pyogenes The normal immune response produces antibodies against streptococcal antigens. However, these anti-streptococcal antibodies can cross-react with heart tissue and cause damage that may result in later heart failure

 

Rheumatoid arthritis is a commonly occuring disease. Although rheumatoid arthritis is usually associated with older people, it often develops early in life. It is a chronic inflammation of the joints, especially the hands and feet It can lead to crippling disabilities. This form of arthritis often begins with a joint inflammation from an infection that causes phagocytic cells to release lysozymes. These degradative enzymes attack and alter certain antigens. B cells make IgM antibodies in response to the antigens and cause more inflammation in the joints.

Treatment of rheumatoid arthritis is designed to relieve the symptoms. There is no cure. Hydrocortisone lessens inflammation and reduces joint damage. Aspirin is also used because hydrocortisone produces side effects. Aspirin also reduces inflammation and pain.

Myasthenia gravis (MG) is an autoimmune disease that affects the neuromuscular system. It is characterized by weakness and rapid fatigue of the skeletal muscles. It affects muscles in the limbs and the muscles used in eye movement, speech, and swallowing. Patients with MG have a high incidence of thyroid abnormality, reduced levels of complement, and antiskeletal muscle antibody. This disease is rare. It affects 3 persons in every 100/000. Twice as many women as men are affected. The disease usually appears in late childhood to middle age.

Normal muscle contraction requires that pores in the membranes of neurons that stimulate muscles be open. It appears that antibodies that react with self-antigens may be blocking these pores in people with miasthenia gravis. When the pores are blocked, the neurons do not release acetylcholine. Acetylcholine initiates muscle cell contraction. Myasthenia gravis is treated with drugs that inhibit the enzyme that freaks down acetylcholine. The slowing of acetylcholine breakdown allows each muscle longer time to act. This compensates for the decreased amount of acetylcholine.

 

 

 

 

 

 

Systemic lupus erythematosus is a widely disseminated, systemic autoimmune disease. Erythematose means red and lupus means wolf. The name of the disease comes from a butterfly-shaped rash that appears on the nose and cheeks. It was thought that the rash looked like a wolf bite. This disease occurs four times as often in women as in men, usually during the reproductive years. Patients have reduced complement levels and high levels of immune complexes in their serum and glomeruli.

In this disease autoantibodies are made primarily against components of chromatin (DNA, RNA, and proteins). Immune complexes are deposited between the dermis and epidermis and in blood vessels, joints, glomeruli of the kidneys, and central nervous system. They cause inflammation and interfere with normal functions wherever they are. The symptoms depend on where the antigen-antibody complexes most interfere with function. Usually there is inflammation of the blood vessels, heart valves, and joints. A skin rash appears. Many victims die from kidney failure as glomeruli fail to remove wastes from the blood.

 

Patients with Graves disease, which include former President and Mrs. George Bush, suffer from overproduction of hormones produced by the thyroid. Normally the pituitary secretes thyroid-stimulating hormone, which controls the amount of thyroid hormone released. Antibodies to thyroid-stimulating hormone receptors are produced in Graves disease patients. These antibodies trigger thyroid cells to produce the hormones. The antibodies are not subject to hormonal feedback control and so the thyroid continues to produce, and overproduce, hormones. Treatment of this disease involves destruction of part of the thyroid. This is often accomplished using the radioisotope ⁿ¹!, which is concentrated in the thyroid gland and subsequently kills thyroid cells.

 

 

 

Multiple sclerosis (MS) occurs in people 20 to 50 years of age. Common signs are sensory and visual motor dysfunction. The etiology of this disease is unknown. It is generally believed, however, that MS is a T-cell-mediated autoimmune disease. Macroscopic lesions called plaques are found in the central nervous systems of MS patients. The lesions contain macrophages and lymphocytes. The term multiple sclerosis was originally used to describe the wide distribution of these lesions. There is also breakdown in the myelin sheath that surrounds nervous tissue.