Usage of immunological tests in diagnosis of infectious  diseases

Usage of immunological tests in diagnosis of infectious  diseases.

Reactions based on Agglutination and precipitation phenomenen

Lysis and complement fixation test.

 

 

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 contaiormal  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.

 

I.                Components: 1. Pair serum from patient (unknown Ab)   2.Specific erythrocytes diagnosticum   3.NaCl solution