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Chapter: Microbiology and Immunology: Antigen-Antibody Reactions

Types of agglutination reactions: Direct, Passive - Antigen Antibody Reactions

Agglutination reactions where the antigens are found naturally on a particle are known as direct agglutination. This is different from passive agglutination, which employs particles that are coated with antigens not normally found on their surfaces.

Types of agglutination reactions

Agglutination reactions where the antigens are found naturally on a particle are known as direct agglutination. This is different from passive agglutination, which employs particles that are coated with antigens not normally found on their surfaces.

Direct agglutination

Direct agglutination reactions can broadly be of the following types: (a) slide agglutination, (b) tube agglutination, (c) heterophile agglutination, and (d) antiglobulin (Coombs’) test.

Slide agglutination test: It is a basic type of agglutinationreaction that is performed on a slide. Identification of bacterial types represents a classic example of a direct slide agglutination that is still used today. In this test, a suspension of bacteria is prepared and is added to a drop of standardized antiserum. A positive reaction is indicated by clumping of bacteria and clearing of the background solution. Clumping occurs instantly or within seconds in a positive test. A control consisting of antigen suspension in saline without adding antiserum is included on the same slide. It is used to validate the results and also to detect possible false positives due to autoagglutination of the antigen.

Tube agglutination test: Tube agglutination test, as the namesuggests, is performed in glass tubes. Typically, in these tests, patient’s serum is diluted in a series of tubes and bacterial antigens specific for the suspected disease are added to it. Antigen and antibody reactions are demonstrated by demonstration of visible clumps of agglutination. It is a standard method used for quantitative estimation of antibodies in the serum. Tube agglutination tests are routinely used for demonstration of antibodies in the serum for serodiagnosis of enteric fever and brucellosis, as follows:

Heterophile agglutination test: This test depends on demons-tration of heterophilic antibodies in serum present in certain bacterial infections:

Antiglobulin (Coombs’) test: Coombs’ test was devisedoriginally by Coombs’, Mourant, and Race for detection of incomplete anti-Rh antibodies that do not agglutinate Rh1 erythrocytes in saline. When serum containing incomplete anti-Rh antibodies is mixed with Rh1 erythrocytes in saline, incomplete antibody antiglobulin coats the surface of erythrocytes but does not cause any agglutination. When such erythrocytes are treated with antiglobulin or Coombs’ serum (rabbit antiserum against human g globulin), then the cells are agglutinated. Coombs’ test is of two types: (a) direct Coombs’ test and (b) indirect Coombs’ test.

·           Direct Coombs’ test: In this test, the sensitization of redblood cells (RBCs) with incomplete antibodies takes place invivo. The cell-bound antibodies can be detected by this testin which antiserum against human immunoglobulin is used to agglutinate patient’s red cells (Fig. 14-9).

·           Indirect Coombs’ test: In this test, the sensitization ofRBCs with incomplete antibodies takes place in vitro. In this test, the patient’s serum is mixed with normal red cells and antiserum to human immunoglobulin is added. Agglutina-tion occurs if antibodies are present in the patient’s serum (Fig. 14-10).

Coombs’ tests are used for detection of (a) anti-Rh antibodies and (b) incomplete antibodies in brucellosis and other diseases.

Passive agglutination

Passive agglutination employs carrier particles that are coated with soluble antigens. This is usually done to convert precipitation reactions into agglutination reactions, since the latter are easier to perform and interpret and are more sensitive than precipitation reactions for detection of anti-bodies. When the antibody instead of antigens is adsorbed on the carrier particle for detection of antigens, it is called reversepassive agglutination.

Until the 1970s, erythrocytes were the major carrier particles used for coating of antigens. Recently, however, a variety of other particles including polystyrene latex, bentonite, and charcoal are used for this purpose. Particle size vary from 7 m for RBCs to 0.05 m for very fine latex particles. The use of synthetic beads or particles provides the advantage of consistency, uniformity, and stability. Reactions are also easy to read visually. Passive agglutination reac-tion, depending on the carrier particles used, can be of the follow-ing types: (i) latex agglutination test, (ii) hemagglutination test, and (iii) coagglutination test.

Latex agglutination test: It is a test that employs latex particlesas carrier of antigen or antibodies. In 1955, Singer and Plotz accidentally found that IgG was naturally adsorbed to the surface of polystyrene latex particles.

Latex particles are inexpensive, relatively stable and are not subject to cross-reactivity with other antibodies. These particles can be coated with antibodies to detect antigen in the serum and other body fluids. Use of monoclonal antibodies has reduced the cross-reactions resulting in reduction of false positive reactions.

Additionally, the large particle size of the latex facilitates better visualization of antigen–antibody reactions by the naked eye observation. The tests are usually performed on cardboard cards or glass slides and positive reactions are graded on a scale of 11 to 41.

Hemagglutination test: RBCs are used as carrier particlesin hemagglutination tests. RBCs of sheep, human, chick, etc. are commonly used in the test. When RBCs are coated with  antigen  to  detect  antibodies  in  the  serum,the  test is  called  indirect  hemagglutination  (IHA)  test.  The IHA  is a most commonly used test for serodiagnosis of many parasitic diseases including amoebiasis, hydatid disease, and toxoplasmosis.

      When antibodies are attached to the RBCs to detect micro-bial antigen, it is known as reverse passive hemagglutination(RPHA). The RPHA has been used extensively in the pastto detect viral antigens, such as in HBsAg in the serum for diagnosis of hepatitis B infection. The test has also been used for detection of antigens in many other viral and parasitic infections.

Viral hemagglutination: Many viruses including influenza,mumps, and measles have the ability to agglutinate RBCs with-out antigen–antibody reactions. This process is called viral hemagglutination. This hemagglutination can be inhibited by antibody specifically directed against the virus, and this phenomenon is called hemagglutination inhibition. This forms the basis of the viral hemagglutination inhibition test, which is used to detect antibodies in patient’s sera that neutralize the agglutinating viruses. To perform this test, patient’s serum is first incubated with a viral preparation. Then RBCs that the virus is known to agglutinate are added to the mixture. If anti-body is present, this will combine with viral particles and prevent agglutination, and a lack of or reduction in agglutination indi-cates presence of antibody in patient’s serum.

Coagglutination test: Coagglutination is a type ofagglutination reaction in which Cowan I strain of S. aureus is used as carrier particle to coat antibodies. Cowan I strain of S. aureus contains protein A, an anti-antibody, that combineswith the Fc portion of immunoglobulin, IgG, leaving the Fab region free to react with the antigen present in the specimens (Fig. 14-11). In a positive test, protein A bearing S. aureus coated with antibodies will be agglutinated if mixed with specific antigen. The advantage of the test is that these particles show greater stability than latex particles and are more refractory to changes in ionic strength.

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