Agglutination

Agglutination

  • Agglutination is an antigen–antibody reaction in which an antigen combines with its antibody in the presence of electrolytes at a specified temperature and pH resulting in formation of visible clumping of particles.
  • Agglutination occurs when antigens and antibodies react in equivalent proportions.
  • Agglutination reactions have a wide variety of applications in the detection of both antigens and antibodies in serum and other body fluids.
  • They are very sensitive and the result of the test can be read visually with ease.

Types of agglutination reactions:

  • Direct agglutination (Active agglutination)

    • Slide agglutination.
    • Tube agglutination.
    • Heterophile agglutination.
    • Antiglobulin (Coombs’) test.
  • Indirect agglutination (Passive agglutination)

    • Latex agglutination test.
    • Hemagglutination test.
    • Coagglutination test.

Direct agglutination (Active agglutination)

Slide agglutination test:

  • It is a basic type of agglutination reaction 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.
  • Control 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 name suggests, is performed in glass tubes or test tube.
  • In this 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 seen 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.

Heterophile agglutination test:

  • This test depends on demonstration of heterophilic antibodies in serum present in certain bacterial infections.

Antiglobulin (Coombs’) test:

  • Coombs’ test was devised originally by Coombs’, Mourant, and Race for detection of incomplete anti-Rh antibodies that do not agglutinate Rh+ erythrocytes in saline.
  • When serum containing incomplete anti-Rh antibodies is mixed with Rh+ 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 γ globulin), then the cells are agglutinated.

Coombs’ test is of two types:

(a) Direct Coombs’ test.

(b) Indirect Coombs’ test.

Direct Coombs’ test:

  • In this test, the sensitization of red blood cells (RBCs) with incomplete antibodies takes place in vivo.
  • The cell-bound antibodies can be detected by this test in which antiserum against human immunoglobulin is used to agglutinate patient’s red cells.

Principle of the direct Coombs’ test

Indirect Coombs’ test:

  • In this test, the sensitization of RBCs 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.
  • Agglutination occurs if antibodies are present in the patient’s serum.

Principle of the indirect Coombs’ test

Coombs’ tests are used for detection of:

(a) anti-Rh antibodies

(b) incomplete antibodies in brucellosis and other diseases.

Indirect agglutination (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 antibodies.
  • When the antibody instead of antigens is adsorbed on the carrier particle for detection of antigens, it is called reverse passive agglutination.

Latex agglutination test:

  • It is a test that employs latex particles as 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.
  • 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 1+ to 4+.

Hemagglutination test:

  • RBCs are used as carrier particles in hemagglutination tests.
  • When RBCs are coated with antigen to detect antibodies in the serum, the test is called indirect hemagglutination (IHA) test.
  • The indirect hemagglutination (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 microbial antigen, it is known as reverse passive hemagglutination (RPHA).
  • The RPHA has been used extensively in the past to detect viral antigens.
  • The test has also been used for detection of antigens in many other viral and parasitic infections.

Coagglutination test:

  • Coagglutination is a type of agglutination 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 combines with the Fc portion of immunoglobulin, IgG, leaving the Fab region free to react with the antigen present in the specimens.
  • 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.

Principle of the coagglutination

Agglutination

THANK-YOU

Also read: 

  1. Precipitation

  2. Antigen Antibody Reactions

 

 

 

 

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