Immunology

Active Agglutination Reaction

Active Agglutination test

When particulate antigens reacts with specific antibody, antigen antibody complex forms visible clumping under optimum PH and temperature. Such reaction is called agglutination. Depending on the nature of antigens, agglutination reaction can be active or passive.

 

Agglutination reactions where antigens are found naturally on a particle are known as direct/active agglutination. In active agglutination, direct agglutination of particulate antigen with specific antibody occurs.

 

Direct bacterial agglutination uses whole pathogens as a source of antigen. It measures antibody level produced by a host infected with that pathogen. The binding of antibodies to surface antigens on the bacteria results in visible clumps.

 

E.g. Blood grouping, Bacterial serotyping such as Salmonella, test serum against Rickettsia diseases, typhoid fever, bacteria that are difficult to grow in culture, such as Tularemia and hemagglutination kits for measles antibody detection.

 

Active Agglutination

 

Active agglutination can be of following types:

Slide/Tile agglutination: Basic type of agglutination reaction that is performed on a slide. Identification of bacterial types is a classic example of slide agglutination. In this method suspension of unknown antigen is kept on slide and a drop of standardized antiserum is added or vice versa. A positive reaction is indicated by formation of visible clumps. E.g. Widal test, RPR test.

 

Slide test may give misleading results, especially with very high serum antibody levels (e.g., prozone phenomenon with brucella antibodies). They are used as initial screening tests.

 

Tube agglutination: It is agglutination test performed in tube and standard quantitative technique for determination of antibody titer.  In this method serum is diluted in a series of tubes and standard antigen suspensions (specific for suspected disease) are added to it. After incubation, antigen-antibody reaction is indicated by visible clumps of agglutination.

 

Tube Agglutination

 

Highest dilution of serum showing visible agglutination is taken as the titer of the serum (E.g. Widal tube test). The titer reflects concentration of antibodies in serum; higher the titer, more is the concentration of antibodies. It is used to quantitate the amount of serum antibodies against an antigen.

  

Heterophile agglutination test: This test depends on demonstration of heterophilic antibodies in serum present in certain bacterial infections. Often referred to as mono-spot test it is a hemagglutination test for detection of antibodies against Epstein Barr (EB) virus that cause infectious mononucleosis disease. The IgM antibodies formed during infectious mononucleosis cross react with equine RBC surface antigens (probably due to antigenic similarity between EB virus and equine RBC surface proteins) and cause hemagglutination.

 

Antiglobulin (Coombs) test: This test was devised 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 surface of erythrocytes but does not cause any agglutination. When such erythrocytes are treated with antiglobulin or Coombs serum (rabbit antiserum against human gamma globulin), then cells are agglutinated. Coombs test can be direct as well as indirect.

 

Antiglobulin or Coombs test

 

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

 

  • In indirect method, sensitization of RBCs with incomplete antibodies takes place in vitro. Patient’s serum is mixed with normal red cells and antiserum to human immunoglobulin. Agglutination occurs if antibodies are present in serum.

 

Coombs test is used for detection of anti-Rh antibodies and incomplete antibodies in brucellosis and other diseases.

 

Uses of Active agglutination:

  1. Identification of microorganisms. E.g. Bacterial typing
  2. Diagnosis of infections by detection of antibodies.
  3. Blood grouping (ABO grouping).

 

Download Link: Active Agglutination Reaction.pdf

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