Agglutination

Mechanism: An antigen composed of a suspension of particles with surface antigen is mixed with the antibody-containing fluid (usually serum, sometimes whole blood). The clumping of the particles by antibody molecules connecting to more than one particle is then visualized (Figure 2.5).

Reagents: The antigen is usually composed of a suspension of killed bacterial cells which are stained with a dye to facilitate the observation of the reaction (for rapid tests and micro-titre tests, see methods below). Agglutination tests are also widely used in blood-typing work. It is possible to create agglutinating antigens for non-particulate antigens by absorbing the antigen on latex particles or blood cells (though this is not commonly used in routine poultry serology at present). The only other reagent required is the serum or blood to be tested (usually undiluted).

Methods: Three distinct types of agglutination tests are used, rapid plate agglutination, slow agglutination in tubes, and slow agglutination in micro-titre plate. The rapid tests are carried out by mixing antigen and serum (initially un-diluted) on a plate, rotation or agitation of the plate to a fixed method, and the reading of the presence of agglutination by macroscopic examination, usually after 2 minutes. If care is taken to mix the antigen and serum in a fixed proportion the intensity of the agglutination reaction is a good indicator of the concentration of antibody in the serum. Strong positive reactions are often confirmed by heat-treating the sera to destroy non-specific agglutinins (56 C. for 30 minutes) and by repeating the test with various dilutions. Slow agglutination in tubes usually involves diluting the serum in a relatively large amount of unstained antigen (usually 1:25 or 1:50) in a tube. After 24 hours positive results are visualized by the presence of a precipitate in the bottom of the tube and a clearing of the supernatant (as compared to antigen without any serum). Micro-agglutination is based on the same mechanism as the two preceding types of agglutination. It is carried out using relatively small amounts of antigen in a micro-titration plate, facilitating the examination of a large number of samples at various dilutions. In this case a positive reaction is manifest as a ragged blanket of coloured antigen covering the bottom of the U-shaped micro-titre well, as opposed to a button of unreacted antigen in the negative wells.

Assays: Various types of agglutination tests were very important in the eradication of Salmonella pullorum from commercial poultry. In most countries this assay is less frequently performed today. Rapid agglutination tests are still widely performed for Mycoplasma gallisepticum, and M.synoviae in chickens and M.g., M.s. and M.meleagridis in turkeys.

Figure 2.5. Rapid plate agglutination tests showing 1 negative test and 3 positives of differing intensity reaction.

Comment: IgM is especially reactive in this test because of the large number of attachment sites on each molecule. The rapid plate agglutination test used in the diagnosis of mycoplasmosis tends to be more sensitive though less specific than the HI test (Avakian et alii, 1988).