Precipitation

Mechanism: Precipitation is a method analogous to agglutination except that the antigen is essentially not particulate (viruses or protein in solution). In order for a visible precipitate to form the antigen and antibody must be in a balanced concentration. This is usually achieved by carrying out a double-immunodiffusion (DID) test in agar.

Reagents: The antigen used requires to have a high concentration of precipitating proteins. For viruses this is usually achieved by inoculating the allantoic cavity of chicken eggs and then collecting the chorio-allantoic membranes. Individual membranes may be checked for activity against a known positive serum and those with good reactivity are homogenized and frozen prior to use. For certain viral infections specific tissues from affected birds may be used to make an antigen (spleens for "Marble Spleen Disease" adenovirus infection of pheasants, or bursae from chickens with Gumboro Disease). The agar used in the test is usually made from 1.25% purified agar (Agar Noble, Difco for example) in 8% saline buffered to pH 7.5. Field sera are usually tested neat, although, where high titres of precipitating antibody are present, testing of doubling dilutions may be used to make the test semi-quantitative (Cullen and Wyeth, 1975). Woernle (1966) provides detailed information on preparing a range of reagents for use in DID.

Methods: Antibody and antigen are allowed to diffuse in the direction of each other from holes cut in an agar gel (Woernle, 1966). By cutting the wells in a circle of six arrayed around a central antigen well it is possible to test 4 sera in each gel (as well as two samples of positive control serum). A valid positive reaction should show as a line of precipitation which is continuous with that produced by the adjacent positive control serum.\par \par Assays: AGP-DID has been used for the detection of antibody to a wide range of antigens, among them Newcastle Disease, Infectious Bronchitis, Avian Influenza, Infectious Laryngotracheitis, Fowl Pox, Adenovirus, and EDS-76 adenovirus, Reovirus and Infectious Bursal Disease/Gumboro Disease.

Comments: Not alone is a highly concentrated antigen required for this test but also the concentration of antibody in the test serum must also be high. For this reason DID positive reactions tend to be of low incidence and short lived, and the test is relatively insensitive compared to most other methods (Woernle, 1966, Beard, et alii, 1991). These factors taken together with the relative difficulty of quantifying antibody titres have led to a decreasing use of this method. On the other hand it is a method which does not require expensive equipment and the reagents can be prepared quite simply if seed strains for the viruses and known positive anti-sera are available.