A known positive antiserum may be collected from a horse previously infected with EIA virus. This serum should yield a single dense precipitation line that is specific for EIA, as demonstrated by a reaction of identity with a known standard serum. It is essential to match the antigen and antibody concentrations in order to ensure the optimal sensitivity of the test. Reagent concentrations should be adjusted to form a narrow precipitation line approximately equidistant between the two wells containing antigen and serum. Immunodiffusion reactions are carried out in a layer of agar in Petri dishes.
Noble agar is used.
Six wells are punched out of the agar surrounding a central well of the same diameter. The wells are 2.4 mm apart and 5.3 mm in diameter. Each well must contain the same volume of reagent. The antigen is placed in the central well and the standard antiserum is placed in alternate exterior wells. Suspect serum samples are placed in the remaining three wells.