Indirect enzyme-linked immunosorbent assay for detection of antibody to a 110,000-molecular-weight hemolysin of Actinobacillus pleuropneumoniae.

An indirect enzyme-linked immunosorbent assay (ELISA) was developed to detect swine antibody to a 110,000-molecular-weight hemolysin (110K hemolysin) of Actinobacillus pleuropneumoniae. Affinity-purified rabbit polyclonal or mouse monoclonal immunoglobulin G to the hemolysin of A. pleuropneumoniae serotype 5 strain J45, followed by hemolysin-rich concentrated culture supernatant, was used to bind swine antibody to hemolysin to microdilution plates. Sixty-nine serum samples from swine that were clinically normal, presented with clinical evidence of pleuropneumonia, were experimentally immunized or challenged, or were free of pleuropneumonia were tested, and their ELISA titers were compared with complement fixation (CF) titers. On the basis of serum samples from swine that were clinically normal and negative by CF, an ELISA titer of 1:320 or greater was considered positive. In comparison with CF, the sensitivity of the ELISA was 98.1% and the specificity was 90%. The two samples negative by CF and positive by indirect ELISA were, however, also positive for antibody to serotype 5 capsule by ELISA. Immunization of normal pigs with whole cells or purified hemolysin boosted titers 4- to 128-fold within 4 weeks. Immunoblotting demonstrated that the affinity-purified immunoglobulin G to hemolysin used for capture in the assay recognized only a 110K protein of A. pleuropneumoniae serotypes 1 to 7, although the reactivity was quantitatively variable between serotypes. Therefore, the indirect ELISA is capable of identifying animals infected with or exposed to most, if not all, serotypes of A. pleuropneumoniae. If an indirect ELISA titer of 1:320 or greater is considered positive, the assay can be a valuable diagnostic tool in both clinical and research laboratories.