Detection of serotype-specific antibodies or capsular antigen of Actinobacillus pleuropneumoniae by a double-label radioimmunoassay

Abstract

Actinobacillus pleuropneumoniae have been problematic because current tests do not use a purified antigen and in most cases measure either antibody or antigen, but not both. We describe a Farr-type double-label radioimmunoassay that utilizes purified, serotype-specific, 3H-capsule to measure antibody to capsule directly or that can measure capsule in a sample indirectly by inhibition of antibody binding. The assay could detect about 1 ng of serotype-specific antibody in serum or at least 100 pg of capsule in a sample. Due to the sensitivity of the assay, false-positive results were common with neat sera (probably due to cross-reacting antibodies to unrelated antigens), but the specificity was improved when the sera were diluted 1:100. The radioimmunoassay should prove to be a useful reference method for research and diagnostic testing and for comparison of new assays for detection of capsule or antibodies to capsule.

Safety, stability, and efficacy of noncapsulated mutants of Actinobacillus pleuropneumoniae for use in live vaccines

“…Phenotypic characterization and stability. Total capsule content was determined by inhibition radioimmunoassay of mid-log-phase bacterial cultures (including bacteria and medium) as described elsewhere (7). Electrophoretic profiles of LPS (one dimension) and total proteins (one and two dimen-NONCAPSULATED A. PLEUROPNEUMONIAE LIVE VACCINES sions) were evaluated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) followed by silver staining (11) or immunoblotting (13).…”

“…x g for 15 min, suspended in 25 RI of PBS, and tested for extracellular capsule by inhibition radioimmunoassay (7). In addition, noncapsulated mutants were passaged in vitro on BHI-N and screened for iridescence and capsule production by latex agglutination (10).…”

“…Sera from sows and piglets used in the study were assayed for antibodies to serotype 1 and 5 capsules by radioimmunoassay (7) and for antibodies to Apx toxins I and II by enzyme-linked immunosorbent assay (ELISA) (13). All pigs used in this study had antibody titers for these antigens less than or within the negative range (7,13). Neutralizing hemolytic antibody titers were kindly determined by Bradley Fenwick, Kansas State University, Manhattan.…”

“…Serum for specific antibody determination was obtained prior to immunization and again immediately prior to challenge. Antibody titers were determined for noncapsulated whole cells by ELISA (9), for Apx toxins I and II by indirect ELISA (13), and for capsule by ELISA (9) or radioimmunoassay (7). Pigs were necropsied as soon as possible after death or immediately after euthanasia with sodium pentobarbital.…”

A Novel Enzyme-Linked Immunosorbent Assay Using the RecombinantActinobacillus pleuropneumoniaeApxII Antigen for Diagnosis of Pleuropneumonia in Pig Herds

“…To date, two biotypes and 14 serotypes of A. pleuropneumoniae have been recognized, and convalescent sera frequently show a serotype-specific reaction (7,12). To control the disease, a variety of serological tests is being employed, with the complement fixation test (CFT) still being used as a reference test (9).…”