ENZYME IMMUNOASSAYS FOR THE RAPID DETECTION OF SALMONELLAE IN FOODS (ELISA, SALMONELLA, METHODS0
Abstract
Enzyme immunoassays (EIA) were developed to detect Salmonella in foods and feeds. Initially, a protocol for an indirect EIA was developed. The assay could be completed within 27 hours and included the use of microtitration plates for immobilization of Salmonella antigens, and a combination of commercial polyvalent Salmonella H antiserum and staphylococcal protein A-alkaline phosphatase conjugate for antigen detection. Before applying the EIA, the samples of products suspected to contain Salmonella were subjected to standard enrichment procedures. The sensitivity of this EIA compared favorably with that of the conventional cultural technique for detection of Salmonella in 40 naturally contaminated food and feed samples. Enteric bacteria and proteins of some food samples were found to cross-react with the polyvalent antiserum. In an attempt to increase the sensitivity of the EIA, covalent binding was used to immobilize Salmonella antigens on the surface of activated microtitration plates. Chemically modified plates retained more antigen after washing than did untreated plates to which the antigens had been physically adsorbed. However, improvement of assay sensitivity depended on the type of plate used for covalent binding of antigen. It was determined that two types of plates commercially available, used without activation, yielded satisfactory results in the EIA for Salmonella. Conjugates of (beta)-galactosidase and a myeloma protein (M467 antibody) prepared with the heterobifunctional reagent N-succinimidyl 3-(2-pyridyldithio) propionate (SPDP) gave better results in the EIA for Salmonella than a conjugate prepared with glutaraldehyde. The sensitivity level for the SPDP conjugate was in the range of 10('3) to 10('5) cells per ml, depending on the serotype of Salmonella. Purification of the SPDP conjugate by gel filtration did not improve assay sensitivity but decreased conjugate titer. Finally, a double-antibody sandwich EIA was developed for the detection of salmonellae in foods or other samples. The test utilizes the M467 antibody-(beta)-galactosidase conjugate and a fluorogenic substrate for enzyme detection. The EIA was sensitive and rapid, and compared favorably with the conventional cultural technique in the analysis of 60 feed samples naturally contaminated with Salmonella. Proteins or natural contaminants from the samples did not interfere in the assay.
Degree
Ph.D.
Subject Area
Food science
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