Microbial Receptor Assay for Rapi d Detection and Identification of Seven Families of Antimicrobial Drugs in Milk: Collaborative Study

Abstract

A microbial competitive receptor assay for detecting residues of antibiotic families in milk was studied collaboratively by 13 laboratories. The drugs and levels (ppb) tested in this study include penicillin G, 4.8; cephapirin, 5.0; cloxacillin, 100; tetracycline, 2000; chlortetracycline, 2000; oxytetracycline, 2000; erythromycin, 200; lincomycin, 400; clindamycin, 400; sulfamethazine, 75; sulfamethoxazole, 50; sulfisoxazole, 50; streptomycin, 1000; novobiocin, 50; and chloramphenicol, 800. In this method, microbial cells added to a milk sample provide specific binding sites for which 14C or 3H labeled drug competes with drug residues in the sample. The 14C or 3H binding to the specific binding sites is measured in a scintillation counter and compared with a zero standard milk. If the sample is statistically different from the zero standard, it is positive. The assay takes about 15 min. The binding reaction occurs between the receptor site and the drug functional group, so all members of a drug family are detected. In this case, beta-lactams, tetracyclines, macrolides, aminoglycosides, novobiocin, chloramphenicol, and sulfonamides, including p-aminobenzoic acid (PABA) and its other analogs, are detectable. The incidence of false negative determinations among samples is about 1%; the incidence of false positives is about 3%. For negative cases, the relative standard deviations for repeatability ranged from 0 to 5% and for reproducibility from 0 to 6%. For positive cases, relative standard deviations ranged from 0 to 13% for repeatability and from 0 to 14% for reproducibility. The method has been adopted official first action.