The Dependence of the Group Specificity of Immunoenzyme Determination of Penicillins in Milk on the Temperature and Duration of Antibiotic Cross Reactions with Polyclonal Antibodies

Abstract

The influence of thermodynamic and kinetic conditions on the interaction of polyclonal antibodies to penicillins with the antibiotics of the penicillin group was studied in the system of a direct enzyme-linked immunosorbent assay (ELISA). Minimal differences in the cross reactions of the polyclonal antibodies with different penicillins were observed when ELISA was carried out at 4°C for 1 h. An increase in temperature and duration of the assay led to an increase in the reactivity of antibodies only to amoxicillin and significantly enhanced differences among the sensitivities of determination of individual penicillins. Under the chosen assay conditions, the following antibody cross-reactivities were obtained: penicillin G, 90%; ampicillin, 100%; and amoxicillin, 110%. The analytical sensitivity was 0.03 ng/mL for ampicillin, and the limit of ampicillin quantification in milk was 0.4 μg/L. The developed group-specific ELISA was used for the determination in milk of seven penicillins that are regulatorily controlled in foods and raw materials of animal origin: penicillin G, ampicillin, amoxicillin, cloxacillin, oxacillin, dicloxacillin and nafcillin.