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

The influence of thermodynamic and kinetic conditions on the interaction of polyclonal antibodies to penicillins with the antibiotics of a penicillin group was studied in the system of a direct enzyme-linked immunosorbent assay (ELISA). Minimum differences in the cross reactions of the polyclonal antibodies with different penicillins were observed when the ELISA was carried out at 4°C for 1 hour. An increase in temperature and duration of the assay led to an increase in antibodies reactivity only to amoxicillin, and significantly enhanced differences among the sensitivities of individual penicillins determination. Under the chosen assay conditions, the following antibodies cross-reactivity values were obtained: to penicillin G — 90%, to ampicillin — 100%, to 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 regulatory controlled in foods and raw materials of animal origin — penicillin G, ampicillin, amoxicillin, cloxacillin, oxacillin, dicloxacillin and nafcillin.