Detection of Penicillinase in Milk by Sandwich ELISA Based Polyclonal and Monoclonal Antibody

Abstract

A sandwich ELISA has been developed using polyclonal and monoclonal antibody for the determination of penicillinase in milk. For this purpose, specific polyclonal and monoclonal antibodies against penicillinase were generated and characterized. Using penicillinase standards prepared from 1–128 ng/mL, the method indicated that the detection limit of the sandwich ELISA, as measured in an ELISA plate reader, was as low as 0.86 ng/mL of penicillinase. For determine the accuracy, raw milk containing 2, 8, 32, and 64 ng/mL of penicillinase were tested by sandwich ELISA. Recoveries were from 93–97.5%, and the coefficient of variation [CV (%)] were from 5.55–8.38%. For interassay reproducibility, recoveries were from 89.5–95.1%, the coefficient of variation [CV (%)] were from 5.26–9.58%. This sandwich ELISA provides a useful screening method for quantitative detection of penicillinase in milk.