Monoclonal Antibody-Based Immunoassay for the Detection of Maduramicin in Chicken Tissues

Abstract

This paper reports on the preparation of a monoclonal antibody (Mab) against maduramicin (MD) and the development of a simple and sensitive ELISA for MD in chicken tissues. MD was conjugated to bovine serum albumin for immunogens and ovalbumin for coating antigens by mixed anhydride (MA) and active ester (AE) methods. Hybridoma cells were generated using spleen cells from a mouse immunized with MD-BSA conjugate. After screening with ELISA, the Mab with high anti-interference ability and high affinity was selected, and it exhibited negligible cross-reactivity with other usual-used polyether antibiotics. After optimization, the developed ELISA showed an IC50 value of 2.12 ± 0.46 ng/ml (n = 20). Chicken muscle and liver samples were extracted with methanol-8% NaCl solution (4:1) and then directly diluted with PBS-10% methanol for analysis. The recoveries of MD from spiked chicken tissues at levels of 40–480 µg/kg ranged from 81.3% to 91.3% with variation coefficients of 5.2–12.1%, and the detection limits were 6.5 µg/kg in muscle and 9.2 µg/kg in liver, respectively (n = 20).