Dual-Label Chemiluminescence Strategy for Multiplexed Immunoassay of 20 Fluoroquinolones, 15 β-Lactams, 15 Sulfonamides, and CAP in Milk

Abstract

In this paper, a novel dual-label time-resolved chemiluminescent multiplexed immunoassay (DLTRC-MIA) based on the distinction of the kinetic characteristics of horseradish peroxidase (HRP) and alkaline phosphatase (ALP) with approximate estimation approach for simultaneous determination of 20 fluoroquinolones (FQs), 15 β-lactams, 15 sulfonamides (SAs), and chloramphenicol (CAP) in milk was developed. The strategy integrated a single-chain variable fragment–alkaline phosphatase fusion protein (scFv-ALP), a recombinant penicillin-binding protein (PBP) 2×*, a monoclonal antibody (MAb), and a polyclonal antibody (PAb) in one immunoassay and in a single well together to fulfill the simultaneous detection of 51 low-molecular weight contaminants (20 FQs, 15 β-lactams, 15 SAs, and CAP). The limits of detection for FQs, β-lactams, SAs, and CAP range from 0.29 μg L−1 for ciprofloxacin (CIP) to 81.6 μg L−1 for trovafloxacin (TRO), 0.27 μg L−1 for ceftiofur (CEF) to 44.1 μg L−1 for cephalexin (CEL), 0.089 μg L−1 for sulfadimethoxine (SDM) to 2.7 μg L−1 for sulfadiazine (SDZ), and 0.028 μg L−1 for CAP, respectively. The results demonstrated that the detection limits of DLTRC-MIA meet the requirement of detection levels for 51 drug residues in milk, suitable for high-throughput screening of low-molecular weight contaminants.