A helper antibody based competitive fluorescence immunochromatographic assay for quantitative detection of florfenicol in poultry eggs.

Abstract

Florfenicol (FF) is a chloramphenicol analogue used in animals, and florfenicol amine (FFA) is the main metabolite of FF. However, their residues in agricultural products are harmful to human health. A highly specific and sensitive assay for FF/FFA detection needs to be developed since the traditional detection methods are low sensitive. In this study, a new method for rapid quantification of FF/FFA in poultry eggs by fluorescent immunochromatographic assay (HAFIA) was established. The triple antibodies including a primary monoclonal antibody (mAb) specific to the targets FF and FFA, a secondary polyclonal antibody (pAb) labeled with Europium nanoparticles (EuNPs) and a helper monoclonal antibody (hAb) reacting with pAb but not react with mAb or target antigen are designed, which can form structural aggregation complexes in microwells with a single step of reactions. By loading the reaction sample solution, the triple-antibodies (mAb-pAb-hAb)-EuNPs complexes migrate to the test (T) line on nitrocellulose membrane of testing strip, which are competitively captured by the immobilized FF-BSA conjugates on the membrane and the FF/FFA targets in sample solution. Fluorescence on T line is read by a portable fluorescent strip reader in 10 min, and the result is given by the ratio of fluorescent intensity on T line than control (C) line. This new fluorescent testing strip with triple-antibody complexes' amplification signal has 50-fold higher sensitivity than conventional CG-LFIAs, and can detect as low as 0.01 ng/mL florfenicol and 0.1 ng/mL florfenicol amine targets from egg samples. The developed competitive fluorescent immunochromatography method based on auxiliary antibodies has the advantages of high sensitivity and specificity for the rapid and quantitative detection of FF/FFA in poultry eggs.