A novel chemiluminescent ELISA for detecting furaltadone metabolite, 3-amino-5-morpholinomethyl-2-oxazolidone (AMOZ) in fish, egg, honey and shrimp samples

摘要

In this study, an indirect competitive enzyme-linked immunosorbent assay with chemiluminescent (CLELISA) detection for 3-amino-5-morpholinomethyl-2-oxazolidone (AMOZ) was developed. A monoclonal antibody (MAb) against AMOZ was prepared through immunizing BALB/c mice with 4-carboxybenzaldehyle derivatized AMOZ (CPAMOZ), conjugated with bovine serum albumin (BSA) as antigen. The effects of the substrates luminol, p-iodophenol and urea peroxide on the performance of the assay were studied and optimized. In addition, the specificity of the MAb, estimated as the cross-reactivity values with 4-nitrobenzaldehyde derivatized AMOZ (NPAMOZ), CPAMOZ and AMOZ, was 100%, 27.45% and 0.18%, respectively. The sensitivity of the developed CLELISA was estimated as 50% inhibitory concentration (IC50) value (0.14μg/l) with a linear working range between 0.03 and 64μg/l, and a limit of detection of 0.01μg/l. The CLELISA described in this study was 5-fold more sensitive than the indirect competitive ELISA previously developed in our laboratory. Finally, this new CLELISA was compared with a commercial kit to detect NPAMOZ in spiked fish, shrimp, honey and egg samples. The recovery values from four spiked fish, shrimp, honey and egg samples with different concentrations of NPAMOZ in CLELISA were 92.1-107.7%. Thus, the immunoassay method described here has a broad detection range and high sensitivity and is a valid and cost-effective means for high throughput monitoring of residual AMOZ levels in fish, shrimps, honey and eggs with potential applications in other animal tissues.