Study On Chemiluminescence Enzyme Immunoassay For Clenbuterol Hydrochloride

In this paper, the chemiluminescence enzyme immunoassay for the determination of Clenbuterol (CLB) residues in animal foods had been developed. We investigated the method of coupling hapten to carrier protein. CLB was coupled with Bovine Serum Albumin (BSA) or Ovalbumin (OVA) or HRP by diazotization, producing the immune antigen (CLB-BSA), the coating antigen (CLB-OVA) and the enzyme trace (CLB-HRP). The anti-CLB antibody produced was specific for CLB. The optimal physical-chemical parameters had been investigated, Under the optimum conditions, the direct competitive enhanced chemiluminescence enzyme immunoassay (dc-CLEIA) for the rapid detection of CLB in various food matrices was developed. The half-maximum inhibition concentration (IC50) and the limit of detection (LOD) of the assay is0.25μg/L and0.02μg/L,respectively. The relative standard deviations (RSD) of intra-and inter-assay were less than20%. Three concentrations (0.1p.g/kg、1μg/kg、5μg/kg) of clenbuterol spiked pig muscles and chicken samples were detected after treated with perchloric acid solution followed by clean-up by solid phase extraction (SPE). The mean recoveried were between80%and100%.Based on polyclonal antibody and enzyme tracer of the chemiluminescence enzyme immunoassay, the enzyme-linked immunosorbent assay (ELISA) for detection of CLB in food samples had been developed. Assay conditions were optimized, and IC50value of the optimized ELISA system was1.0μg/kg, with the limit of detection (LOD) of the assays was0.1μg/kg. The correlation of the dc-CLEIA with the ELISA was investigated, and the result showed that their relativity was very good, R2=0.999.In order to validate veracity of the dc-CLEIA, the fortified samples were analyzed by high-performance liquid chromatography (HPLC). Sample extracts were analyzed by dc-CLEIA and HPLC, and the results obtained by two methods correlated well, the correlation coefficient was0.9647.The stability of antibody and enzyme tracer was tested, and the result indicated that the reliability of the assay was good within6months when the two reagents were placed at4℃. The dc-CLEIA method and the rapid extraction method developed in this paper is feasible for preparing CLEIA kits, which can determine CLB residues in animal foods rapidly.