Study On The Detection Of Clenbuterol Hydrochloride By Immunochromatographic Assay

Clenbuterol hydrochloride（CL）is a representative of the class ofβ-adrenergic agonist.Since 1980s,CL was used as a feed additive to increase the lean rate of animals,as it was found to be able to alter etabolic pathways,reduce fat deposition and increase protein accretion.However,with steady chemical property and long plasma half-life,CL is easy to accumulate in animals’body.But then countries all over the world have banned the use of CL as animal feed additive,because once animal products containing excessive CL are taken by people,it will cause food poisoning and even death among consumers.Even so,CL is still being used illegally,which is a great hidden danger to food safety.In order to ensure the food safety and protect human health,it’s urgent to build a fast and reliable detection method for this food safety hazard.In this study,clenbuterol hydrochloride was used as the detection target to prepare monoclonal antibody.With the antibody as the key substance,two kinds of immunochromatographic assay（ICA）strips were successively developed for rapid detection of CL in pork,milk,bacon,et al.The following results were obtained in this study:1.Preparation of the anti-CL monoclonal antibody（mAb）.Prepared by induction method in mice,the ascites containing anti-CL monoclonal antibodies were purified and identified.The results showed that the titer and the 50%inhibitory concentration(IC₅₀)of the mAb were 1:32000 and 0.69 ng mL^-1,respectively.The anti-CL mAb was a critical material for the construction of ICA strips.2.Construction and characterization of the traditional immunochromatographic assay strip based on gold nanoparticles（GNPs）for the rapid detection of CL.As signal labels,the prepared GNPs were combined with the anti-CL monoclonal antibody to form the GNP-mAb probe.Then,after optimizations,the optimal labeling amount of the antibody,the optimal coating concentration of the antigen on the NC membrane,the optimal blocking condition of the sample pad and the optimal volume of the probe are determined.Under the optimal conditions,the visual detection limit（VDL）of the traditional ICA strip for CL standard solution was 5 ng mL^-1.In addition,the strip showed satisfactory specificity with no cross-reaction with the other five structural analogues of CL.For the residual monitoring of CL in food samples,its VDL for CL in pork,bacon and milk was 10 ng g^-1,10 ng g^-1 and10 ng mL^-1,respectively.3.Construction and characterization of a new lateral flow assay（LFA）strip based on Prussian blue nanoparticles（PBNPs）for the rapid detection of CL.The PBNPs were synthesized by a typical procedure.The Prussian blue nanoparticles were combined with the anti-CL monoclonal antibody by electrostatic adsorption.PBNP is quite larger than most of the commonly used labels and has bright colors,meaning much fewer antibodies are needed in the probe to produce clearly visible results,which greatly saves the applied amount of antibody.In addition,the extremely small quantity of antibody can trigger fierce competition between the free CL and the immobilized antigen to improve the detection sensitivity of the strip test.A series of experimental parameters were optimized.Under the optimal conditions,the visual detection limit of the PBNP-based LFA strip for the CL standard solution was 1.0ng mL^-1,5-fold improvement in sensitivity compared with that of the traditional gold nanoparticles based strip.The immunochromatographic assay had no cross-reaction with salbutamol and ractopamine.The assay method was successfully applied to the residual detection of CL in pork,pig kidney,bacon and milk samples with the VDL of 3 ng g^-1,5 ng g^-1,5 ng g^-1,and 5 ng mL^-1,respectively.