Establishment Of ELISA For Clenbuterol And Screening Of Nanoantibody

With the continuous improvement of people’s living standards, People started topay attention to food safety problems. After feeding the animals a certain dose ofclenbuterol, the percentage of animal lean will be significantly increased. Therefore, alot of breeders add CL to the animal feed. In the consumption of meat and meat prod-ucts containing clenbuterol, people will be poisoned by CL, it is a serious conditionthat can result in death. In the detection of clenbuterol residues, the enzyme-linkedimmunosorbent Assay (ELISA) has many advantages. It is a convenient, fast andquantitative method. It is the key to preparation of antibodies for ELISA. Polyclonalantibody preparation process is simple, but the antibody purity is not high enough, thespecificity is poor, and the incidence of cross-reaction is high. The specific of monoc-lonal is high, but the preparation process is too cumbersome, difficult, long operationcycle, expensive and it requires eukaryotic cells for expression. Nanobody is the anti-gen-binding site of the heavy chain antibodies produced by llamas, and its molecularweight is only15KDa. Nanobodies contain many unique structural and functionalproperties compared to other types of antibody, including high stability, high antigenspecificity and affinity, low immunogenicity, high soluble expression in prokaryotes.In this study firstly we use the diazotization-coupling method to synthesize CLcompletely antigen, and the coupling ratios of OVA and CL is28.7:1. We also usedaffinity chromatography to purificated polyclonal antibody. After purified, the serumtiter is finally up to1:80000, the concentraton is0.667mg/mL.Using the syntheticcomplete antigen and affinity purification of antibody to eatablishment of ELISA forCL, according the optimization of antigen concentration, antibody concentration,blocking concentration and the second antibody concentration, finally, the ELISA me-thod for detecting CLB was established with the sensitivity6.4ng/mL.At last, utilizes phage display technology to pan against the CL completely anti-gen with a naive phage library of nanobodies for three rounds. After the three-roundpanning, three positive CL nanoantibody was obtained. The final goal of this projectis to provide a new kind of small molecular antibody fragment for ELISA to detectCL, which is highly effective, low immunogenic, and affordable.