Development Of ELISA For Screening Clenbuterol And Study On Panning The Mimotope Of Clenbuterol By Phage Display

Clbuterol (CLB) is one of theβ—Adrenergic agonist. It has beenused as bronchiodilators in human medicine for over 30 years.Unfortunately, it has been illegally used toenhance the growth, ratesof livestock and to promote the repartition from fat into muscle. Itmay lead to toxic effects after consumption of meat products containingCLB. A lot of countries in the world have banned the use of CLB so arapid and reliable method to detect CLB is needed. ELISA, which is arapid and sensitive detection method compared to other ones and can beused to screen CLB. Phage display is a new technology and has a rapiddevelopment. Itcan be applied in panning the Mimotopes of CLB as thesurrogate of CLB. This use of peptide libraries as sources of reagentsin immunoassays could be applicable in ELISA tests for other low-weighttoxic chemicals.The main results are as follows:1 Development of the indirect competitive ELISA of CLB.2 Panning mimotopes of CLB by phage display peptide library.Part one includes conjugating of CLB—BSA, detection of monoclonalantibody titers and purity, selection of ELISA factors, and evaluationof ELISA, etc. The method of ELISA was established by optimizing.parameters. The experiment results showed that the optimumconcentrations of BSA-CLB, monoclonal antibody against CLB andanti-mouse IgG were 0.5ng/mL, 1:250000 and 1:5000 respectively. Theindirect competitive ELISA curves were established, linear range of theinhibition curves was between 0.38ng/mL and 50ng/mL, IC₅₀ was 6.25 ng/mL,and the detectioh limit was 0.19ng/mL. The variation coefficients ofintra-assay and inter-assay were 8.3ï¼…and 9,97ï¼…respectively. Averagerecovery of CLB samples was 93.14ï¼….In part two, the anti-CLB monoclonal antibody was used as ligand to pan the binding peptide from the Ph. D.-7^TM phage display peptide library.After four rounds of panning, 18 Clones were randomly picked from thefourth round. Those phage plaques were amplified and purified. Six of.the eighteen clones were identified positive by indirect ELISA. Thesequences of all. clones were His—His—Thr—Phe—Phe—Lys—His; Theindirect competitive ELISA curves were established for clone phage PS.Finally, ELISA was eastablished byclone phage which substitutes forCLB-BSA. Recovery of CLB samples was tested.