Developments Of Rapid Immunoassay For Illicit Growth-promoting Drugs In Animal Derived Food

With the rising of food safety issues, more demands are required on detection technology.Fast, accurate, highly sensitive and high-throughput become the trends. Immunoassays, whichhas been widely used in food safety, must meet the demands of country. Ractopamine,salbutamol, testosterone, methyltestosterone and19-nortestosterone, the drug of abuse, waschosen for this research, with the aim of development of rapid immunological detectiontechniques by design and synthesis of antigens to prepare sensitivity and high affinitymonoclonal antibodies.This research was started from design and synthesis of a new type of ractopamine haptenOAA. High purity product hapten was obtained, and immunogen OAA-KLH and coatingantigen OAA-OVA was successful synthesized by UV-Vis and electrophoresis identification.The OAA-KLH showed good immune effect. By using pig urine samples in the cell line screenstage, ractopamine monoclonal antibody3F11and9C2with some anti-matrix effect wasobtained. The affinity constant of monoclonal antibody9C2is9.69u109L/mol, and theantibody subtype is IgG2b. Meanwhile, the screening method is applied to screening ofmacromolecules monoclonal antibody for lactoferrin, and high specificity antibody ofactoferrin was obatined, proving the feasibility of this screening methods.An indirect competition ELISA(ic-ELISA) was established for ractopamine. The optimumconditions of the ic-ELISA method are as follows: coating concentration of antigen is0.25Pg/mL; monoclonal antibody concentration was0.025Pg/mL; coating buffer is0.05M pH9.6CB, standard dilute solution is0.01M pH7.4PBS with1.6%NaCl and10%methanol. And theIC50is0.229ng/mL, the detection limit (IC10) is0.012ng/mL, the linear quantitative detectionrange (IC20~IC80) is0.037ng/mL~1.412ng/mL. The average recoveries at different spikelevels are between60%-100%, and the coefficient of variation is less than10%.Rapid colloidal gold test strip for ractopamine was developed. The optimum conditions fortest strip are as follows:2PL0.1mol/L K2CO3+1mL colloidal gold solution;2.4Pg/mL antibody+1mL colloidal gold solution×The coating antigen concentration on NC membrane is0.8mg/mL. The detection limits for ractopamine in swine urine is2ng/mL by color comparisonmethod,5ng/mL by T line disappearance. And a semi-quantitative detection limit0.1ng/mLwas obtained by acquiring the T line gray values.Two salbutamol haptens with different epitopes was designed and synthesized. A sensitivitybroad-specific (1E1) and a sensitivity high specific (3G6) salbutamol monoclonal antibodywere obtained respectively. And an indirect competition ELISA(ic-ELISA) was established for1E1and3G6monoclonal antibody.The optimum conditions of the ic-ELISA method for1E1are as follows: coatingconcentration of antigen is0.25Pg/mL; monoclonal antibody concentration was0.125Pg/mL;coating buffer is0.05M pH9.6CB, standard dilute solution is0.01M pH7.4PBS with1.6%NaCl and10%methanol. And the IC50is0.2ng/mL, the detection limit (IC10) is0.02ng/mL, thelinear quantitative detection range (IC20~IC80) is0.047ng/mL~0.856ng/mL. The averagerecoveries at different spike levels are between80%-130%, and the coefficient of variation is less than10%.The optimum conditions of the ic-ELISA method for3G6are as follows: coatingconcentration of antigen is0.25Pg/mL; monoclonal antibody concentration was0.0625Pg/mL;coating buffer is0.05M pH9.6CB, standard dilute solution is0.01M pH7.4PBS with1.6%NaCl and10%methanol. And the IC50is0.185ng/mL, the detection limit (IC10) is0.018ng/mL,the linear quantitative detection range (IC20~IC80) is0.0367ng/mL~0.935ng/mL. And therecovery in spiked swine urine is between90%-115%.A testosterone hapten with the five-membered ring as epitope was designed andsynthesized. And the high specific monoclonal antibody (3G7) for testosterone was obtained.The IC50of icELISA after optimization is0.11ng/mL èthe detection limit (IC10) is0.016ng/mL,the linear quantitative detection range is0.034ng/mL~0.394ng/mL. The cross-reactivity of thisantibody is less than0.7%with other hormone analogs. And the recovery in spiked tissuesample is between85%~110%.A methyltestosterone hapten was designed and synthesized. And the high specificmonoclonal antibody (1E12) for methyltestosterone was obtained. The IC50of icELISA afteroptimization is0.22ng/mL èthe detection limit (IC10) is0.037ng/mL, the linear quantitativedetection range is0.067ng/mL~0.738ng/mL. And the recovery in spiked aquatic sample isbetween90%~110%.A broad-sepcific monoclonal antibody (8F5) was obtained using19-nortestosterone hapten.The monoclonal antibody (8F5) can recognize testosterone, estradiol, and methyltestosteroneand ethinyl estradiol. The IC50of icELISA after optimization is0.12ng/mL for19-nortestosterone èthe detection limit (IC10) is0.014ng/mL, the linear quantitative detection rangeis0.03ng/mL~0.448ng/mL. And the recovery in spiked swine urine sample is greater than75%which is acceptable in field detection.