| In this paper,the direct competitive enzyme-linked immunosorbent assay(cdELISA) method for the determination of 17β-19-Nortestosterone(17β-NT) in functional foods had been developed.This method is generally applicable to the synthesis of 17β-NT haptens.We synthesized a hapten of 17β-NT and investigated the method of coupling the hapten to carrier albumens.The anti-17β-NT antibody with high affinity and specificity were obaitned.The cdELISA established in this study exhibited high sensitivity.The IC50 and IC15 were 0.08μg/kg and 0.0055μg/kg respectively.The IC50 value for the five compounds were all above 3.2 ng/mL,and the variations of inter-assay and intra-assay reproducibilities were all lower than 20.The effect of different matrices on the ELISA methods were investigated detailedly,the matrix removal methods for different samples and the simple extraction procedures were optimized.C18 cartridges were investigated for removing the matrix effects in foods and the sample purification protocol was optimized.Under the developed SPE-ELISA method, good recoveries of functional samples were obtained in the range of 71.0%-91.5%. Furthermore,the ELISA methods developed above were validated by HPLC method respectively,and the results of these methods were correlated well(R2＞0.9) with HPLC. The developed immunoassays are suitable for the rapid and reliable screening of sulfonamide residues in animal products. |
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