Preparation Of Monoclonal Antibodies Against Heavy Meltal Chromium Ion And Establishment Of Blocking ELISA Method

The heavy metal chromium(Cr) ion had toxic action such as liver toxicity, renal toxicity,carcinogenesis, reproductive toxicity, teratogenesis and other toxic effects to human. Immunoassays forCr3+ion pollution were new methods in recent years, which were of time-saving, labor-saving, simple toperform, reasonably portable and cost-effective, and gradually replaced traditional detection methodscompared to the traditional physical and chemical methods. This study aimed at blocking ELISA rapiddetermination of Cr3+ion pollution residues in animal food, including the immunogenicity analysis of Cr3+ion, synthesis and identification of artificial antigen, preparation and immunological propertiesidentification of Cr3+ion monoclonal antibody(mAb), establishment and performance measurement ofblocking kits. The main contents and experimental results of this study were as follows:Artificial hapten Cr3+-ITCBE was synthesized by ITCBE method. The artificial antigens weresynthesized through the coupling the hapten with carrier protein BSA and OVA. The protein concentrationof the antigens were assayed with ultraviolet spectrophotometry, antigens were verified with SDS-PAGE,the concentration of Cr3+in antigens were determined with ICP-AES, the immunogenicity were identifiedwith antiserum. The results indicated that artificial antigens were synthesized successfully. The protein andCr3+concentration was5.80mg/mL,140.08μg/mL for Cr3+-ITCBE-BSA, and7.69mg/mL,175.40μg/mLfor Cr3+-ITCBE-OVA. The Cr3+-EDTA pAb had the high titers of1∶5.12×104by indirect ELISA, a goodsensitivity with IC50of28.81μg/L and no cross-reactivity to other heavy metal ions chelating-compoundexcept Mo6+by blocking ELISA.One Balb/C mouse was chosen from five Balb/C mice immunized with Cr3+-ITCBE-BSA for cellfusion by the titer of indirect ELISA and blocking ELISA. Five hybridoma lines of2A3C11,2A3D9,2A3E5,2A3F2and2A11G5that secreted Cr3+mAb were screened by indirect ELISA and blockingELISA. The indirect ELISA titers of them were1∶2.56×103,1∶1.28×103,1∶2.56×103,1∶3.2×102and1∶2.56×103in supernatant,1∶1.28×105,1∶2.56×105,1∶2.56×105,1∶1.28×105and1∶5.12×105inascites respectively, the isotypes of them were IgG1/κ, IgG2a/λ, IgG1/λ, IgG2a/κ and IgG1/κrespectively., The best one of them was2A11G5, and the affinity constant(Ka) of2A11G5was2.69×109(L/mol). Cr3+mAbof2A11G5showed good sensitivity with an IC50of25.12μg/L to Cr3+and22.13%CR to Mo6+and little or no CR to other compounds by blocking ELISA. Cr3+-EDTA pAb and Cr3+-EDTAmAb obtained were both used to establish immunoassay of Cr3+residues in animal food, but Cr3+mAbwas better than Cr3+pAb.The calibration curve of Cr3+Kit with standard Cr3+inhibitor was typical sigmoid curve fitted to thefour parameters logistic equation with the linear determination of5.0to512.0μg/L, the sensitivity of3.23μg/L and the determination limit of5.0μg/L. The recoveries of Cr3+spiked in pig feed were85.97%, in pigurine were84.18%, in pig serum were81.13%, in water were90.21%. The precision and accuracy of theassay as determined by inter-assay and intra-assay coefficient variation were below15%. The Cr3+Kitgenerally had25.43%CR to Mo6+and little or no CR to other compounds. The dilution solution of Cr3+had no effect on results of Cr3+Kit. The validity of Cr3+Kit in4℃was above six months.