Preparation And Estbilished Primary Immunoassay Of Monoclonal Antibodies Against Ion Lead And Chromium

Objectives:To produce the McAbs against Pb2+and Cr+, and initially est ablish indirect-competitive enzyme-linked immunoassay and colloidal gold probe-based immunochromatographic assay for the detection of the heavy metal, and make a basis for the ultimate establishment of immunological detection metho ds of Pb2+and Cr3+.Methods:Ion lead and chromium were respective chelated with p-SCN-Bn-DTPA and isothiocyanobenzyl-EDTA (iEDTA) and linked with BSA to gain the complete antigen (Pb-DTPA-BSA and Cr-iEDTA-BSA). Samely, the Pb-DT PA-OVA and Cr-iEDTA-OVA were prepared for dectecting antigen. Five BAL B/c mice were immunized with Pb-DTPA-OVA and Cr-iEDTA-OVA conjugates. The McAbs against heavy metal were produced through general hybridoma tec hnology. The titer, subtype and affinity of the antibodies were characterized in the way of indirect enzym-elinked immunosorbent assay, and the specificity w ere identified in the way of indirect competetive enzyme-linked immunosorbent assay. Indirect competitive enzyme-linked immunsorbent assay and colloidal go Id immunochromatographic strip was established primarily.Results:The complete antigen of Pb2+and Cr3+were prepared successfull y which were detected by ICP-AES. After immunization and cell fusion, each of which has three hybridomaes which can stably secrete McAbs against ion le ad or chromium were obtained. The best hybridoma of which can stably secret e McAbs against ion lead is 2A11D11, which secrete antibodies is IgGl, the r elative affinity is 3.34±0.24×109M-1, no cross-reactivity exists between the ant ibodies and the carrier protein OVA. The cross reactivity with other heavy ion s was less than 1%, except the CR with Fe(III) was nearly to 5%. The IC50 of indirect competitive enzyme-linked immunsorbent assay is 5 ng/mL respecti vely, the detectionlimit is 0.1 ng/mL, the LOD of colloidal gold probe-based i mmunochromatographic assay is 50 ng/mL. The best hybridomas of which can stably secrete McAbs against ion chromium is 5G12C5, which secrete ant ibodies is IgG1.Conclusion:The initially establish indirect-competitive enzyme-linked imm unoassay and colloidal gold probe-based immunochromatographic assay for the detection of the ion lead, each of which was meet the national standard of wa ter quality monitoring(GB/T 14848-93). The prepared McAbs against the ion ch romium, which was the basis of establishing related immunoassay.