Preparation Of Monoclonal Antibodies And Development Of Immunoassay For Copper

Copper is a common heavy metal in our daily life, which exists in the nature environment asmetal irons. Because of it's small-molecule, it has no antigenicity, can not directly stimulate thebody to produce antibodies against copper ions. In order to detect the copper ions in the waterenvironment, the bio-functional chelating agents were used as the bridge to make copper ionscoupled to a carrier protein. The preparation of a complete antigen was used to stimulate thebody's immune system to produce antibodies against metal ions.Bio-functional chelating agent1-(4-Isothiocyanobenzyl) ethylenediamine N,N,N',N'-tetraacetic acid (ITCBE) used in the experiment, is coupled with divalent copper ions and bovineserum albumin (BSA), preparation of complete antigen Cu-ITCBE-BSA, for the establishment ofan indirect ELISA and colloidal gold strip, the carrier protein OVA was chelated withBio-functional chelating agent ITCBE and linked with copper irons to gain the completeddetection antigen Cu-ITCBE-OVA. The identification of the complete antigens was carried out byNondenaturing Polyacrylamide gel electrophoresis method.The8-week-old BALB/C mice were immunized by subcutaneous injection in a hind footpad,After the third immunization, a indirect competitive ELISA was carried out to detect the serumtiter of the mice. Traditional approaches were used to make cell fuse, the positive hybridoma celllines were screened by the indirect competitive ELISA, A hybridoma cell line designated4A6steadily secreting monoclonal antibody(MAb) against copper ion was obtained based on "limitingdilution". After subclass identification, the subclass of the antibody was IgG1, and the affinityconstants of the MAb was2.98×10~8L/mol. Hybridoma cells were injected into abdomens of theBALB/C mice to obtain the ascites,which were purified by CA-AS and affinity chromatographymethods. The indirect competitive ELISA method had been established and developed usingascites. Throught the standard sample of copper ion was detected by the indirect competitiveELISA, we got the regression equation and correlation coefficients of this method: y=-51.9x+153.02, R~2=0.9870. The detection range was from21ng/mL to488ng/mL, and the minimum detection limit was found to be21ng/mL.Nanometer colloidal gold was prepared by controlled reduction of gold chloride withtrisodium citrate. The average diameter of obtained gold colloidal particles was20nanometers.Identified by transmission electron microscopy, the uniform particles size of colloidal goldsolution can be used for the preparation of colloidal gold probes. Under optimal conditions, thismethod shows high detecting sensitivity with a LOD (limit of detection) of75ng/ml. Stability testindicates that the immunochromatographic strips are stable for60days at4℃.