The Preparation Of Specific Monoclonal Antibodies Against Copper Complexes And Construction Of Three Dimensional Model Of Single Chain Antibody

Heavy metals are toxic and persistent environmental contaminants. They are the most insidious pollutants because of their nonbiodegradable nature and ability to persist for long periods. Heavy metals often persist in the environment for long periods of time bound to soils or sediments. Unfortunately, changes in weather, in the pH of the soil or water, or in other combinations of environmental factors can mobilize bound metals and greatly increase their availability and effective toxicity. Therefore, the quantitative analysis of trace heavy metals is extremely important in environmental and agricultural food monitoring.Monoclonal antibodies (MAbs) against the chelated Cu2+ were prepared. Based on the identification of their affinity, specificity and function, clone the variable region genes of the McAb and construct recombinant anti-copper and anti-mercury single Fv (ScFv) gene. According to variable region gene a three-dimensional model of ScFv of anti-copper and anti-mercury single chain antibody was mimiced.The immunoconjugatewas prepared by coupling of Cu2+ to keyhole limpet hemocyanin (KLH) with bifunctional chelators. Stable hybridoma cell lines were produced by fusion of murine splenocytes and SP2/0 myeloma cells. Purified McAbs were prepared. The titer and affinity of McAbs were determined by indirect ELISA, the specificity of McAbs was determined by competitive inhibition ELISA. Under the optimization of the ionic strength and pH, the standard curve was established by indirect competitive ELISA.The total RNA of hybridoma cell line (DF4) was isolated with Trizol reagent, and the variable region genes were amplified with variable region primers by Reverse transcriptase polymerase chain reaction (RT-PCR). The variable region genes of heavy chain and light chain were strung together by SOE-PCR and the ScFv gene was constructed. After cloned into T-Easy vector, the recombinant ScFv gene was identified by endonuclease digestion, PCR and sequencing.On Discovery Studio, with homologous protein-structure-prediction, we constructed and analyzed molecular models of McAb.Stable hybridoma cell lines were produced by fusion of murine splenocytes and SP2/0 myeloma cells. The total RNA of hybridoma cell lines (30) was isolated with Trizol reagent, and the variable region genes were amplified with variable region primers by RT-PCR. The variable region genes of heavy chain and light chain were strung together by SOE-PCR and the ScFv gene was constructed. After cloned into pGEM-T Easy vector, the recombinant ScFv gene was identified by endonuclease digestion, PCR and sequencing.On Discovery Studio, with homologous protein-structure-prediction, we constructed and analyzed molecular models of McAb.Three hybridoma strains that can secrete anti-heavy metal McAbs were established. The anti-heavy metal McAbs with high purity were prepared. The affinity of antibody is 5.69 x 1010L/mol. The cross-reactivities in this ELISA were less than 2% for other metal ions. The standard curves were obtained by the antibodies with higher affinity. The IC50 of DF4 was 0.671μg/ml and the lowest detection limit of 0.014μg/ml. The recoveries from ultrapure water, tap water were in the range of 86.0% to 111.5%. The variable region genes were amplified from hybridoma cell lines by RT-PCR, and recombinant ScFv gene was constructed. The sequencing result shows that the ScFv gene consists of about 750 bp, with more than 300 bp of heavy chain variable region gene and light chain variable region gene, which were linked by a 45 bp peptide. The structure model of antibody is constructed successfully.