Studies On The Preparation For Anti-Pb～(2+) Monoclonal Antibody (McAb) And The Method Of DTC Chelating ELISA Detection For Hg～(2+)

The methods of tranditionally instrument-intensive detection for heavy metals have many inconveniences in practice, and can't satisfy the needs of the rapid detection, so the development of rapid, high-performance and low-cost heavy metals detection teniques is a necessary supplement for the tranditional detection.According to the status of heavy metal pollution in our country, the present study chose largely residual heavy metals Pb²⁺ and Hg²⁺ as research objects, and tried to develop the rapid detection techniques for the two metals.This paper consists of two parts as follows:Part One: The preparation of anti-Pb²⁺ monoclonal antibody (McAb)By selecting bifuntional chelator, the complete antigen (Pb-CHX-A"-DTPA-KLH) could be prepared by the chelator chelating Pb²⁺ and covalently linking itself to KLH. The concentration of Pb²⁺ in the complete antigen was 38.02mg/L; After 5 times' mouse immunizations with the helps of freund's complete and incomplete adjuvants, the antiserum of mouse was assessed by indirect ELISA, and the results showed the titer of antiserum was above 1 : 163840 and the OD values of Pb-loaded antigen(Pb-CHX-A"-DTPA-BSA) were much larger than those of metal-free antigen (CHX-A"-DTPA-BSA); then synthesizing and secreting anti-Pb²⁺ McAb cell line 1D4 was obtained after a series of steps of cell fusion, hybridoma screening and positive hybridoma subcloning, etc; cell fusion rates of two 96-holes-culture plates were 51.04 % and 63.54 %, respetively; the positive rate of hybridoma cells was 2.04 %; At last, ascites fluids were produced in freund's incomplete adjuvant-primed BALB/c mice by intraperitoneal injection of 1-2×10⁶ hybridoma cells, and its titer was above 1:204800 assessed by indirect ELISA, and the concentration of protein in ascites fluid purified by saturated ammonium sulfate was 19mg/ml. Part two: the method of DTC chelating ELISA detection for Hg²⁺The rapid analyse for Hg²⁺ was devised depending on the principles of dithiocarbamate's higher avidity for Hg²⁺ and the signal amplification of ELISA. On one aspect, the detection antigen coated on the ELISA plate was constructed by dithiocarbamate bound to carrier protein (BSA); On the other aspect, the competitive antigen contained Hg²⁺ and reporter enzyme (HRP) was synthesized, which is competitive with the Hg²⁺ in the environment samples for the dithiocarbamate coated on the ELISA plate; the concentrations of Hg²⁺ in samples could be obtained by...