Synthesis Of Antigens For Heavy Metal Ions, Preparation Of Polyclonal Antibodies And Indirect ELISA Analysis

Immunoassay for metal ions is an interesting and challenging field that combines chemistry, biology, immunology and other disciplines together. This method could provide a sensitive, low-cost, rapid and high throughput analytical method for heavy metal ions in environment and food samples.Study on‘immunoassays for heavy metal ions’including following procedures: designing and synthesizing hapten of metal ions, synthesizing antigen, immunizing animals, evaluating serums by ELISA, optimizing analytical methods. The most important step of the whole study is the hapten designing which determines whether we can achieve ideal results. We innovatively synthesized two hapten for heavy metal ions, which were characterized and applied to further experiments.Mercuric mercaptide of penicillenic acid (MMPA), a stable derivative of penicillin, has been used for a quantitative analysis of penicillin for nearly fifty years (Bundgaard H., et al., J. Pharm. Pharmacol., 1972, 24, 790 and Chinese Pharmacopoeia (Ed. 2), 1995, 334). In this paper, MMPA was successfully used as a novel hapten for mercuric antigen. Accordingly, the hapten of MMPA was synthesized and conjugated with bovine serum albumin (BSA) or ovalbumin egg (OVA) for the preparation of mercuric antigen, the relevant rabbit immunization was performed for production of antibody and the ELISA was used for the analyses of antibody titer in rabbit serum. The experiments manifest the following interesting results. At first, the synthesized antigen was safe for the immunized animals, no obvious toxic response was observed in the animals. Secondly, MMPA could vigorously simulate animal immune system to produce affinity antibody, the antibody titer exceeded 83000 indicating the strong immunogenicity of the hapten used. Thirdly, immunoglobulins (IgGs) have higher reactivity to OVA-GSH-HgCl than OVA-GSH, indicating a specific affinity to the mercuric ion. In addition, it was easily to synthesize the hapten and conjugate it with a carrier protein, such as BSA or OVA. The hapten and its relevant antigen have potential use in immunoassay of mercuric ion in environment and food sample matrixes.Meanwhile, we synthesized a new hapten copper mercaptide of penicillenic acid (CMPA) for copper antigen using the method developed by ourselves. We systematic evaluated the copper antigen according to the procedures similar to that of BSA-MMPA. The results indicated that this new copper antigen adhere to the required criterion for an excellent heavy metal ions antigen.Monoclonal antibodies for mercuric and copper ions are preparing at present.