Preparation Of Anti-Zinc Monoclonal Antibodies

Heavy metal residues on farm and stock products have become a big threat to human health. It is necessary to develop some fast and efficient detection methods. Heavy metal immunoassays are new methods for detection of heavy metal ions. Comparing to the traditional chemical methods, immunoassays are not only fast, cheap, simple, but also reasonably portable, highly sensitive and selective. It can be used as preliminary screening for rapid determination of heavy metal ions. There are two prototype assays based on polyclonal antibody immunoassay and monoclonal antibody immunoassay. The detection methods include fluorescence polarization immunoassay, indirectly competitive enzyme linked immunosorbent assay (ELISA), one-step competitive immunoassay and KinExA immunoassay.The purpose of the paper was to synthesize and identify the zinc-chelated antigen and prepare the specific anti-zinc monoclonal antibody (McAb) in order to establish the rapid detection method.In this paper, zinc ion was conjugated to keyhole limpet hemocyanin (KLH) via bifuctional chelate reagent diethylene triamine penlaacetic acid (DTPA). After purification, it was used as immunogen to immunize six-week-old to eight-week-old BALB/c mice. The hybridoma celllines that can secrete anti-zinc McAb were established by hybridoma technique. In vivo, ascites fluid was produced in BALB/c mice, and in vitro, hybridoma celllines secreted monoclonal antibodies were main- tained in a membrane-based disposable cell cultivation system. Purified McAbs were prepared by salt precipitation and protein chromatography.The following five primary results were included in this research:1 Protein concentration in isolated and purified Zn-DTPA-OVA, Zn-DTPA-KLH, DTPA-OVA and DTPA-KLH was determined by BCA method, 25.155±0.038, 4.195±0.014, 9.242±0.015 and 6.609±0.025mg/mL sequencely. The optimum reaction condition was set when the ratio of the amount of Zn2+ in solution A and DTPA in solution B was 5:1, while the ratio of the amount of substance of chelating agent and lysine in protein was 2:1. 2 The contents of total Zn in Zn-DTPA-OVA, Zn-DTPA-KLH, DTPA-OVA, DTPA-KLH and HEPES were determined to be 295.0±0.3, 49.5±0.5, 0, 0 and 0μg/mL sequencely by graphite-furnace atomic absorption spectrometry (AAS).3 Three hybridoma celllines, designated C1-1-1, 1A1 and 1A5 can secrete anti-zinc monoclonal antibodies were established. The titer of hybridoma supernatants are 1:160, 1:640 and 1:640 sequencely. The types of the three monoclonal antibodies were all IgG3,κ.4 The synthesis of antigen was successful. Three hybridoma celllines that can screte anti-zinc McAb were established and this made it possible to establish ELISA for zinc.