| With the growth of cyanobacteria (blue-green algae), the bloom often occur. MCs are produced by Microcystis aerugnosa, Anabaena and Oscillatoria. There are more than 70 congeners of MCs. MC-LR is one of the most toxins which can accumulate in fish, bivalves and other water living animals. The toxicity of MCs can not be subducted by present water-treatment.In the past 30 years, more than 10,000 people were poisoned by ingestion of toxin food and water. In some region, where the source of the drinking water comes from river, lake and reservoir, the poisoning often occurs because of the limited detection technology for MCs. In China, the limited concentration of MCs is 1.0μg/L in drinking water.Some detection methods of MC-LR have been developed, such as mouse bioassay, chemical instrumental method and immunological detection. HPLC is a standard method for detection of MCs. However, it is unsuitable for rapidly screening great number of samples, since the sample preparation is time-consuming and expensive standard substance of toxin are needed. The immunological detection based on a monoclonal antibody is thought to be a sensive and simple method for the detection of the toxins.MC-LR were coupled with human IgG and BSA by using Gultaralaehyde mehtod to be immunogen and detecting antigen. The conjuates were analyzed by Agarose Gel Electrophoresis and Polyacrylamide Gel Electrophoresis and the molecule coupling ratio of MC-LR with human IgG and BSA were 17:1 and 9:1. The electrophoretic result showed that small molecular has been coupled to carrier protein. The female BALB/c mice aged 8 weeks were immunized by MC-LR-IgG using foot pad injection protocol.The mice whose serum titer was 104 or higher were used for node cell donor. Spleen cell from mice immunized with MC-LR-IgG were fused with myeloma cells SP2/0. Two hybridomas cell lines named 2G3 and 2B11, which secret McAb against MC-LR were selected. The secialties of McAb secreted by 2B11 whose titer was higher were studied, the titer of ascites was1:2.5×106, the subclass and the affinity were IgG1 and 9.65×109 M-1.The MC-LR-McAb secreted by 2B11 was used to develop the ic-ELISA. An ic-ELISA was developed for the quantitative detection of MC-LR and the optimized condition as follow: The MC-LR-BSA was added to microtiter plates at a concentration of 2.5μg/mL and incubated overnight at 4℃.The plates were wash three times with PBST for 90 seconds, and 100μL 1% BSA was added to each well to eliminate nonspecific binding by blocking the plastic surface where protein was not bound. After 1h of incubation at 37℃, the plates was washed three times with PBST. The MC-LR-McAb (1:3.2×105) and varying concentrations of standard MC-LR (50μL/well) were added toghter to anther blocked plates and incubation at 37℃for 30min. The commixture of MC-LR and McAb were added to the blocked plates which coating antigen MC-LR-BSA incubated at 37℃for 30min. Then the plates were washed three times with PBST, and 100μL/well goat anti-mouse IgG-HRP(1:4000) was added and incubated for 1h at 37℃. The plates were washed four times, and 100μL/well OPD substrate solution was added, followed by the addition of stopping solution (2M H2SO4) after 15 minutes of incubation in the dark at 37℃. Absorbance values at 490 nm were determined by an enzyme immunoassay reader. The inhibiting rate (A/A0×100%) was calculated from the absorbance value obtained in the presence (A) and absence (A0) of MC-LR. A liner dose-response standard curve was prepared by plotting log[MC-LR]versus inhibiting rate. The regression equation of the standard curve was y = -29.929x + 51.381. The correlation coefficient, the lower limit detection and a linear range were R2=0.9748, 0.1μg/L and 0.1-10μg/L respectively.Water samples which collected from our laboratory and the organ samples which extracted form shellfish and johnny carp (the innards and muscle ) were detected by icELISA. The average recovery rate of standard MC-LR were 96.3% (from water), 89.4% (from innards), 80.25%( from muscles) and 85.05% (from shellfish) respectively. The heavy and light chain variable genes of monoclonal antibody against MC-LR were amplified by RT-PCR from the total RNA of 2B11 hybridona cell strain. Single chain variable fragment gene (VH-Linker-VL)was assembled by SOE-PCR,and then was cloned with pMD18-T vector. The sequence analysis showed that MC-LR-ScFv was 741 bp and encoded a deduced amino acid sequence of 256 residues. The flexible linker connection between heavy and light chain were 15 amino acids. Gene sequence of antibody heavy and light chain was analyzed by IgBLAST, which was accord with the variable region gene characteristics of the mouse antibody. Amino Acids homology had the typical domains (IGV) of immunoglobulin and had high homology with single chain antibody registered, which was the mouse antibody variable gene sequence of the functional rearrangement.On the whole, a monoclonal antibody against MC-LR was produced and an indirect competitive inhibition ELISA was developed. The ScFv gene (VH-linker-VL) based on monoclonal antibody against MC-LR was assembled. This experiment provided a foundation for future study of the bivalent and the fusion ScFv.
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