Preparation Of Monoclonal Antibody Against Brevetoxin And Establishment Of ELISA Detected Method

Brevetoxins(BTX) are lipid-soluble cyclic polyether neurotoxins produced by Gymnodinium breve. They are thermostable and stable at acid pH. Brevetoxins can cause significant mortalities of fish and other aquatic animals, including marine mammals, through direct exposure during harmful algal blooms, or indirectly in the food web. Many filter-feeding molluscan shellfish are known to accumulate brevetoxins with no obvious adverse effects. However, brevetoxins in shellfish do pose a significant human health risk. Consumption of brevetoxin-contaminated molluscan shellfish causes neurotoxic shellfish poisoning(NSP). NSP results primarily from cell depolarizing actions of brevetoxins, through activation of voltage-gated sodium channels. Symptoms and signs of NSP are mainly gastrointestinal (e.g., abdominalpain, nausea, vomiting, diarrhea) and neurological (e.g.,ataxia, myalgia, paresthesia, and reversal of temperature sensation). Symptoms develop within minutes to hour safter consumption of contaminated shellfish, and resolve within a fewdays. The toxic effects of BTX are embryotoxicity, developmental toxicity, immunotoxicity, inherent toxicity and carcinogenesis effect. It is diffficult to remove by means of heating in high tempreture. Thus it is necessary to develop a high performance, sensitive and fast method to determine BTX to satisfy practical requirement.At present, the detection methods of BTX mainly are mouse bioassay, cytotoxicity spectro assay, receptor binding assay, immunoassays and high performance Liquid chromatography(HPLC). Mouse bioassay is less utilized because of its lower specificity and sensitivity. HPLC are commonly used, but it is unsuitable to detect large scale sample, because the sample pre-processing procedure is tedious, the equipment and standard substance are expensive, and operants who can operate expertly are needed. The immunological method which is sensitive, specific and high-performance is widely applied in detecting micromolecule shellfish toxin. In this study, PbTx-2, the most widely distributed in BTX, is the research object. PbTx-2 was reformed by succinic anhydride coupling method, and coupled with BSA and OVA respectively by mixed anhydride coupling method, acquire the immunizing antigen PbTx-2-BSA and the detecting antigen PbTx-2-OVA. The conjuates were analyzed by Agarose Gel Electrophoresis and Polyacrylamide Gel Electrophoresis. The female BALB/c mice were immunized by PbTx-2-BSA, the mice whose serum titer was 6 400 or higher were used for popliteal lymph nodes donors. The popliteal lymph nodes were fused with myeloma cells SP2/0. PbTx-2-OVA was used for screening positive hybridoma cell.Two hybridoma cell lines steadily secreting monoclonal antibodies against PbTx-2, coded 2E1 and 4B5, was obtained by subcloning for 3 cycles. The hybridoma cell line 2E1 was selected to identify the characteristics of McAb. The titer of purified ascites was 1: 64 000, the purity of antibody was 77.46%, the protein level of the ascites was 4.052mg/mL, the antibody subclass was IgM, affinity constant was 1.774×10~7 L/mol。The indirect competitive ELISA was established with McAb 2E1 which against PbTx-2, the optimized condition as follow: The PbTx-2-BSA was added to microtiter plates at a concentration of 1.0μg/mL and incubated overnight at 4℃; blocking condition is 0.1 mol/L NH4Cl incubating in 37℃for 1 h; the precise working concentration of McAb in the competition environment is 1:8 000; the action time of competition, goat anti-mouse IgG-HRP, OPD substrate solution is 30 min, 1 h, 15 min, respectively. A linear dose-response standard curve was prepared by plotting log[PbTx-2] versus inhibiting rate. The regression equation of the standard curve was y=-8.1998x+81.796. The correlation coefficient, the lower limit detection and a linear range were R~2=0.9912, 1 ng/mL and 1～200 ng/mL respectively. The shellfish samples which extracted from Crassotrea ariakensis, Mytilus galloprovincialis Lamarck, Dosinia japonica, Ruditapes philippinarum, Nassarius variciferus and Rapana venosa were detected by icELISA, and the average recovery rate of standard PbTx-2 were 82.91%,80.16%,78.46%,74.11%,80.95% and 79.43%, respectively.