Preparation Of Monoclonal Antibody Against Okadaic Acid And Development Of Detection Method Based On Monoclonal Antibody

Okadaic acid(OA)has been reported to be one of the major constituent of DSP toxins.It is a kind of lipophylic biotoxin and produced by Dinophysis and Prorocentrum.OA can be transmitted to the humans via food chain.Human consumption of the contaminated shellfish with OA can cause severe disease symptoms such as diarrhea,nausea,and vomiting.Moreover,a number of studies have confirmed that OA can induce cell apoptosis and cancer in human patients.The aime of this study is d to develop a high affinity and specific monoclonal antibody(mcAb)against OA toxin,and to establish ELISA and colloid gold immunochromatographic strip immunoassay methods for rapid detection of OA.OA is a characteristic hapten and cannot induce immunogenic response in animals.Therefore,complete antigens OA-BSA and OA-OVA were successfully prepared by carbodiimide method.OA-BSA was used as the immunogen to administer Balb/c mice,and OA-OVA was used as the detect antigen for the determination of the mice serum titer and the screening of anti-OA antibody.Three cell lines of positive hybridoma were screened out,and named 10E8,10D1 and 8G12 respectively,among which 10E8 showed the highest titer and the subtype was IgG1.The 10E8 cells were cultured in vitro,and ascites were developed by injecting hybridoma cells into the abdomen of Balb/c mice.The ascites fluid harvested from mice was further purified by octanoic acid-ammonium sulfate precipitation method and Protein G purification to obtain high purity IgG antibody.The affinity of the purified mcAb was about 2.66×109 L/mol,and IC50 was 1.225 ng/mL determined by ELISA.The limit of detection(LOD)was 0.244 ng/mL.The colloidal gold immunochromatographic strip was prepared by sodium citrate reduction method.The prepared colloidal gold strip was labeled with the purified anti-OA mcAb.After purification,the colloidal gold was solidified on the release pad.The complete antigen OA-OVA was coated at the T-line of the NC membrane,and the goat anti-mouse IgG antibody was coated on the C-line of the NC membrane to prepare the colloidal gold test strip.At the same time,the condition of the test strip was optimized,and optimized the release pad and NC membrane,GL0194 and Millipore 135S were used respectively.The anti-OA mcAb labeled colloidal gold was diluted at 1:4,and the original concentration was 0.3 mg/mL.Antigen OA-OVA was diluted at 1:6,and 10 mg/mL goat anti-mouse IgG was diluted at 1:120.After the test strips were assembled,different concentrations of standard OA samples were used to determine the detection limit.The lowest detection concentration of OA was 2.5 ng/mL and the strip exhibited high specificity.No significant cross-reactivity to other marine toxins was observed.The mcAb developed in this study was successfully used for the establishment of ELISA and colloidal gold strip detection methods.The immunoassays were significant to achieve the rapid detection of OA in sea food samples.