Preparation Of Polyclonal Antibody Against Okadaic Acid And Eestablishment Of Competitive Indirect ELISA For Detecting Shellfish

Diarrhetic Shellfish Poisoning (DSP) is a serious and globally widespread toxic syndrome caused by consumption of shellfish contaminated with algal toxins produced by marine dinoflagellates. DSP toxins are a group of thermostable, polyether and lipophilic compounds causing of diarrhoea and vomiting with no known medical treatments. Okadaic acid (OA) and its derivatives named dynophysisitoxins (DTXs) are main pathogenic factor of DSP. DSP has become key scientific research project in global marine progarammes for its worldwide distribution, high-frequency outbreak and potential harmfulness. Every country work hard to establish and improve analytical methods, is based on the principle of high sensitivity, high accuracy, and simple operability, but the research and technology of DSP in China is relatively backward. Therefore, establishment rapid assays of DSP are necessary for sustainable development of aquaculture and personal safety of consumers. In our study, OA is choosen to be the research object to develop competitive indirect enzyme-linked immunosorbent assay (ciELISA). The study can provide theoretical foundation for developing rapid and economical detections.Preparation of immunity and detection antigenThe artificial antigen was made by coupling hapten of OA with keyhole limpet hemocyanin (KLH) and Ovalbumin (OVA) using activation ester method, the result was tested by SDS-polyacrylamide gel electrophoresis and IR spectrum. The resulte showed that OA was coupled with carrier proteins and the artificial antigens were prepared successfully.Antibody productionNew Zealand rabbits were immunized by using artificial antigen (OA-KLH) and increase the immune dosage in every booster immunization. The titer and sensitivity of antiserum were tested by ciELISA. Whole blood was collected from cardiac vein after six booster immunization. The antiserum was purified by Chromatography on ProteinA Sepharose CL-4B gel column, and the purification efficiency was verified by ultraviolet scanning and ciELISA. The resulte showed that the antiserum had high titer (12800) and sensitivity (the value of IC15 was 3.41 ng/mL). After purified, lots of interfering substances were removed and the sensitivity was improved.Establishment of analytical conditions by ciELISADifferent analytical conditions of ciELISA were optimized by carefully screening the performance of each step, and the optimal protocol was obtained by appropriate maximum absorbance (OD_max) and IC₅₀.The ciELISA was established and optimized as follows: the concentration of coating antigen was 1 mg/L, coating buffer solution was 0.05 mol/L CBS and the plates were incubated at 4°C over night, purified antiserum was diluted 300-fold in PBS, competitive time was 1h and secondary antibody was diluted 5000-fold in PBS. A standard curve ranging from 0.977 to 250 ng/mL was obtained with the optimized analytical conditions, y=13.886Ln(x)+11.744,R²=0.9871, IC₁₅=(1.26±0.32) ng/mL. The cross-reactivity of anti-OA polyclonal antibody with DTX-1 was 46.94 %.Removal of matrix interferenceTwo extraction methods were employed to remove shellfish matrix interference. In dilution protocol, shellfish mussel was extracted by methanol/H₂O (80/20, v/v) at the ratio of 1:1 (w/v), the methanol extraction could be analyzed after 40-flod dilution in PBS with recovery of 72.67%¹18.23%. The correlation of linear regression equation with HPLC-FLD was y=1.0064x-10.234, R²= 0.9347. In dryness protocol, blank sample was extracted by MeOH: water (80:20) at the ratio of 1:10 (w/v). After dryness under nitrogen, the residue was reconstituted in PBS with recovery of 77.78%⁸6.04%. The results of HPLC-FLD and ciELISA with dryness protocol were similar by detecting spiked and six natural simples.