Preparation Of Monoclonal Antibody Against Domic Acid And Establishment Of ELISA For Domic Acid

Domoic acid (DA), an important marine ASP toxin , is mainly generated by Pseudonitzschia diatom. Its structure is similar to glutamic and kainic acid. It often contaminates halobios such as sea food of fish and seashell. The Amnesic Shellfish Poisoning is caused in human beings by the consumption of the DA-contaminated food. DA was detected in seafood worldwide. As the frequent occurrence of red tide, more and more studies on DA were carried out.For protection against ASP, it is very important to detect DA in edible shellfish. At present, high performance liquid chromatography (HPLC) with good accuracy and reproducibility is most widely employed for detecting DA.Therefore strict inspection systems for DA have been established in many countries, and the relevant detection methods have also been established, such as mouse bioassay, chemical instrumental method, immunological detection and other specific detection methods according to the specialities of the different toxins. In China, the study on DA, the detection of DA was quite late.At present, HPLC is widely used to detect DA. However, it is unsuitable for rapidly screening large numbers of samples, since the treating with samples prior to analysis is time-consuming.EIA method based on a monoclonal antibody, which can be obtained in unlimited quantities, is thought to be much appropriate in comparison with other methods.DA as a low molecular weight substance, it was respectively coupled with human IgG and BSA as immunity antigen and detection antigen by active ester method. The man-made antigen was successfully identified by the agarose gel electophoresis and denaturalized polyacrylamide gel electrophoresis, and molecule conjugation ratio of DA with human IgG and BSA were 29:1 and 11:1 respectively by the indirect competitive ELISA which was established in this experiment.BALB/C mice were immunized with IgG-DA by different injection routes (foot pad, intraperitioneal, and spleen). The titer and the specificity of the DA McAb against DA were detected by ELISA. The best results is obtained in the group of the foot pad injection route. The antibody titers was 1:6400 14 days after injecting with a immune dose of 60μg antigen.The mouse with higher antibody titer were used for cell fusion. The hybridomas was selected, screened, and cloned for 3 times after cell fusion. Three hybridomas cells were obtained by conventional method. One hybridoma cell line which secreted IgG1 monoclonal antibody against DA was identified and named 9C1.The number of chromosome of the hybridoma cell line was 84 ~90, the affinity constant was 2.83×10~7 M-1, and the titer of the monoclonal antibody was 1: 148,000 . Hybridoma cells were cloned for 2 times with positive percent of 100%. The cell was injected into BALB/C through subcutaneous injections. Hybridoma cell was selected and cultured when Solid tumor grown up after 14 days.The positive percent of hybridoma was 100% by use of micromanipulation after sub-cloning of two times. The absence of some nutriments in the culture medium resulted in much lower proliferation of hybridoma cells than SP2/0 cells. So,each mice was injected with a number of 2×106 live hybridoma cells. After tumors were detected by unaided eye, the mice was killed for sterilely obtaining hybridoma cells which were in good state of growth.The sterilized wax cantaining hybridomas was injected into the mouse abdomens. About 7 days later, ascites with monoclonal antibodies were obtained.Monoclonal antibodies in the ascites were indentified by SDS-PAGE and purified by CA-AS. A standard curve of protein molecular weight markers was determine by use of scanning photography. The albumen of ascites was 5.17mg/mL and the relative molecular weight of McAb was 156,390Da.The coating antigen DA-BSA was diluted to 1μg/mL and incubated overnight at 4℃. Plates were washed three times with PBST for 3 min per times, and 100μL 1% gelatin was added to each well to eliminate nonspecific binding by blocking the plastic surface where protein was not bound. After 1 h of incubation at 37℃, Plates were washed three times with PBST, and 1:8000 McAb and varying concentrations of standard DA (50μL/well) were added. After 1 h of incubation at 37℃, 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℃. Plates were washed four times, and 100μL/well OPD substrata solution was added, followed by the addition of stopping solution(2M H2SO4) after incubation of 20 min in the dark at 37℃. Absorbance at 492nm was determined by an enzyme immunoassay reader. The inhibiting (b/b0×100%) was calculated from the absorbance obtained in the presence (b) and absence (b0) of DA. A linear dose-response standard curve was prepared by plotting log [DA] versus inhibiting. The regression equation of standard curve was y = -13.094x + 106.19,The correlation coefficient R2 = 0.9962,the limit of detection was 2.43 ng/mL and the linear range was 2.43～600 ng/mL. The average recovery rate of standard DA was 98.197% and the coefficients of variation was 6.91%.It was the first report in China that the McAb against DA was applied to monitor the DA residues in the seafood by the ELISA method. The success of this experiment providing a foundation for future study of the test kits for DA and other ocean toxin pollution.