Preparation Of Polyclonal Antibody Against Domoic Acid And Establishment Of Indirect ELISA For Domoic Acid

Domoic is a kind of marine biotoxin produced by pseudo-nitzschia diatoms, wihch can be accumulated by marine organism (i.e. shellfish and crab).People who take in the contaminated sea foods could suffer from gastrointestinal symptoms such as vomiting and diarrhea, memory loss, even coma and death. Thus it is also called amnesic shellfish poison (ASP). The single outbreak of ASP in Canada caused great illness among people and therefore it was regarded as a serious threat to human health. Although there was no report of intoxication events in China, it should be taken seriously by the government. Regarding the frequent occurrences of red tide and the increase of international sea food transaction, more attention should be paid on the monitoring of ASP in seafood.Domoic acid is usually detected by mouse bioassay, HPLC-UVD and immunoassay etc. Mouse bioassay becomes to fade out gradually for its failure to reach the limit of national standard for domoic acid in sea foods (20μg/g). Meanwhile, it is also unsuitable for primary organization to detect a great number of samples by HPLC-UVD method for high cost. In contrast, immunoassay has low detection limits and is very efficient for analysis of large amount of samples in a short time. However, there is still no available immunoassay method for ASP in our country. It is necessary for us to establish rapid immunoassay.In this study, DA was coupled to keyhole limpet hemocyanin (KLH) and bovine serum albumin (BSA) using activation ester method to prepare complete immunogen and coating antigen. The conjugates were identified by UV absorption. Polyclonal antibodies against domoic acid were generated from rabbits after the animals had been immunized by DA-KLH. The titers of serum were measured by indirect ELISA from the third injection. The blood was got by heart extraction and the antibodies were purified by an octanoic acid-(NH4)2SO4 method. The specificity of antibody was assessed. An indirect competitive enzyme-linked immunosorbent assay (ic-ELISA) was established primarily. The working concentration of artificial antigen, antibody and HRP-IgG were measured. The best blocking solution, blocking time and best incubation condition were decided respectively. Several shellfish were detected by both ic-ELISA and HPLC-UVD. The main achieved results are as follows:1. DA was coupled to keyhole limpet hemocyanin (KLH) and bovine serum albumin (BSA) using activation ester method to prepare complete immunogen and coating antigen. The conjugates were proved that it was successfully conjugated.2. Polyclonal antibodies against domoic acid were generated from rabbits after the animals had been immunized by DA-KLH for 19 weeks. The titer of antiserum was 1:1600, detected by an indirect ELISA. After purified, the titer of antiserum became 1:800. There is no cross reaction with L-Glu and D-Asp.3. An ic-ELISA was established and optimized as follows: The working concentration of artificial antigen, antibody and HRP-IgG were 6400, 800 and 3000 times dilution respectively. The best blocking solution was decided to be 1% PVA, with blocking time for 2 hours. The optimized incubation condition was 37℃, 1 hour with a 4% PEG dilution for both antibody and HRP-IgG incubation.4. A standard curve by ic-ELISA was plot ranging from 10 ng/mL to 10μg/mL. The regression equation of standard curve was y = 109.40 - 15.45x and the correlation coefficient R2 was 0.9682. The detection limit was about 18 ng/mL.5. Three kinds of shellfish were detected by HPLC-UVD and all results showed negative. Adding standard recovery ranged from 68.0% to 84.0% for a low adding after extract, while a lower adding standard recovery was gained by adding standard before extract.6. The samples were also measured by ic-ELISA and it turned out to be the same results. Adding standard recovery ranged from 53.0% to 63.2% and RSD ranged from 8.5% to 11.8% for a low adding after extract,while the results was 47.2% ~ 94.2% and 12.3% ~ 17.0% respectively for adding before extract. It showed a discrepancy among different matrix.7. A method comparison between these two methods was studied. The correlation coefficient was 0.6375. The results which were measured by ic-ELISA were lower than by HPLC-UVD.