Development Of Direct Competitive Enzyme-Linked Immunosorbent Assay And The Kit For The Determination Of ASP

Amnesic Shellfish Poisoning (ASP) is produced by certain diato, the main constituents of ASP is domoic acid (DA) which one is a kind of Biotoxins. Its typical victims' symptoms include nausea, vomiting, dizziness, bad stomachache, diarrhea and amnesia. The worst pollution area of the ASP is in the Europe and America. The polluted situation in china is slight and poisoning incidents have not been found until now, but precaution and the monitoring work can not be slothful.The researchers have already established many methods to detect the concentration of DA in samples, for instance, mouse bioassay, high performance liquid chromatography (HPLC), immunological methods. The test period of mouse bioassay is long and the detection limit is high, so it is already eliminated by most of countries; the HPLC method is sensitive enough, high accuracy, but the equipment of this method is expensive and hard to move, the pretreatment of this method is cockamamie too, so it is not suitable for the rapid analysis in dealing with poisoning incidents; the immunological methods is easy to carry and operate, with low detection limit. Batch of data can be got in one detect period. So the immunological method is the right way to develop the rapid site monitoring. Development of the immunological methods in China lag behind the western wrold, and did not have the mature rapid ELISA kit. So the development of immunological methods are important and has a wide business prospects.In this study, develop the direct competitive enzyme linked immunosorbent assay (ELISA) kit by taking advantage of the specific immune responses. Most of the kits in the market is produced according to the indirect competitive enzyme linked immunosorbent assay (icELISA) method. The method needs the step that linking the bovine serum albumin (BSA) to DA, adding the secondary antibody marked by enzyme. The test period will insist 2-3h. The method of direct competitive enzyme linked immunosorbent assay (cdELISA) method can shorten the test period to 1.5h because the step of adding the secondary antibody marked by enzyme can be saved. Link the enzyme to the DA directly can make the circle shorter. And then the sensitive of cdELISA is as low as the icELISA. The main processes of the research in below:1. Link the BSA and the horseradish peroxidase ( HRP ) to DA successfully. Analyze the conjugate concentration by the method of coomassie brilliant blue colorimetric. The concentration of DA-BSA is 6.32 mg·mL^-1 and DA-HRP is 8.36 mg·mL^-1; analyze the titer of monoclonal antibody by icELISA. The data of P/N is 5.66, when the titer reaches 1:64000. Determine the best reaction concentration of antibody and the DA-HRP of cdELISA method though the chessboard method. The best degree of dilution of antibody is 1:200, and DA-HRP is 1:400 on the basis of method sensitivity and saving antibody; choose 1% gelatin as the best blocking solution, blocking at 37℃for 1h, incubate 1h at 37℃; on the situation above, set up the standard curve y=-0.15608x+0.9866,R2=0.98297,IC50 is 1.32μg·mL^-1, linear region is 5¹000 ng·mL^-1, the lowest detect limit is 3.58ng·mL^-1; the recovery of this method can reach 88.7%¹36.15%, and the cross reactivity between glutamate and aspartate is low. The result proves that the specificity of antibody and the cdELISA is high.2. Assemble the cdELISA kit according to the former study results, the kit include DA-HRP solution, ELISA plate blocked, detergent, chromogenic agent and end stopper. Analyze the durability time of the kit, and 6 months and -20℃is the durability time.3. Detect the DA concentration of shellfish which is bought in Tongchuanlu aquatic market in Shanghai. The result shows that the situation of DA pollution is slight, none of the sample was detected polluted by DA. In order to ensure the result, use the HPLC to test the samples. Analyze the data of cdELISA and HPLC by using SPSS 13.0. The Pearson correlation coefficient is 0.92, and T-test correlation coefficient is 0.892, p=0.108>0.08. So Based on the data above, the cdELISA method and HPLC have good correlation coefficient. At last comparing the cdELISA method to the icELISA method and colloidal gold immunochromatographic strip and the result shows that the correlation coefficient is good. Through the comparison, the shortage and advantage of these methods is obvious. All of the three methods are developed by the research group.The method of cdELISA reduces the test period further with stronger sensibility and lower detect limit by using high quality antibodies. The cdELISA kit is easy to operate and carry which is suitable for rapid monitoring in poisoning incidents. So the business prospect of the research is wide.