| Snowdrop Lectin (Galanthus nivalis agglutinim, GNA) is active against chewing pests and sap-sucking insects, while safe to higher animals. And it is a suitable component to complement and strengthen the insecticidal properties of Bt-toxins and trypsin inhibitors and so on. Therefore GNA becomes the most widely used plant lectin in genetically modified plants. At present, the detection of GNA protein mainly is based on genetic testing. Enzyme-linked immunosorbent assay (ELISA) can detect proteins which are directly related to the safety of genetically modified food (GMF). It is not only fast and sensitive for detection, but also suitable for field monitoring and a large number of samples’screening. The development technology of rabbit monoclonal antibody (McAb) is one of the relatively advanced biological technologies. There is no report involved the application of rabbit McAb in detection of GMF proteins at home and abroad. Compared with mouse McAb, rabbit McAb can identify more epitopes, owns higher affinity and specificity and so on. Above all, the rabbit McAb possesses stronger immune and captured ability in the research employing the recombinant antibody technology.As a result, this purpose of the research is to establish a sandwich ELISA detection system detecting GNA protein based on rabbit McAb. The main contents and results are as follows:(1) The preparation and identification of anti-GNA rabbit polyclonal antibodyThe molecular weight and the purity of GNA protein were studied by SDS-PAGE. The results showed its molecular weight was14.3kDa and its purity surpassed90%. Therefore, GNA protein can be the immunogen to bring the immune reaction. The concentration of GNA was1.64mg/mL using BCA kit. Then applying immunological technique, two rabbits (NO.3365, NO.3366) were immunized by GNA. After the fifth immunization, the rabbit (NO.3366) which had higher serum titer was got the whole blood. The serum containing anti-GNA PcAb was purified by Sepharose CL-4B Protein A. The purity and titer of the rabbit PcAb were tested respectively by SDS-PAGE and indirect ELISA. It is said that there were not any other proteins except the target antibodies and its titer was8000-32000.(2) The preparation and identification of anti-GNA rabbit monoclonal antibodyTwo rabbits (NO.3365, NO.3366) were immunized, and the spleen of the rabbit (NO.3366) owned higher serum titer was got into the cell fusion stage. The spleen cells of the rabbit were fused with the240E myeloma cells. After primary-screening and primary-confirm screening through indirect ELISA,6double positive clones (zju-17-2、zju-17-3、zju-17-5、zju-17-6、zju-17-1、zju-17-17) were screened. Three clones (zju-17-3、zju-17-5、zju-17-6) were chosen for sub-clone process. The best match (zju-17-5(2L/2H)) which secreted more McAb and has the highest affinity was got to produce McAb. At last, the McAb were purified by Sepharose CL-4B Protein A.The result of SDS-PAGE showed the purity of the rabbit McAb was high. And the concentration of rabbit McAb was2.28mg/mL by IgG detection. The results of indirect ELISA showed that the titer of rabbit McAb was between32000and128000and the affinity constant was0.387×109L/mol.(3) The establishment and application of an sandwich ELISA system detecting GNABiotin was conjugated to rabbit PcAb. Some indexes of the rabbit PcAb-Biotin were that the mark rate was2.40, the concentration of the PcAb was7.67×10"6mol/L and the concentration of the biotin was1.84×10-5mol/L.Tried for several times, the sandwich ELISA system detecting GNA was established.4μg/mL anti-GNA rabbit McAb was as the first antibody to coat the enzyme label plate, the rabbit PcAb-Biotin was twenty times dilution to do as the second antibody. Anti-biotin conjugated with enzyme diluted400times to join into the plate. Then the color reaction lasted for9min and then was terminated to get the OD450. Otherwise, GNA protein, anti-GNA rabbit McAb and Anti-biotin conjugated with enzyme were all used3%skim milk to dilute. As a result, the limit of detection (LOD) of sandwich ELISA was3.9-7.8ng/mL, and the linear detection range was0.975-62.5ng/mL.In order to validate the practical applicability of the sandwich ELISA detection system, GNA genetically modified rice was applied to detect. After the pre-treatment of rice, the sandwich ELISA was used to detect, at the same time the negative control and blank control were set, so was the standard curve. The results showed that OD450(0.41) of the sample was obviously higher than the negative control (0.17) and blank control (0.14). If don’t consider the loss of the target protein in extraction process, the content of GNA protein in the sample was around0.08μg/g (content of GNA protein/weight of rice). |
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