| GTX2,3 is a kind of carbamate toxins of paralytic shellfish poison(PSP), which has a wide distribution, strong toxicity, and serious harms to human body. It is one of the major toxic poisons of red tide algae and the infected shellfish of China’s southern coast. It poses a serious threat to food security of Guangdong coastal shellfish. This experiment mainly used glyoxal as a coupling agent to conjugate hapten GTX2, 3 with carrier protein BSA and KLH to make the immunizing and detected antigen of GTX2,3, respectively. Through the identification of complete antigen, animal immunity, cell fusion, hybridoma cell technology and the means of intraperitoneally injecting female BALB/c mice, the GTX2,3 monoclonal antibody ascites were prepared and the reaction conditions of ic-ELISA were optimized to establish the rapid detection of indirect competitive ELISA of GTX2, 3. The experimental results are as following:(1) In this paper, the GTX2,3 was conjugated with BSA and KLH by the aldehyde, which was modified for use as an immunosizing and detected antigen in an indirect ELISA. The complete antigen were confirmed by BCA, UV spectral scanning, sodium dodecylsulfate-polyacrylamide gel electrophoresis(SDS-PAGE) and the production of antiserum with titer of 1:51200 from mice immunized with the immunogen in indirect ELISA. The conjugates had strong immunogenicity. The concentration of GTX2,3 with BSA and KLH was 8.52 mg/m L and5.72 mg/m L, respectively. And the molecule coupling ratio of immunogen was 15:1.(2) The female BALB/c mice aged between 6 to 8 weeks were immunized by GTX2,3-BSA using four different immunizing schemes, such as foot-pad and intraperitoneal injection, subcutaneous immunization, intraperitoneal injection and intramuscular immunization. The serum titer had reached 1:25600, 1:51200, 1:102400 and 1:51200 by the four immunizing protocol, respectively. The spleen cells from the mice with the highest titer in the four immunizing schemes were fused with SP2/0 cells, respectively. Three hybridomas cell lines named 2-2H11, 3-2F2 and 3-3C9, which the subclass of IgG1, with the cell supernatant titer of 1:128, 1:64 and 1:256 of each, were selected. Being Manufactured by intraperitoneally injecting BALB/c mice, purified by CA-AS and Protein A affinity chromatography, and identified by BCA, indirect ELISA and the subtype of McAbs identification, the GTX2,3-McAb secreted by 3-3C9, which the subclass of IgG1, with the titer of 1:102400, the affinity of 1.05×108 L/mol and the great speciality with GTX2,3, were finally used to establish the ic-ELISA of GTX2, 3.(3) The GTX2,3-McAb secreted by 3-3C9 was used to develop an ic-ELISA, which was used for the quantitative detection of GTX2,3 in seashell samples. A liner doseresponse standard curve was prepared by plotting log [GTX2,3] versus inhibiting rates. The regression equation of the standard curve was y =-14.821 x + 98.544,with the correlation coefficient R2 = 0.9942. The sensitivity(IC50), the linear range(IC20-IC80) and the detection limits of GTX2,3-ic-ELISA were 0.277-9.852μg/m L, 1.653μg/m L and 0.136μg/m L, respectively. The recoveries of GTX2,3 in the sellfish samples, including Ostrea rivularis and Paphia undulata were detected in both GTX2,3-ic-ELISA and HPLC. The results of GTX2,3-ic-ELSA showed that recoveries of Crassostrea hongkongensis were 90.05%-113.7% and Paphia undulata were 94.55%-106.0%, while were 97.98%-103.3% and 96.90%-101.9% in HPLC, respectively. The recoveries of GTX2,3-ic-ELISA and HPLC were both in the range of 80%-120%, which showed that in some concentration range, the GTX2,3-ic-ELISA had the same accuracy with HPLC. |
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