膝沟藻毒素GTX2，3单克隆抗体的制备、鉴定及酶免疫检测方法的初步建立

发布时间：2018-01-20 01:47

本文关键词： 膝沟藻毒素单克隆抗体酶免疫检测法　出处：《暨南大学》2006年硕士论文　论文类型：学位论文

【摘要】：目的：制备膝沟藻毒素2，3(GTX2，3)的单克隆抗体(McAbs)，并对其进行鉴定，初步建立起GTX2，3的间接及直接竞争酶免疫学检测方法(ELISA)，为该毒素酶免疫学快速检测试剂盒的研制奠定基础。方法：利用醛化法将GTX2，3与载体蛋白血蓝蛋白(KLH)偶联，制备完全抗原。以GTX2，3-KLH作为免疫原免疫Balb／C小鼠，同时用高碘酸盐氧化法将该毒素与葡萄糖氧化酶(GOX)偶联，得到的GTX2，3-GOX偶联物作为检测抗原。应用杂交瘤技术制备抗GTX2，3的单克隆抗体。常规方法鉴定该单克隆抗体的亚类及亲和力，间接ELISA检测腹水中抗体效价和抗体的特异性，Protein A亲和层析纯化腹水中单克隆抗体。初步建立检测该毒素的间接竞争、直接竞争酶免疫学检测方法，比较两种方法的灵敏度、检测限、工作范围。结果：分别用醛化法和高碘酸盐氧化法成功制备出GTX2，3-KLH和GTX2，3-GOX偶联物；获得一株稳定分泌抗GTX2，3单克隆抗体的杂交瘤细胞株CD1，该细胞株分泌的抗体属于IgG1，腹水中抗体效价为2.5×10~5，亲和力为1.2×10~(-8)mol／1，该抗体与载体蛋白BSA、KLH和GOX均无交叉反应，特异性好。初步建立了检测麻痹性贝类毒素的间接、直接竞争酶免疫学检测方法，两种方法的灵敏度分别为15μg／ml和6μg／ml，检测限分别为6μg／ml和0.6μg／ml，工作范围分别为6-50μg／ml和0.6-50μg／ml。结论：运用醛化法或高碘酸盐氧化法能够将毒素GTX2，，3与载体蛋白偶联。利用小鼠杂交瘤技术获得了一株分泌抗GTX2，3单克隆抗体的杂交瘤细胞株，该单克隆抗体亲和力高，特异性好。利用该单克隆抗体初步建立了GTX2，3的间接和直接竞争酶免疫学检测方法，但两种检测方法的灵敏度尚未达到海产品中毒素残留最低限量(80μg／100g)的要求，还需进一步优化。
[Abstract]:Objective: to prepare and identify the monoclonal antibody McAbsN of geniculatoxin 2, 3, GTX2, and to establish a preliminary GTX2. 3. The indirect and direct competitive enzyme immunological detection method (ELISAA) lays a foundation for the development of the rapid detection kit for the toxin enzyme immunology. Methods: the complete antigen was prepared by coupling GTX2H3 with carrier protein hemocyanin KLH. GTX2O3-KLH was used as immunogen to immunize Balb/C mice. At the same time, the toxin was coupled with glucose oxidase (GOX) by periodate oxidation method, and the GTX2O3-GOX conjugate was used as the detection antigen. The hybridoma technique was used to prepare anti-GTX2O3-GOX. Routine method was used to identify the subclass and affinity of the monoclonal antibody, and indirect ELISA was used to detect the titer and specificity of the antibody in ascites. Protein A affinity chromatography was used to purify monoclonal antibody in ascites. Indirect competition and direct competition enzyme immunoassay were established to detect the toxin. The sensitivity and detection limit of the two methods were compared. Scope of work Results: GTX2O3-KLH and GTX2O3-GOX coupling compounds were successfully prepared by aldehyde method and periodate oxidation method, respectively. A hybridoma cell line CD1 was obtained, which secreted monoclonal antibody against GTX2H3 stably. The antibody secreted by the cell line belonged to IgG1, and the titer of antibody in ascites was 2.5 脳 10 ~ (5). The affinity was 1.2 脳 10 ~ (-8) mol / 1, and the antibody had no cross reaction with the carrier protein BSA-KLH and GOX, and had good specificity. The indirect detection of paralytic shellfish toxin was established. The sensitivity of the two methods were 15 渭 g / ml and 6 渭 g / ml, respectively, and the detection limits were 6 渭 g / ml and 0.6 渭 g / ml, respectively. The working ranges were 6-50 渭 g / ml and 0.6-50 渭 g / ml, respectively. Conclusion: the toxin GTX2O3 can be conjugated to the carrier protein by the method of formaldehyde or periodate oxidation. A strain secreting anti-#en0# was obtained by using mouse hybridoma technique. (3) the hybridoma cell line of monoclonal antibody has high affinity and good specificity. The indirect and direct competitive enzyme immunoassay method of GTX2H3 was established by using this monoclonal antibody. However, the sensitivity of the two methods is not up to the minimum limit of 80 渭 g / 100 g of residual toxin in seafood, which needs further optimization.
【学位授予单位】：暨南大学
【学位级别】：硕士
【学位授予年份】：2006
【分类号】：R392

【参考文献】