膝沟藻毒素GTX2，3完全抗原的合成与鉴定

Preparation and Identification of Complete Antigen of Gonyaulax Parlaytic Shel fish Toxin 2 and 3(GTX 2,3)

膝沟藻毒素GTX 2,3是一类小分子物质,不具备免疫原性,欲制备其免疫血清及抗体,则必须与偶联剂及载体蛋白偶联形成完全抗原。运用乙二醛作为偶联剂,分别与牛血清白蛋白(BSA)、血蓝蛋白(KLH)合成免疫抗原GTX 2,3-glyoxal-BSA和检测抗原GTX 2,3-glyoxal-KLH,并对经超滤纯化浓缩后的偶联产物进行BCA蛋白浓度测定、全波段紫外扫描、变形SDS-PAGE凝胶电泳和抗原免疫原性的鉴定。结果表明,免疫抗原和检测抗原的蛋白质量浓度分别为8.52 mg/m L和5.72 mg/m L;经全波段紫外扫描和变形SDS-PAGE凝胶电泳鉴定,半抗原GTX 2,3-乙二醛与载体蛋白BSA、KLH偶联成功,偶联比约为1︰16和1︰20,并制备出纯度较高的免疫抗原;用制备的人工抗原GTX 2,3-glyoxal-BSA对Balb/C小鼠进行免疫,通过间接ELISA检测6免后小鼠血清效价达到1︰20 000。成功制备了具有较强免疫原性的膝沟藻毒素GTX 2,3人工抗原。

Preparing complete antigens of paralytic shellfish poisoning toxins GTX 2, 3, a GTX 2, 3-glyoxal was proposed to be used as an intermediate cross-linking with BSA or KLH, by which two artificial complete antigens GTX 2, 3-glyoxal-BSA and GTX 2, 3-glyoxal-KLH, as an immunizing and detected antigen respectively, were then prepared. The suecc＇ssful preparation of the two complete antigens was identified by BCA, UV spectral scanning and SDS-PAGE. Four female Balb/C mice were immunized with GTX 2, 3-glyoxal-BSA conjugates by intmperitoneal immunization and the antibody titration in immunized mice serum was measured by indirect ELISA. The concentration of the complete antigen of GTX 2, 3-glyoxal-BSA and GTX 2, 3-glyoxal-KLH were 8.52 mg/mL and 5.72 mg/mL. The UV spectral manning and SDS-PAGE confirmed that the hapten GTX 2, 3-glyoxal and carrier protein had been conjugated, with the coupling ratio of 1 ： 16 and 1 ： 20 of each protein and hapten, along with the high purity of the immunizing complete antigens. The titer of 1 ： 20 000 after 6 times immunization of antibody against GTX 2, 3 in mice serum was detected in indirect ELISA. In conclusion, the synthesized complete antigens, which have strong immunogenicity, have been successfully prepared.