河豚毒素(TTX)論文河豚毒素特异性单克隆抗体的制备.docVIP

河豚毒素(TTX)论文：河豚毒素特异性单克隆抗体的制备 【中文摘要】河豚毒素(Tetrodotoxin, TTX)是毒性最强的非蛋白类神经毒素之一,分布广泛,因最早从河豚鱼体内分离而得名。在众多河豚毒素的检测技术中,免疫检测技术因其灵敏度高和操作简便等优点受到人们的青睐,而免疫学检测的关键要素是目标物质的特异性抗体。本研究首先以牛血清白蛋白(Bovine Serum Albumin,BSA)、卵清白蛋白(Ovalbumi,OVA)、匙孔血蓝蛋白(keyhole limpet hemocyanin,KLH)为载体蛋白,分别与TTX进行偶联以制备人工完全抗原。采用非变性聚丙烯酰胺凝胶电泳及紫外分光光度分析方法,优化了河豚毒素人工完全抗原(BSA‐TTX)的合成条件,并筛选出较佳的反应条件,即反应时间为72h、偶联剂添加量为12%、TTX与BSA质量比为1:5时河豚毒素免疫抗原偶联率较大。同时应用聚丙烯酰胺凝胶电泳、紫外分光光度法、红外光谱检测技术以及Quantity One凝胶分析软件计算偶联率等方法建立了人工完全抗原的表征体系。然后利用制备出的两种免疫抗原OVA‐TTX和KLH‐TTX免疫Balb/C小鼠,通过杂交瘤技术成功制备出两株杂交瘤细胞OVA 1E3和KLH CB2。两株细胞分泌的单克隆抗体均为IgM型,对两种单克隆抗体的特异性鉴定结果显示两种单抗均与TTX有特异性结合。此外,本研究还针对不同载体蛋白对单克隆抗体制备产生的影响机理进行了初步探讨。以疏水相互作用理论为基础,结合不同蛋白的疏水性能,推理认为KLH是一种较适合于TTX特异性单抗制备的载体蛋白。本课题的研究为河豚毒素的免疫学分析检测奠定了物质基础,对推动河豚毒素免疫学检测技术的发展具有重要意义。 【英文摘要】Tetrodotoxin (TTX) is one of the most toxic non‐protein neurotoxin and can be found in a variety of organisms. TTX was named because it was isolated from puffer fish originally. During many detection technologies of tetrodotoxin, immunoassay attracts scientists for its high sensitivity and easy operation, etc. Preparation of monoclonal antibodies, the foundation of Immunoassays, is focused on at the same time. In this paper, Bovine Serum Albumin (BSA), Ovalbumin (OVA) and keyhole limpet hemocyanin (KLH) were used as carrier proteins for immunization antigens (OVA‐TTX and KLH‐TTX) while BSA‐TTX as detection antigen. Effect of the coupling time, the amount of coupling agent formaldehyde (FA), and the mass ratio of tetrodotoxin (TTX) to bovine serum albumin (BSA) on the coupling rate in the immune antigen synthesis system of TTX‐FA‐BSA was studied by non‐denaturing polyacrylamide gel electrophoresis and ultraviolet spectrophotometry analysis. Meanwhile, the reaction conditions were optimized, that is 72h of coupling time, 12% of coupling agent, 1:5 mass ratio of TTX to BSA. Applying gel analysis software Quantity One, the calculation method of immunogen coupling rate was e