抗软骨藻酸单克隆抗体制备及ELISA检测方法建立

发布时间：2018-01-14 14:26

本文关键词：抗软骨藻酸单克隆抗体制备及ELISA检测方法建立　出处：《吉林大学》2008年硕士论文　论文类型：学位论文

【摘要】： 软骨藻酸由海洋中的硅藻产生后常通过食物链蓄积于海洋贝类、蛤类等海产品体内,进而引起海洋动物乃至人的食物中毒,国外由于误食有毒海产品而引发中毒的事件时有发生,严重影响了人们的身体健康和对海产品的开发利用。本实验通过活泼酯法将软骨藻酸同牛血清白蛋白及人免疫球蛋白进行偶联,成功制备了免疫原及检测原,通过足垫快速法免疫BALB/C小鼠,常规方法进行融合,间接ELISA方法筛选阳性克隆,同时采用有限稀释法、显微操作法及皮下荷瘤方法进行杂交瘤细胞的亚克隆和培养,建立了9C1杂交瘤细胞株,并对其单克隆抗体的效价、分子量、抗体亚类、有效浓度、染色体众数、纯度、蛋白质含量、亲和力、特异性等方面进行了研究,利用优化后的实验条件进行间接竞争ELISA检测并建立标准曲线,标准曲线的回归方程为y = -13.094x + 106.19,相关系数R2=0.9962,最低检测限2.43ng/mL。
[Abstract]:After being produced by diatoms in the ocean, alginic acid often accumulates in marine shellfish, clams and other marine products through the food chain, which leads to food poisoning of marine animals and even people. Poisoning has occurred from time to time in foreign countries due to the accidental consumption of toxic seafood. In this experiment, the alginic acid was coupled with bovine serum albumin and human immunoglobulin by active ester method, and the immunogen was prepared successfully. BALB/C mice were immunized with foot pad fast method, fusion was performed by routine method, positive clones were screened by indirect ELISA method, and limited dilution method was used at the same time. The hybridoma cell line 9C1 was subcloned and cultured by micromanipulation and subcutaneous tumor-bearing method. The monoclonal antibody titer, molecular weight, antibody subclass and effective concentration of the monoclonal antibody were obtained. Chromosome mode, purity, protein content, affinity, specificity and other aspects were studied. The optimized experimental conditions were used for indirect competitive ELISA detection and the establishment of standard curve. The regression equation of the standard curve is y = -13.094x 106.19, the correlation coefficient R _ 2N _ 2 is 0.9962and the minimum detection limit is 2.43ng / mL.
【学位授予单位】：吉林大学
【学位级别】：硕士
【学位授予年份】：2008
【分类号】：R155.3;R392

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