小鼠抗蛋清提取物单克隆抗体的制备与初步鉴定
发布时间：2018-10-23 16:34
【摘要】： 目的:制备和初步鉴定小鼠抗鸡蛋清提取物单克隆抗体。 方法:以纯化后的蛋清提取物为免疫原,经背部皮下多点注射免疫BALB/c小鼠。采用棋盘滴定法确定抗原的最佳包被浓度,并通过间接ELISA法检测免疫小鼠血清效价,选取血清效价高的小鼠用于单克隆抗体的制备。无菌分离免疫小鼠脾脏,制成单个细胞悬液,与小鼠骨髓瘤NS-1细胞融合。通过间接ELISA法筛选10株阳性融合孔中的融合细胞,采用有限稀释法进行克隆与亚克隆培养;间接ELISA法筛选可特异性分泌抗蛋清提取物单克隆抗体(monoclonal antibody, McAb)的杂交瘤细胞;增量培养法制备单克隆抗体; Ig类与亚类鉴定试剂盒鉴定McAb的Ig类与亚类;间接ELISA法检测杂交瘤细胞冻融前后产生McAbs的效价;Western blot鉴定McAbs的特异性。 结果:采用棋盘滴定法确定抗原的最佳包被浓度为100 ng?mL-1。经克隆及3～4轮亚克隆,8株克隆失去产生抗体的能力,最终获得2株可稳定分泌抗蛋清提取物McAb的杂交瘤细胞,分别命名为5E12和4F1;其分泌的McAb均为IgG1。Western blot分析表明,McAb 4F1可与转印至膜上的蛋白形成单一条带,所识别的靶分子的相对分子质量(Mr)约为44KD。间接ELISA法检测两株细胞冻融前后所产生的McAb5E12、4F1的效价分别为1:5000、1:300。 结论:成功地制备了两株抗蛋清提取物McAb 5E12和4F1,为下一步制备、纯化特异性抗蛋清提取物单克隆抗体提供了杂交瘤细胞,并为进一步建立快速检测、提取、纯化及鉴定食物中蛋清变应原的方法,奠定了有效的实验基础。
[Abstract]:Objective: to prepare and identify mouse monoclonal antibody against egg white extract. Methods: BALB/c mice were immunized by subcutaneous injection of purified egg white extract. The best coating concentration of antigen was determined by chessboard titration and the titer of immunized mice was detected by indirect ELISA method. The mice with high serum titer were selected for the preparation of monoclonal antibody. The spleen of mice was isolated and immunized with aseptic method. A single cell suspension was made and fused with mouse myeloma NS-1 cells. The fusion cells from 10 positive fusion cells were screened by indirect ELISA method, cloned and subcloned by limited dilution method, and hybridoma cells secreting monoclonal antibody (monoclonal antibody, McAb) against egg white extract were screened by indirect ELISA method. Monoclonal antibodies were prepared by incremental culture, Ig classes and subclasses of McAb were identified by Ig and subclass identification kit, and the specificity of McAbs was identified by indirect ELISA assay for detection of McAbs production before and after freezing and thawing of hybridoma cells. Results: the optimum coating concentration of antigens was determined by chessboard titration for 100 ng?mL-1.. After cloning and three rounds of subcloning, 8 clones lost the ability to produce antibodies. Finally, two hybridoma cells secreting McAb stably from egg white extract were obtained. They were named 5E12 and 4F1, respectively, and the McAb secreted by them were both IgG1.Western blot analysis. The results showed that McAb 4F1 could form a single band with the protein transferred to the film, and the relative molecular weight (Mr) of the target molecule recognized was about 44kD. The titer of McAb5E12,4F1 produced by indirect ELISA assay before and after freezing and thawing was 1: 5 000 and 1: 300, respectively. Conclusion: two strains of anti-egg white extract (McAb 5E12 and 4F1) were successfully prepared, which provided hybridoma cells for further preparation and purification of specific monoclonal antibodies against egg white extract. The method of purification and identification of egg white allergens in food has laid an effective experimental foundation.
【学位授予单位】：南昌大学
【学位级别】：硕士
【学位授予年份】：2007
【分类号】：R392
本文编号：2289834
[Abstract]:Objective: to prepare and identify mouse monoclonal antibody against egg white extract. Methods: BALB/c mice were immunized by subcutaneous injection of purified egg white extract. The best coating concentration of antigen was determined by chessboard titration and the titer of immunized mice was detected by indirect ELISA method. The mice with high serum titer were selected for the preparation of monoclonal antibody. The spleen of mice was isolated and immunized with aseptic method. A single cell suspension was made and fused with mouse myeloma NS-1 cells. The fusion cells from 10 positive fusion cells were screened by indirect ELISA method, cloned and subcloned by limited dilution method, and hybridoma cells secreting monoclonal antibody (monoclonal antibody, McAb) against egg white extract were screened by indirect ELISA method. Monoclonal antibodies were prepared by incremental culture, Ig classes and subclasses of McAb were identified by Ig and subclass identification kit, and the specificity of McAbs was identified by indirect ELISA assay for detection of McAbs production before and after freezing and thawing of hybridoma cells. Results: the optimum coating concentration of antigens was determined by chessboard titration for 100 ng?mL-1.. After cloning and three rounds of subcloning, 8 clones lost the ability to produce antibodies. Finally, two hybridoma cells secreting McAb stably from egg white extract were obtained. They were named 5E12 and 4F1, respectively, and the McAb secreted by them were both IgG1.Western blot analysis. The results showed that McAb 4F1 could form a single band with the protein transferred to the film, and the relative molecular weight (Mr) of the target molecule recognized was about 44kD. The titer of McAb5E12,4F1 produced by indirect ELISA assay before and after freezing and thawing was 1: 5 000 and 1: 300, respectively. Conclusion: two strains of anti-egg white extract (McAb 5E12 and 4F1) were successfully prepared, which provided hybridoma cells for further preparation and purification of specific monoclonal antibodies against egg white extract. The method of purification and identification of egg white allergens in food has laid an effective experimental foundation.
【学位授予单位】：南昌大学
【学位级别】：硕士
【学位授予年份】：2007
【分类号】：R392
【引证文献】
相关硕士学位论文 前1条
1 朱晓琳;猪传染性胃肠炎病毒重组N蛋白的表达纯化及其单克隆抗体的研究[D];福建农林大学;2012年
，本文编号：2289834
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