抗黄曲霉毒素B1单克隆抗体的制备及其性质的鉴定

发布时间：2018-04-05 23:10

本文选题：黄曲霉毒素B1　切入点：单克隆抗体　出处：《福建农林大学》2013年硕士论文

【摘要】：黄曲霉毒素（Aflatoxin，AFT）主要是由黄曲霉（Aspergillus flavus）和寄生曲霉（Aspergillus parasiticus）产生的毒性极强的次生代谢产物，黄曲霉毒素是一类结构类似的化合物的总称。在黄曲霉毒素家族中，常见的有黄曲霉毒素B1、B2、G1、G2、M1和M2等。早在1988年黄曲霉毒素B1被国际肿瘤研究机构（IARC）定为一级致癌物。传统的检测方法不但耗时耗力，成本偏高，而且还需要专业技术人员对分析仪器进行操作。采用免疫化学分析法进行检测则可以克服以上缺点。本文旨在筛选出能分泌抗AFB1的高亲和力的单克隆抗体细胞株，为建立免疫化学检测方法打下基础。首先用碳二亚胺法（EDC）构建了两种完全抗原AFB1-BSA和AFB1-OVA，其中AFB1-BSA作为免疫原免疫两只8周龄BALB/C雌性小鼠四次，AFB1-OVA作为包被原包被酶标板，iELISA法测定免疫小鼠血清效价。加强免疫后，两只小鼠的效价分别达到了128000和32000。采用PEG（4000）法将SP2/0骨髓瘤细胞和免疫小鼠脾脏细胞进行融合，HAT筛选培养基筛选培养，经过3~4轮筛选得到三株能稳定分泌抗AFB1单克隆抗体的杂交瘤细胞株：1B7、5A8、5B8。其中，1B7和5A8细胞株分泌的单克隆抗体为IgM亚型，5B8细胞株分泌的抗体为IgG1亚型。为便于后期纯化，本次实验采用5B8细胞株进行后续实验。经测定，5B8细胞株分泌的单克隆抗体效价达到20,0000，亲和力为7.96×108。将该细胞株进行扩大培养并注射到13周龄雌性Balb/c小鼠腹腔中，一周后取腹水，采用辛酸-硫酸铵法纯化腹水，经SDS-PAGE检测可以看到明显的重链和轻链条带，，证明单克隆抗体纯化成功。
[Abstract]:Aflatoxin (AFT) is a highly toxic secondary metabolite produced by Aspergillus flavusand Aspergillus parasiticus. Aflatoxin is a class of compounds with similar structure.Among the aflatoxin families, aflatoxin B _ (1) B _ (2) G _ (1) G _ (2) G _ (2) M _ 1 and M _ 2 are common.As early as 1988, aflatoxin B1 was designated as a carcinogen by the International Cancer Research Institute (IARC).The traditional detection method is not only time-consuming and expensive, but also needs professional technicians to operate the analytical instrument.The above shortcomings can be overcome by immunochemical analysis.The aim of this study was to screen monoclonal antibody cell lines which secreted high affinity to AFB1, which laid the foundation for the establishment of immunocytochemical assay.Two complete antigens (AFB1-BSA and AFB1-OVA) were constructed by carbodiimide method. Two 8-week-old BALB/C female mice were immunized with AFB1-BSA for four times.The titers of the two mice reached 128000 and 32000 respectively after immunization.The SP2/0 myeloma cells and the spleen cells of immunized mice were screened and cultured by the method of PEGN 4000. after 3 rounds of screening, three hybridoma cell lines were obtained which could stably secrete the monoclonal antibody against AFB1, namely: 1B7A8A8A8B8B8.The monoclonal antibody secreted by 5A8 cell line was IgG1 subtype of IgM subtype 5B8 cell line.In order to facilitate the later purification, 5B8 cell line was used to carry out the follow-up experiment.The titer of monoclonal antibody secreted by the cell line was 20: 00000.The affinity was 7.96 脳 10 ~ (8).The cell line was cultured and injected into the abdominal cavity of 13 week-old female Balb/c mice. Ascites were extracted one week later, and ascites were purified by caprylic acid-ammonium sulfate method. The heavy chain and light chain band could be seen by SDS-PAGE detection.It was proved that the monoclonal antibody was purified successfully.
【学位授予单位】：福建农林大学
【学位级别】：硕士
【学位授予年份】：2013
【分类号】：R392

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