卵清蛋白单克隆抗体的研制及应用
发布时间：2019-04-23 23:40
【摘要】：目的：制备和鉴定卵清蛋白特异性单克隆抗体，建立双抗体夹心ELISA法检测流感疫苗中的卵清蛋白残留量。 方法：以提纯的卵清蛋白免疫Balb/c小鼠，第四次加强免疫后取脾淋巴细胞与NS－1骨髓瘤细胞按常规方法融合、筛选、克隆化及腹水注射制备McAb，采用间接ELISA方法进行McAb的鉴定，Western blot进行特异性鉴定；使用ProteinA亲和层析柱纯化腹水中的卵清蛋白单克隆抗体，用改良过碘酸钠法对单抗进行辣根过氧化物酶标记。经抗体配对实验确定包被抗体和酶标抗体，再用方阵滴定法确定抗体工作浓度，制备针对卵清蛋白的双抗体夹心ELISA检测试剂，分析其稳定性、灵敏度和特异性。用卵清蛋白标准品制备定量标准曲线，对待检标本进行检测并与国外试剂进行比较。 结果：经过细胞融合、多次克隆化获得3株特异性分泌抗卵清蛋白的单克隆抗体杂交瘤细胞株，经3次复苏传代仍能稳定分泌抗体，分别命名为1C2、2F7和3B2。3株McAbs腹水ELISA效价在1：105~1：106；3株McAbs的亚型均为IgG1；相对亲合力为2F73B21C2；初步建立了双抗体夹心ELISA检测方法，其中以1C2与3B2混合后作为包被抗体，，2F7为酶标抗体时为ELISA最优体系，最低检测极限为31.25ng/ml，且检测结果与卵清蛋白含量呈现良好的线性相关，R2=0.910，试剂盒有很好的特异性、稳定性和精密度。对样品测定结果与Serazym Ovalbumin试剂测定结果相比较（P0.05），无显著差异。 结论：已成功制备出了卵清蛋白McAb，并建立了一种灵敏度较好、特异性强的测定流感疫苗中卵清蛋白残留量的ELISA捕获法，可应用于流感疫苗生产过程卵清蛋白的监测及疫苗的检定。
[Abstract]:Aim: to prepare and identify specific monoclonal antibodies against ovalbumin and to establish a double antibody sandwich ELISA method for the detection of ovalbumin residues in influenza vaccine. Methods: Balb/c mice were immunized with purified ovalbumin. The spleen lymphocytes were fused with NS-1 myeloma cells by routine method after the fourth enhanced immunization. The McAb, was prepared by cloning and ascitic injection. Indirect ELISA method was used to identify McAb., Western blot was used for specific identification. Monoclonal antibodies against ovalbumin in ascites were purified by ProteinA affinity chromatography and labeled with horseradish peroxidase by modified sodium periodate method. The coated antibody and enzyme labeled antibody were determined by antibody pairing test, and the working concentration of the antibody was determined by square titration. The double antibody sandwich ELISA assay for ovalbumin was prepared and its stability, sensitivity and specificity were analyzed. The quantitative standard curve of ovalbumin standard sample was prepared and compared with foreign reagent. Results: three hybridoma cell lines secreting monoclonal antibodies specifically secreting ovalbumin were obtained after cell fusion. The hybridoma cells could still secrete antibodies stably after 3 times of resuscitation. They were named 1C2, 2F7 and 3B2.3, respectively, and the ELISA titers of ascitic fluid of McAbs strain were 1? 10 5 and 1? 10 6, respectively. The subtypes of McAbs were 2F73B21C22and 2F73B21C2respectively. A double antibody sandwich ELISA method was established, in which the mixture of 1C2 and 3B2 was used as the coated antibody, and 2F7 as the enzyme labeled antibody was the optimal system for ELISA, and the lowest detection limit was 31.25 ng / ml, and the detection limit was 31.25 ng / ml. There was a good linear correlation between the detection results and the content of ovalbumin. R2, 0.910, the kit had good specificity, stability and precision. There was no significant difference between the results of sample determination and that of Serazym Ovalbumin reagent (P0.05). Conclusion: ovalbumin McAb, has been successfully prepared and a sensitive and specific ELISA capture method for the determination of ovalbumin residues in influenza vaccine has been established. It can be applied to the monitoring of ovalbumin in the production process of influenza vaccine and the verification of vaccine.
【学位授予单位】：吉林大学
【学位级别】：硕士
【学位授予年份】：2012
【分类号】：R392
[Abstract]:Aim: to prepare and identify specific monoclonal antibodies against ovalbumin and to establish a double antibody sandwich ELISA method for the detection of ovalbumin residues in influenza vaccine. Methods: Balb/c mice were immunized with purified ovalbumin. The spleen lymphocytes were fused with NS-1 myeloma cells by routine method after the fourth enhanced immunization. The McAb, was prepared by cloning and ascitic injection. Indirect ELISA method was used to identify McAb., Western blot was used for specific identification. Monoclonal antibodies against ovalbumin in ascites were purified by ProteinA affinity chromatography and labeled with horseradish peroxidase by modified sodium periodate method. The coated antibody and enzyme labeled antibody were determined by antibody pairing test, and the working concentration of the antibody was determined by square titration. The double antibody sandwich ELISA assay for ovalbumin was prepared and its stability, sensitivity and specificity were analyzed. The quantitative standard curve of ovalbumin standard sample was prepared and compared with foreign reagent. Results: three hybridoma cell lines secreting monoclonal antibodies specifically secreting ovalbumin were obtained after cell fusion. The hybridoma cells could still secrete antibodies stably after 3 times of resuscitation. They were named 1C2, 2F7 and 3B2.3, respectively, and the ELISA titers of ascitic fluid of McAbs strain were 1? 10 5 and 1? 10 6, respectively. The subtypes of McAbs were 2F73B21C22and 2F73B21C2respectively. A double antibody sandwich ELISA method was established, in which the mixture of 1C2 and 3B2 was used as the coated antibody, and 2F7 as the enzyme labeled antibody was the optimal system for ELISA, and the lowest detection limit was 31.25 ng / ml, and the detection limit was 31.25 ng / ml. There was a good linear correlation between the detection results and the content of ovalbumin. R2, 0.910, the kit had good specificity, stability and precision. There was no significant difference between the results of sample determination and that of Serazym Ovalbumin reagent (P0.05). Conclusion: ovalbumin McAb, has been successfully prepared and a sensitive and specific ELISA capture method for the determination of ovalbumin residues in influenza vaccine has been established. It can be applied to the monitoring of ovalbumin in the production process of influenza vaccine and the verification of vaccine.
【学位授予单位】：吉林大学
【学位级别】：硕士
【学位授予年份】：2012
【分类号】：R392
【参考文献】
相关期刊论文 前10条
1 高和;流感疫苗的研究与应用[J];解放军保健医学杂志;2004年04期
2 祝振鑫,吴立明,胡晓s
本文编号：2463910
本文链接：https://www.wllwen.com/xiyixuelunwen/2463910.html
最近更新
教材专著
