摘要
将1-氰基-4-二甲胺吡啶四氟硼酸盐(CDAP)活化后的透明质酸(HA)与牛血清白蛋白(BSA)共价偶联制备完全抗原BSA-HA纯化后免疫BALB/c小鼠,采用杂交瘤技术制备HA特异性单克隆抗体,考察单抗的生物学特性,并建立HA定量免疫分析方法——间接竞争ELISA法。研究表明,完全抗原BSA-HA免疫BALB/c小鼠后,能诱导HA特异性抗体的分泌;经细胞融合、选择性培养、筛选和亚克隆,获得一株稳定分泌HA特异性抗体的杂交瘤细胞8B6,其腹水抗体效价达1:256 000以上,亲和常数为6.71×10~9mol^(-1),独特型为IgGl;以10.0 ng/mL HA包被酶标板,1.0%BSA为封闭剂,建立HA间接竞争ELISA法,其线性范围介于0.01~10.0μg/mL,特异性好、灵敏度高。
This study was to develop a quantitative immunoassay for hyaluronic acid （ HA）. We firstly prepared a monoclonal antibody （MAb） against it. HA, activated by 1-cyano-4-dimethyl-aminopyridinium tetrafluoroborate （CDAP）, was coupled to bovine serum albumin （BSA）. BALB/c mice were immunized with purified complete antigen BSA-HA and the monoelonal antibody was prepared by hybridoma technique. To characterize the specificity of the antibody, inhibition enzyme-linked immunosorbent assay （ELISA） was conducted and the cross reactivity along ,with affinity constant was determined. The results showed that the substitution degree of BSA-HA （ mBSA/ WHA） was about 9. Also, a hybridoma cell line named 8B6 was obtained by hybridoma technology. The indirect ELISA titer of the ascetic fluid was 1 ： 256 000; the isotype of MAb 8B6 was IgG1 and the affinity constant was K, =6. 71 × 109 mol-1. To establish the indirect competitive ELISA method, polystyrene microwell plates were coated with 10. 0 ng/mL HA and blocked with 1.0% BSA. The linear range of indirect competitive ELISA was between 10. 0 ng/mL to 10. 0 μg/mL which confirmed that the method was specific and sensitive.
出处
《中国药科大学学报》
CAS
CSCD
北大核心
2015年第6期740-744,共5页
Journal of China Pharmaceutical University
基金
国家"重大新药创制"科技重大专项资助项目(No.2012ZX09502001-004)
江苏高校优秀科技创新团队资助项目~~
