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透明质酸单克隆抗体的制备及免疫分析方法的建立

Preparation of a monoclonal antibody against hyaluronic acid and its application in immunoassay

  • 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6.71×109 mol-1,独特型为IgG1;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0.01~10.0 μg/mL,特异性好、灵敏度高。

     

    Abstract: 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 monoclonal 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 Ka=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.

     

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