抗人甲状腺球蛋白单克隆抗体的制备与鉴定

Production and characterization of monoclonal antibodies against human thyroglobulin.

作者信息

Piechaczyk M, Chardes T, Cot M C, Pau B, Bastide J M

出版信息

Hybridoma. 1985 Winter;4(4):361-7. doi: 10.1089/hyb.1985.4.361.

DOI:10.1089/hyb.1985.4.361

Abstract

Spleen cells of Biozzi-HR mice immunized with human thyroglobulin (hTg) were fused with P3-X63-Ag8.653 mouse myeloma cells. Twenty monoclonal antibodies (MAbs) selected by an enzyme immunoassay (indirect ELISA) were produced, purified and characterized. The equilibrium association constant (Ka) of one of the MAbs, determined by Scatchard analysis of the ELISA data, was found to be 2 X 10(9) M-1; the Ka of the other MAb, estimated from titration curves by comparison with the aforementioned MAb, ranged from 8 X 10(9) M-1 to 6 X 10(7) M-1. The reaction between the MAb and hTg was not inhibited by thyroxin (T4), triiodothyronine (T3) and triiodothyropropionic acid (DT3). Species specificity of the MAb was studied using bovine and porcine Tgb. The topology of the MAb was investigated by competitive inhibition immunoassays. Seven distinct antigenic regions were identified.

摘要

用人类甲状腺球蛋白（hTg）免疫的Biozzi-HR小鼠的脾细胞与P3-X63-Ag8.653小鼠骨髓瘤细胞进行融合。通过酶免疫测定法（间接酶联免疫吸附测定）筛选出20种单克隆抗体（MAb），并进行生产、纯化和特性鉴定。通过对酶联免疫吸附测定数据进行Scatchard分析，发现其中一种单克隆抗体的平衡缔合常数（Ka）为2×10⁹ M⁻¹；通过与上述单克隆抗体比较，根据滴定曲线估算出另一种单克隆抗体的Ka范围为8×10⁹ M⁻¹至6×10⁷ M⁻¹。甲状腺素（T4）、三碘甲状腺原氨酸（T3）和三碘甲状腺丙酸（DT3）均未抑制单克隆抗体与hTg之间的反应。使用牛和猪的甲状腺球蛋白（Tgb）研究了单克隆抗体的种属特异性。通过竞争性抑制免疫测定法研究了单克隆抗体的拓扑结构。鉴定出7个不同的抗原区域。