可溶性白细胞介素-6受体结合链免疫放射检测方法的建立及应用

Establishment and application of immunoradio assay for detecting human soluble IL-6Rα

目的 建立一种可溶性白细胞介素 6受体结合链 (IL 6Rα)免疫放射检测方法。方法采用B淋巴细胞杂交瘤技术获取分泌鼠抗人可溶性IL 6Rα单抗的杂交瘤细胞株 ;经体内诱生腹水法制备单抗 ;免疫亲合层吸法纯化单抗 ;用 12 5I标记单抗后 ,采用竞争抑制分析法对单抗识别的抗原表位进行鉴定 ,选择识别不同抗原表位的 2株单抗H12 6和SI10分别作为包被单抗和检测单抗 ,建立双单抗夹心的可溶性IL 6Rα免疫放射检测方法。分析该检测方法与可溶性IL 6Rα相关的细胞因子白细胞介素 6 (IL 6 )及可溶性IL 6信号传导链 (IL 6Rβ ,也称gp130 )的交叉反应性 ;采用加入 回收法对检测方法的准确性进行鉴定 ,并绘制标准工作曲线 ,然后测定正常人及多发性骨髓瘤患者血清中可溶性IL 6Rα的含量。结果 建立的可溶性IL 6Rα免疫放射检测方法的灵敏度为 10ng/ml,具有良好线性关系的检测范围是 10～ 4 0 0ng/ml,与IL 6及可溶性gp130 (sgp130 )无交叉反应性 ,加入重组可溶性IL 6Rα的回收率在 92 %～ 10 8%之间。正常人血清中可溶性IL 6Rα的含量为 (81 96± 7 2 3)ng/ml,多发性骨髓瘤患者血清中可溶性IL 6Rα的含量为 (2 37 5 8± 70 96 )ng/ml,明显高于正常对照组 (P <0 0 1)。结论 该检测方法能够对血液中可溶性IL 6Rα进行?

Objective To establish the sensitive,specific,stable and convenient immunoradio assay for detecting human soluble IL-6Rα.Methods The hybridoma cell lines were obtained by fusing spleen cells of BALB/c mice that had been immunized with soluble IL-6Rα protein to mouse myeloma cells sp2/0. Ascites were used to produce the monoclonal antibodies (mAbs). The mAbs were purified by protein G immunoaffinity method. The mAb SI10 was used as coating antibody, the other mAb H126 recognized different epitope from SI10 was labeled by 125I. Results The immunoradio assay for detecting soluble IL-6Rα was set up. It has high stability and accuracy. The detecting limit is 10 ng/ml. The serum concentration of soluble IL-6Rα is (81.96 ± 7.23) ng/ml in healthy donors and (237.58±70.96) ng/ml in patients with multiple myeloma. Significant difference was founded between two groups (P<0.01). Conclusion The method may be of value in basic studies and contribute to soluble IL-6Rα related disease′diagnosis, evaluation of curative effect and prognosis.