抗Ig融合蛋白Fc段单克隆抗体的制备、鉴定与应用研究

Preparation, characterization and application of monoclonal antibodies to Fc fragment of Ig fusion protein

目的 :制备并鉴定抗Ig融合蛋白Fc段的单克隆抗体 (mAb) ,建立用于检测Ig融合蛋白的夹心ELISA法和纯化Fc融合蛋白的亲和层析法。方法 :以hBCMA Ig融合蛋白为抗原免疫BALB/c小鼠 ,通过细胞融合制备抗Fc段mAb ,用ELISA等方法鉴定mAb的Ig亚类、表位以及种属特异性 ,建立用于检测Ig融合蛋白的夹心ELISA法 ;Westernblot检测mAb对变性Ig融合蛋白的反应性。将mAb与Sepharose4B交联 ,制备亲和层析柱 ,对LAIR1 Ig融合蛋白进行纯化。 结果 :获得 7株稳定分泌抗Fc段mAb的杂交瘤 (FMUFc1～FMUFc7)。利用FMUFc4作为包被mAb ,FMUFc5作为酶标记mAb ,成功地建立了检测Ig融合蛋白的ELISA法 ,敏感度达到 2 μg/L ;在 7株mAb中 ,FMUFc6可用于Ig融合蛋白的Westernblot检测。用FMUFc6mAb制备的亲和层析柱 ,可有效地纯化LAIR1 Ig融合蛋白。结论 :成功地制备了抗Ig融合蛋白Fc段的mAb ,建立了可用于Ig融合蛋白检测和纯化的方法 。

AIM: To prepare and characterize mAb to Fc fragment of Ig fusion protein, and to establish sandwich ELISA for detecting Ig fusion proteins and affinity chromatography method for Ig fusion protein purification. METHODS: hBCMA Ig was used as antigen to immune BALB/c mice. The lymphocyte hybridoma technique was used to establish hybridoma cell lines stably secreting anti Fc mAb. ELISA was employed to detect the isotype, epitopes, and species specificity of mAbs. Western blot was used to assess the reactivity between anti Fc mAbs and denatured Ig fusion protein. LAIR1 Ig fusion protein was purified through affinity column anti Fc mAb cross linked to sepharose 4B. RESULTS: Seven hybridoma cell lines(FMUFc1～FMUFc7)were acquired. A sandwich ELISA was successfully established using FMUFc4 as coating mAb and FMUFc5 as enzyme labeled mAb. FMUFc6 could be used for Western blot. LAIR1 Ig fusion protein was effectively purified through FMUFc5 sepharose affinity chromatography column. CONCLUSION: mAb against Fc fragment of Ig fusion protein has been prepared successfully, and methods to detect and purify Ig fusion protein are established. These mAbs provide useful tool for further application of Ig fusion protein.