抗组氨酸标签单克隆抗体的制备、鉴定及应用

摘要

目的：制备特异性抗组氨酸标签（His-tag）的单克隆抗体（mAb）并进行鉴定。方法以合成的15个组氨酸的多肽耦联牛血清清蛋白（BSA）为抗原免疫Balb/c小鼠，按常规方法进行细胞融合。采用间接酶联免疫吸附试验（ELISA）筛选阳性克隆及有限稀释法进行克隆化，用Protein G柱亲和纯化抗体及ELISA 检测纯化抗体的效价、相对亲和力及抗体亚类，用ELISA及Western blotting对mAb的特异性进行鉴定，并与含His-tag的重组蛋白特异性抗体配对建立双抗夹心ELISA定量检测对应的重组蛋白。结果筛选出5株能稳定分泌抗 His-tag的杂交瘤细胞株，抗体亚类均为IgG1（κ），其中1-G2亲和力最高（1．27×1010），并且1-G2在ELISA及 Western blotting中能与不同类型的含 His-tag的重组蛋白结合，能与降钙素原（PCT ）抗体构建双抗夹心ELISA定量检测含 His-tag的PCT重组蛋白，灵敏度达到4．6 ng/mL。结论成功制备了特异性好、亲和力高、能稳定分泌抗His-tag的mAb ，该抗体能应用于ELISA及 Western blotting等多种 His-tag的检测方法，为蛋白质研究检测、定位、相互作用等提供了工具。%Objective To prepare and identify monoclonal antibodies (mAb) against histidine-tag (His-tag ) .Methods Balb/c mice were immunized with polypeptides containing 15 histindine(His)-coupled BSA and this fusion was prepared according to con-ventional methods .Indirect ELISA was used to screen the positive clones and limited dilution for further cloning .After purified with Protein G affinity chromatography ,these antibodies for His-tag were detected for antibody titer ,relative affinity as well as subtypes by using ELISA .The specificity of these mAb was identified by ELISA and Western blotting analysis .Double-antibody sandwich ELISA was composed of these mAb paired with the corresponding recombinant protein of specific antibody ,which was used to de-tect the recombinant protein quantitatively .Results 5 hybridoma cell lines stably secreting anti-His-tag IgG1(κ) were screened .A-mong these antibodies ,1-G2 was of the highest affinity ,reaching 1 .27 × 1010 ,which could combine with different recombinant pro-teins containing His-tag in the experiment of ELISA and Western blotting .1-G2 also could be used to detect recombinant PCT pro-tein containing His-tag quantitatively by using double-antibody sandwich ELISA with antibody of PCT ,the sensitivity of detection reached 4 .6 ng/mL .Conclusion The mAb against for His-tag with high specificity ,affinity and secreted stably are successfully prepared .This prepared antibody could be used in ELISA and Western blotting to detect a variety of His-tag .