CAJ | 학술논문

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。
목적：제비특이성항조안산표첨（His-tag）적단극륭항체（mAb）병진행감정。방법이합성적15개조안산적다태우련우혈청청단백（BSA）위항원면역Balb/c소서，안상규방법진행세포융합。채용간접매련면역흡부시험（ELISA）사선양성극륭급유한희석법진행극륭화，용Protein G주친화순화항체급ELISA 검측순화항체적효개、상대친화력급항체아류，용ELISA급Western blotting대mAb적특이성진행감정，병여함His-tag적중조단백특이성항체배대건립쌍항협심ELISA정량검측대응적중조단백。결과사선출5주능은정분비항 His-tag적잡교류세포주，항체아류균위IgG1（κ），기중1-G2친화력최고（1．27×1010），병차1-G2재ELISA급 Western blotting중능여불동류형적함 His-tag적중조단백결합，능여강개소원（PCT ）항체구건쌍항협심ELISA정량검측함 His-tag적PCT중조단백，령민도체도4．6 ng/mL。결론성공제비료특이성호、친화력고、능은정분비항His-tag적mAb ，해항체능응용우ELISA급 Western blotting등다충 His-tag적검측방법，위단백질연구검측、정위、상호작용등제공료공구。
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 .