Preparation identification and application of monoclonal antibodies against histidine-tag / 重庆医学

ABSTRACT

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 .