Aim: To prepare the rabbit antibody against the alpha3 domain of the human CD1d (hCD1d-alpha3).
Methods: The gene fragment coding for hCD1d-alpha3 was amplified by PCR and cloned into prokaryotic expression vector pET28, then expressed in E.coli BL21(DE3). The recombinant protein hCD1d-alpha3 was purified with Ni(2+)-NTA agarose column and used as immunogen to immunize the rabbit. The titer and specificity of the anti-hCD1d-alpha3 antibody from the rabbit were analyzed by ELISA, Western blot and immunohistochemistry, respectively.
Results: The recombinant hCD1d-alpha3 was successfully expressed and purified, and the polyclonal anit-hCD1d-alpha3 antibody was successfully prepared. The titer of the antiserum was 1:6 400 by ELISA. Western blot analysis showed this antiboday reacted specifically with hCD1d. Immunohistochemistry analysis showed the antibody could recognize the native hCD1d in the human intestinal tissue.
Conclusion: The anti-CD1d-alpha3 antibody from the rabbit with high titer and specificity has been prepared with purified recombinant hCD1d-alpha3 as immunogen, which lays a foundation for further research into detection and functional study of CD1d.
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