[Preparation and characterization of the rabbit polyclonal antibody against the alpha3 domain of human CD1d].
OBJECTIVE
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.


CONCLUSIONS
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.
Chen
Zhang-quan
ZQ
Department of Clinical Immunology, Guangdong Medical College, Zhanjiang 524023, China. czqhk@126.com
Huang
Zhen
Z
Liang
Xiao-dong
XD
He
Tian-wen
TW
Lu
Tian-tian
TT
D000818:Animals; D000906:Antibodies / Q000378:metabolism*; D055610:Antigens, CD1d / Q000235:genetics / Q000276:immunology* / Q000378:metabolism; D015153:Blotting, Western; D004797:Enzyme-Linked Immunosorbent Assay; D006801:Humans; D007150:Immunohistochemistry; D017434:Protein Structure, Tertiary / Q000235:genetics; D011817:Rabbits
D000906:Antibodies; D055610:Antigens, CD1d; C526877:CD1D protein, human