title: [Preparation of rabbit anti-recombinant human calreticulin antibody and its characterization].
abstract: OBJECTIVE
  To prepare the rabbit anti-recombinant human calreticulin (hCRT) antibody and its characterization.
  
  
  METHODS
  The gene coding for hCRT was amplified by PCR and cloned into prokaryotic expression vector pET32a. Then the recombinant plasmid pET32a/hCRT was transformed into E.coli BL21 (DE3) and expressed under IPTG induction. The recombinant hCRT was purified through Ni(2+) -NT agarose gel column and the purified hCRT used as immunogen to immunize the rabbit.The titer and specificity of the rabbit anti-hCRT antibody were analyzed by ELISA, Western blot and immunohistochemical staining, respectively.
  
  
  RESULTS
  The recombinant hCRT was successfully expressed and purified, and the polyclonal anit-hCRT antibody was successfully prepared. The titer of the antiserum was 1:51 200 by ELISA. Western blot analysis showed that the antibody reacted specifically to hCRT. Immunohistochemical staining detection manifested the antibody could recognize the native hCRT.
  
  
  CONCLUSIONS
  The rabbit anti-hCRT antibody with high titer and specificity has been successfully prepared, which lays the foundation for further research on detection of hCRT and its clinical application.
authors.affiliation: Department of Clinical Immunology, Guangdong Medical College, Dongguan 523808, China.
authors.forename: Xiao-lei
authors.initials: XL
authors.lastname: Tang
authors.forename: Xiao-dong
authors.initials: XD
authors.lastname: Liang
authors.forename: Rui-huan
authors.initials: RH
authors.lastname: Xu
authors.forename: Zhang-quan
authors.initials: ZQ
authors.lastname: Chen
mesh_terms: D000818:Animals; D000906:Antibodies / Q000737:chemistry / Q000302:isolation & purification*; D000918:Antibody Specificity / Q000276:immunology*; D037282:Calreticulin / Q000276:immunology*; D003001:Cloning, Molecular / Q000379:methods; D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism; D005822:Genetic Vectors; D006801:Humans; D007114:Immunization; D010957:Plasmids; D011817:Rabbits; D011994:Recombinant Proteins / Q000737:chemistry / Q000302:isolation & purification
chemical_list: D000906:Antibodies; D037282:Calreticulin; D011994:Recombinant Proteins