title: [Application of recombinant GPⅢa combined Luminex beads for the detection of HPA-1a antibody].
abstract: OBJECTIVE
  To generate recombinant GPⅢa as an alternative source for HPA-1a antigen and combine it with Luminex xMAP beads for the detection of HPA-1a-specific alloantibody.
  
  
  METHODS
  The full coding region of ITGB3 gene was amplified and ligated with pcDNA3.1. The recombinant plasmid was transfected into CHO cells, and those with stable expression were screened with G418. Expressed protein was identified and coupled with Luminex xMAP beads, which were then reacted with sera samples. Subsequently, phycoerythrin-labeled anti-species IgG antibody was added to the reaction wells and the median fluorescence was determined on a Luminex-100 analyzer.
  
  
  RESULTS
  DNA sequencing confirmed that the cloned ITGB3 gene was HPA-1aa. The recombinant GPⅢa was coupled with Luminex xMAP beads. The sensitivity of Luminex beads assay to detect HPA-1a antibody was dilution 1/32 (3.125 U/mL). The Luminex beads assay could specifically identify the HPA-1a antibody from the test sera, and the results were consistent with that of monoclonal antibody-specific immobilization of platelet antigens (MAIPA) technology. Cross-reactivity was not observed with the samples containing HLA, ABO and other HPA antibodies (HPA-3a and HPA-5b). The results illustrated that to detect HPA antibody with Luminex xMAP beads technology is feasible.
  
  
  CONCLUSIONS
  Recombinant GPⅢa was successfully obtained and used to establish a Luminex technology-based method for the detection of HPA antibodies.
authors.affiliation: Blood Center of Zhejiang Province, Key Laboratory of Blood Safety Research of the Ministry of Health, Key Laboratory of Blood Safety Research of Zhejiang Province, Hangzhou, Zhejiang 310006, China. zfm00@hotmail.com.
authors.forename: Sudan
authors.initials: S
authors.lastname: Tao
authors.forename: Ying
authors.initials: Y
authors.lastname: Liu
authors.forename: Yanming
authors.initials: Y
authors.lastname: He
authors.forename: Yanling
authors.initials: Y
authors.lastname: Ying
authors.forename: Ji
authors.initials: J
authors.lastname: He
authors.forename: Faming
authors.initials: F
authors.lastname: Zhu
mesh_terms: D000818:Animals; D016824:Antigens, Human Platelet / Q000276:immunology*; D001323:Autoantibodies / Q000276:immunology*; D001483:Base Sequence; D015153:Blotting, Western; D016466:CHO Cells; D006224:Cricetinae; D003412:Cricetulus; D006801:Humans; D007118:Immunoassay / Q000379:methods; D039661:Integrin beta3 / Q000235:genetics / Q000276:immunology* / Q000378:metabolism; D008863:Microspheres; D011994:Recombinant Proteins / Q000276:immunology* / Q000378:metabolism; D015203:Reproducibility of Results
chemical_list: D016824:Antigens, Human Platelet; D001323:Autoantibodies; C061248:ITGB3 protein, human; D039661:Integrin beta3; D011994:Recombinant Proteins