title: [Preparation of polyclonal antibody of human endothelial-overexpressed lipopolysaccharide-associated factor 1].
abstract: OBJECTIVE
  To prepare the polyclonal antibody of human endothelial-overexpressed lipopolysaccharide-associated factor 1 (EOLA1), and to determine the expression of EOLA1 in human umbilical vein endothelial cell (HUVEC).
  
  
  METHODS
  The protein samples (sample 1 and 2) expressing EOLA1 were purified and renatured. The protein concentrations were determined with bicinchoninic acid assay. The protein samples were identified with peptide mass fingerprinting (PMF) analysis. Protein sample with higher coincidence rate of amino acid sequence with theoretic protein was used to inoculate 4 mice; another 4 mice inoculated with adjuvant were used as control. Serum was isolated from collected mice blood. Polyclonal antibody of EOLA1 was purified with saturated ammonium sulfate precipitation, and was determined with ELISA for the titer (data were denoted by absorbance value). The expression of EOLA1 in HUVEC was determined with Western blot.
  
  
  RESULTS
  The concentration of protein sample 1 and 2 was respectively 0.124 16 mg/mL and 0.132 15 mg/mL. According to PMF analysis, the coincidence rate of amino acid sequence between protein samples and theoretic protein were 32% (protein sample 1) and 24% (protein sample 2). The polyclonal antibody of EOLA1 with titer more than 1:10 000 was obtained from mice inoculated with protein sample 1. The expression of EOLA1 protein in HUVEC was determined with polyclonal antibody of EOLA1.
  
  
  CONCLUSIONS
  The polyclonal antibody of EOLA1 can be prepared by inoculating mice with EOLA1 prokaryotic expressing protein, which can be used for determination of EOLA1 protein.
authors.affiliation: Department of Burns and Plastic Surgery, the First Affiliated Hospital of Chengdu Medical College, Chengdu 610500, China.
authors.forename: Yue-Ming
authors.initials: YM
authors.lastname: Liu
authors.forename: Hai-Rong
authors.initials: HR
authors.lastname: Liu
authors.forename: Zhen
authors.initials: Z
authors.lastname: Cai
authors.forename: Bing
authors.initials: B
authors.lastname: Ma
authors.forename: Yi-Lun
authors.initials: YL
authors.lastname: Liu
authors.forename: Wei
authors.initials: W
authors.lastname: Zhang
mesh_terms: D000818:Animals; D000906:Antibodies; D002478:Cells, Cultured; D061307:Human Umbilical Vein Endothelial Cells / Q000378:metabolism; D006801:Humans; D008070:Lipopolysaccharides / Q000378:metabolism; D008565:Membrane Proteins / Q000276:immunology* / Q000378:metabolism; D051379:Mice
chemical_list: D000906:Antibodies; C495142:EOLA1 protein, human; D008070:Lipopolysaccharides; D008565:Membrane Proteins