title: [Establishment and application of double-antibody sandwich ELISA for determination of 3-nitrotyrosine].
abstract: OBJECTIVE
  To prepare the working standards of 3-nitrotyrosine (3-NT) and establish a two-antibody-sandwich ELISA for determining the concentration of peroxynitrite in the tissue.
  
  
  METHODS
  Nitrated bovine serum albumin was prepared by additions of an alkaline stock solution of peroxynitrite which was synthesized by a quenched-flow reactor. The monoclone anti-3-NT antibody from mouse was used as coating antibody and the polyclone anti-3-NT antibody from as labeling antibody to prepare the standard work curve by orthogonal design. The concentrations of 3-NT in cardiac tissue from rats subjected to myocardial ischemia and reperfusion (MI/R) were analyzed.
  
  
  RESULTS
  A two-antibody-sandwich ELISA method for measuring 3-NT content in biological fluids and homogenates was successfully established. The detecting limit was 0.1 ng x ml(-1) and the linear range of standard work curve was 0.15 - 7.50 ng x ml(-1) (r2 = 0.995). The 3-NT concentration in cardiac tissue from rats subjected to MI/R (1022.42 +/- 97.35 ng x mg pro(-1)) was significantly higher than that in the sham group (246.58 +/- 56.52 ng x mg pro(-1), P < 0.01).
  
  
  CONCLUSIONS
  A two-antibody-sandwich ELISA was established for determining the 3-NT concentration in the tissue and conveniently, quickly, accurately quantitative analysis of the content of 3-NT. The assay provides a new method for quantitative analysis of the peroxyinitrite in the future.
authors.affiliation: Department of Physiology, Shanxi Medical University, Taiyuan 030001, China.
authors.forename: Li
authors.initials: L
authors.lastname: Yan
authors.forename: Yan-Wu
authors.initials: YW
authors.lastname: Xu
authors.forename: Xiao-Liang
authors.initials: XL
authors.lastname: Wang
authors.forename: Ye
authors.initials: Y
authors.lastname: Wu
authors.forename: Jue
authors.initials: J
authors.lastname: Tian
authors.forename: Guang-Zhao
authors.initials: GZ
authors.lastname: Yang
authors.forename: Xiu-Rui
authors.initials: XR
authors.lastname: Ma
authors.forename: Hui-Rong
authors.initials: HR
authors.lastname: Liu
mesh_terms: D000818:Animals; D000911:Antibodies, Monoclonal; D004797:Enzyme-Linked Immunosorbent Assay / Q000379:methods*; D008297:Male; D015428:Myocardial Reperfusion Injury; D009206:Myocardium / Q000737:chemistry; D030421:Peroxynitrous Acid / Q000032:analysis; D051381:Rats; D017208:Rats, Wistar; D012680:Sensitivity and Specificity; D014443:Tyrosine / Q000031:analogs & derivatives* / Q000032:analysis
chemical_list: D000911:Antibodies, Monoclonal; D030421:Peroxynitrous Acid; C002744:3-nitrotyrosine; D014443:Tyrosine