[Establishment and application of double-antibody sandwich ELISA for determination of 3-nitrotyrosine].

Aim: 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·ml-1 and the linear range of standard work curve was 0.15～7.50 ng·ml-1(r2=0.995).The 3-NT concentration in cardiac tissue from rats subjected to MI/R(1022.42±97.35 ng·mg pro-1) was significantly higher than that in the sham group(246.58±56.52 ng·mg pro-1,P0.01).Conclusion: 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.