AIMTo 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.

METHODSNitrated 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.

RESULTSA 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).

CONCLUSIONA 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.