Quantitative Measurement of Endothelial Nitric Oxide Synthase in Rat Brain Tissue Homogenates Using an Enzyme‐Linked Immunosorbent Assay Kit

Abstract

A reliable method of endothelial nitric oxide synthase (eNOS) protein measurement in brain tissue samples would enable the direct study of its regulation and expression. Current eNOS ELISA methods are designed for cell lysates (Aberle et al, Nitric Oxide, 1997, 1: 226). We investigated a commercially available human eNOS ELISA kit (R&D Systems, Minneapolis, MN) for use with rat brain tissue homogenates.Brain tissue homogenate samples from three normal male Sprague‐Dawley rats were tested for dilutional linearity and spike‐and‐recovery characteristics. Assayed in quadruplicate, a measurable amount of endogenous eNOS was detected in all unadulterated samples (40.0±1.6, 28.2±0.9, and 50.0±1.9 pg/mg wet tissue). All samples were fractionally diluted 9/10, 4/5, 7/10, 3/5, 1/2, and 2/5, with the kit calibrator diluent and displayed good dilutional linearity (linear regression correlation coefficients of 1.0, 0.97, and 0.98). Low spike‐and‐recovery rates (less than 37.5%) were obtained with neat samples; however, prediluting the sample 1/20 before spiking resulted in a 70.3% recovery suggesting eNOS detection is affected by matrix differences between the kit standard and these samples.With the addition of a correction of recovery, these results suggest the potential usage of this eNOS ELISA kit with rat brain tissue samples.Support: Pennsylvania Department of Health (Tobacco Settlement Fund)