Prototype reagents for an automated serum inducible nitric oxide synthase assay

Introduction

Recent reports indicate that inducible nitric oxide synthase (iNOS) can be detected in the serum of sepsis patients. In order to study the potential clinical utility of iNOS, both an analytical mass standard and an automated, specific immunoassay have been developed. A subcloned cell-line derived from DLD-1 cells has been developed that can be induced to express high levels of human iNOS. A prototype Access^®-based immunoassay has also been developed.

Methods

DLD-1-5B2 mammalian cells were grown in tissue culture using DMEM + 10% FCIII media. Cells were grown to confluency and changed to induction media containing DMEM plus interferon gamma, IL-1β and tumor necrosis factor alpha. Following induction, cells were incubated in 5% CO₂ at 37°C for 18–30 hours. INOS expression was evaluated with the prototype immunoassay consisting of dual iNOS-specific monoclonal antibodies and by production of nitric oxide (NO) as determined by nitrite detection using the Griess assay. iNOS was extracted from induced DLD-1 cells by freeze–thaw disruption.

Results

Production of NO was not detected by the Griess assay until 10 hours after induction. The production of NO increased proportionally from 2.6 μM NO at 10 hours to 18.7 μM at 30 hours. Using the prototype immunoassay, iNOS was detected in cell extracts at 2 hours after induction at a concentration of 2.4 ng per 10⁶ cells. Levels increased steadily until reaching a plateau of approximately 150 ng per 106 cells from 18 to 30 hours. While the iNOS immunoassay was much more sensitive in detecting the early stages of iNOS induction than the measurement of NO production, the increase in iNOS expression was proportional to NO production over the first 18 hours post induction, once NO became measurable.

Summary

DLD-1-5B2 cells can provide a stable source of human iNOS. NO production is proportional to mass of iNOS produced up to 18 hours in confluent, induced cells. Nominal values of iNOS detected by immunoassay were based upon calibration with commercial murine iNOS standards. Additional work is required to optimize the prototype for clinical use. Purification, characterization, and analytical mass assignment of iNOS from DLD-1-5B2 cells are in progress to provide mass standardization for the iNOS immunoassay.