A new quantitative nitroblue tetrazolium reduction assay based on kinetic colorimetry

A new method for the quantitative assay of nitroblue tetrazolium (NBT) in which the reduction is measured by kinetic colorimetric analysis is reported. The assay is conducted along standard conditions as far as neutrophil isolation and stimulation, except that the test is performed on microtiter plates and the change of color corresponding to NBT reduction is monitored on a kinetic enzyme immunoassay (EIA) reader for 25 min at 490 nm. The results are expressed as mOD/min/in. The influence of several parameters on the results of the assay was studied, including cell concentration, the nature and concentration of the stimulus, and the freshness of the reagents. Cell concentrations of 5 x 10(6) and 1 x 10(7)/ml were found to be optimal, and IgG-coated immunobeads, at a concentration of 1 mg/ml, were found to be the ideal stimuli. NBT reduction for nine normal volunteers studied at 5 x 10(6) cells/ml ranged from 1.80 to 7.30 mOD/min (mean +/- SD = 3.66 +/- 1.69). NBT reduction values at 1 x 10(7) cells/ml in six normal individuals ranged from 2.59 to 7.41 (4.73 +/- 1.89). In contrast, NBT reduction in a child with clinical symptoms suggestive of chronic granulomatous disease was 0.31 mOD/min. This method is considerably simpler than any alternative method for the performance of quantitative NBT assays.