Evaluation and improvement of a rapid microassay for measuring superoxide anion production by phagocytes. 1. Spectrophotometric aspects.

Phagocyte synthesis of superoxide anion can be reliably measured as superoxide dismutase-inhibitable reduction of ferricytochrome C (Cytc). The original assay involved separation of the Cytc solution from the cells before estimating the extent of reduction spectrophotometrically. More recently, a rapid microplate assay has been devised, in which the cells are left in situ and reduction is measured on a multi-channel photometer. To date, no direct comparison of the two methods has been reported. The present study, which compares superoxide anion production by human neutrophils in both assays, indicates that different results may be obtained by the two methods and that an important contributory factor to the discrepancy may be the types of photometry employed. The lower values observed in the microplate assay were attributed to the reduced sensitivity of the multi-channel plate reader in detecting the absorbance of reduced Cytc, which in turn could be ascribed to the bandwidth of the interference filter employed in wavelength selection. The association between filter bandwidth and sensitivity is analysed and methods of correction are proposed.