A semiautomated hemolysis microassay for membrane lytic proteins

Abstract

A simple, rapid, semiautomated microassay for hemolysis using a microtiter plate spectrophotometric system is described. The assay relies on the differences in light scattering (turbidity) properties of intact and of lysed erythrocytes. Lysis of erythrocyte suspensions in 96-well plates is determined by absorbance at 690 nm. A linear correlation between the percentage of hemolysis and the turbidity decrease is observed, indicating that this assay may be used for both rapid screening and quantitation of the hemolytic activity. This assay allows screening of 300 samples in less than 6 min. Small samples derived from protein fractionation columns (HPLC, for example) can be rapidly screened. This assay has been used in the successful isolation of a cytolytic membrane-lytic protein from the granules of cloned cytotoxic T lymphocytes and NK cells.