The quantitation of alternative pathway complement function by timed lysis assay.

Abstract

A simple timed lysis assay is described for quantifying haemolytic complement activity in human serum. Classical pathway complement function was determined by measuring the time taken to lyse 50% of a standard suspension of antibody-coated sheep erythrocytes; the time required for 50% lysis of a standard rabbit erythrocyte suspension was similarly used to evaluate alternative pathway function. Because target erythrocytes prepared on different days gave slightly different 50% lysis times, it was necessary first to construct a series of calibration curves for converting 50% lysis times into CH50 U/ml. For this purpose, a range of dilutions of the standard human serum, of known haemolytic activity, was tested against erythrocytes prepared on 10 separate occasions. The standard serum was subsequently included with each batch of unknown sera and used to select the appropriate calibration curve for direct conversion of the 50% lysis time into CH50 U/ml. Eleven samples of normal human serum were tested by both the timed lysis assay and by the dilution methods of Mayer (1971) (classical) and Platts-Mills and Ishizaka (1974) (alternative pathway). Comparable results were obtained in all cases.