Kinetic Assessment of Alternative Complement Pathway Activity in A Hemolytic System

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Abstract
Rabbit erythrocytes (RaRBC) can serve as both the initiator and target of alternative pathway activity. We describe here a turbidimetric assay of RaRBC hemolysis that depends on the observation that, within the limits applied, absorbance at 700 nm of an erythrocyte suspension is directly proportional to cell concentration. Absorbance of multiple samples can be measured at frequent short intervals, which permits kinetic analysis. Sera to be studied are chelated with 0.008 M EGTA in the presence of 0.002 M Mg++, which prevents lysis of antibody-coated sheep erythrocytes through the classical pathway, even by concentrated serum. The time required to lyse one-half the RaRBC (t½) is directly proportional to the reciprocal of the serum dilution (1/S). RaRBC lysis by human serum appears to require factor B in that sera heated to 50°C for 30 min to inactivate this protein lost all activity in the assay, and activity could be restored in a dose-response fashion with purified factor B. Sera from two humans with homozygous C2 deficiency and approximately half-normal serum levels of factor B were mildly deficient in the assay; this abnormality could be removed by the addition of purified factor B but not C2. C4-deficient serum functioned normally. Serum from guinea pigs, mice, and chickens also demonstrated alternative pathway activity by this technique. The relationship between t½ and serum dilution can be expressed by a time-dependent von Krogh equation for complement-mediated lysis, which extends the usefulness of the assay in analyzing rate-limiting components of alternative pathway activation. The maximum rate of hemolysis reported for antibody-coated sheep erythrocytes is about 50-fold greater than that calculated by this equation for RaRBC lysis, in agreement with observations in other systems that biologic activity is generated more slowly through the alternative pathway than through the classical pathway.

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