Simplified assays of hemolytic activity of the classical and alternative complement pathways

Simplified hemolytic assays for the classical (CP) and alternative (AP) pathways of complement (C) were developed. The CP function was tested with sensitized sheep erythrocytes in a diluent containing Ca2+ and Mg2+, while AP was tested with unsensitized rabbit erythrocytes in a diluent containing Mg2+-EGTA. In contrast to the commonly used hemolytic titration (CH50) assays, the present techniques tested the activity in reaction mixtures containing C at final dilutions which would not affect its function. These ranges fell between 1/1 and approximately 1/20 for CP and between 1/1 and approximately 1/3 for AP. With the adopted assay techniques single aliquots of serum were tested at single final dilutions of 1/8 for CP and 1/2 for AP, in the presence of excess target cells. Hemolysis was allowed to take place at 37 degrees C for 20 min. The number of cells lysed by CP and AP under these conditions was directly proportional to the dose of serum and unaffected by the presence of a large excess of target cells. Each pathway was tested independently of the other. Serum C levels, measured as described, correlated strongly with those determined by standard hemolytic titration (CH50) assays. The modified assays should offer less laborious alternatives for the functional assay of C than current routine procedures.