A simple radial immunohemolysis assay for the measurement of functional complement C2 activity

1. This study describes a simple radial immunohemolysis method for determining the hemolytic activity of the second component of complement (C2) in human serum. The assay is based on the recovery of hemolytic activity of normal serum which has been pretreated to inactivate endogenous C2 and then mixed with test serum containing an unknown amount of C2. 2. The pretreated serum, designated R2 reagent, is obtained by heating normal human sera under carefully standardized conditions of temperature, time, volume and type of test tube. 3. R2 reagent is incorporated into agarose together with hemolysin-sensitized erythrocytes, and spread on a plate. The test serum is placed in wells cut in the agarose and, after appropriate incubation, the diameters of the hemolytic areas are measured. The area of hemolysis is directly proportional to the logarithm of the serum concentration. As a standard for C2 functional activity, dilutions of a pool of normal sera are tested on the same plate. 4. The method is specific for C2 and can detect as little as 20% of the C2 in normal serum (about 6 micrograms C2 protein/ml). The error in reproducibility is about 3% of the mean. In normal serum, the lower confidence limit of the distribution of the C2 values (based on a sample of 80 individuals) corresponded to 70% of undiluted serum. 5. This method is suitable for use in clinical laboratories since it is simple, rapid, quantitative and inexpensive, and does not require special equipment.