A simple radial immunohemolysis assay for the measurement of functional complement C2 activity / O ensaio simples de imunohemolise radial para medir da atividade funcional do complemento 2

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 anactivate endogenous C2 and thenmixed with test serum containing an unknown amount of C2. 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. R2 reagent is incorporated into agarose together with hemolysin-sensitized erythrocytes, and spread om a plate. The test serum is placed in wells cut in the agarose and, after appropriate incubation, the diameters of the hemolytic areas are measuremed.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. The method is specific for C2 and can deted as little as 20% of the C2 in normal serum (abouth 6 *g 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 undiluited serum. This method is sultable for use in clinical laboratories since it is simple, rapid quantitative ans inexpensive, and does not require special equipement

Critical study of the effect of temperature on total serum complement determination

Basic rules establish that the total serum complement determination (CH50) should be done after quick serum separation at 4 degrees Celsius. When there is a long period between blood sample collection and laboratory tests, the results may not be exact, with findings being below normal level due to thermolability and dysfunction of some of the components. Sera from 15 normal individuals and 15 patients with Systemic Erithematous Lupus (SEL) were studied. CH50 determinations were performed by the radial immunohemolysis technique according to the above basic rules and compared to determinations performed after 4, 8, 12 and 24 h in sera maintained at room temperature and after 24 h in those maintained at 4 degrees Celsius. Five samples (2 controls and 3 lupoid) were stored at -2O degrees Celsius and -7O degrees Celsius. CH50 determinations were performed weekly in sera kept at -2O degrees Celsius and after l month in those stored at -7O degrees Celsius. Analysis of the results suggest that, if blood samples are kept at room temperature and the determination is performed within about 8 h, the results are still reliable, that is, they will be within normal levels. The same will happen if the sera are stored at -2O degrees Celsius or -7O degrees Celsius for up to l month.