DETECTION OF COMPLEMENT ACTIVATION BY COUNTERIMMUNOELECTROPHORESIS (CIE)

Counterimmunoelectrophoresis (CIE) was used to detect activation of the 3rd component of the complement system (C3). Highly purified C3, normal human serum (NHS), EDTA-treated plasma and serum activated with aggregated human immunoglobulin (agg-IgG) or inulin were used as sources of C3 and/or C3 split products. Activation of the alternative pathway of complement was assayed in the presence of EGTA [ethylene glycol bis (.beta.-aminoethyl ether) N,N,N'',N'' tetraacetic acid] (10 mM) and MgCl2 (0.3 mM), conditions which block activation of the classical pathway. When purified native C3, fresh NHS and fresh EDTA-plasma were tested in CIE against antisera to whole C3 or to C3 split products, only 1 precipitin line was found, which was identified as native C3. When serum activated with agg-IgG or inulin were tested against the same reagents, 2 precipitin lines were seen. The 1st, with more cathodal mobility was identical to that of native C3. The 2nd line had a more anodal mobility, was distinctly separated from the 1st and contained C3c and C3d, as shown immunochemically with specific antisera. Native C3 and split products of C3 were identified by this CIE method in patients showing evidence of activated complement by having subnormal total complement (CH50) levels. When C3 split products were identified, the C3c-C3d precipitin line could always be distinguished from native C3 by its different electrophoretic mobility, even when C3 concentrations in serum varied from 0.25 mg/ml-1.5 mg/ml. The sensitivity of CIE was compared to that of CH50 by assaying at different time intervals after agg-IgG was added to fresh NHS. C3c-C3d split products were detected by CIE before any fall in CH50 and at all times when a significant decrease in CH50 was present. This study shows that the CIE technique is a highly sensitive, specific and rapid method for detecting activation of the complement system via classical or alternative pathways in human disease.