Measurement of fragments of the third component of human complement on erythrocytes by a new immunochemical method

This paper describes a new antiglobulin consumption method to quantitate the fragments of the third component of human complement (C3) on red blood cell (RBC) membranes. Zymosan-bound C3, which can be stored frozen at -80 degrees C for years, was used as a standard in these tests. Using anti-C3c antibody, zymosan-bound C3 could be calibrated against soluble converted C3 (beta 1A), but not against soluble, native C3 (beta 1C). Calibration with several commercial serum standards yielded virtually identical values. Approximately 79.8 +/- 28.2 C3d molecules (mean +/- 1 SD, n = 50) were detected on normal, freshly collected RBC by this method, whereas no C3c fragments were noted. EC43, prepared by dilution of blood samples with low ionic strength solution, had between 650 and 3,100 C3 molecules/RBC when measured with anti-C3c and between 1,140 and 6,500 C3 molecules when measured with anti-C3d. These data indicated that part of the C3b molecules on EC43 had cleaved to C3d. EC43 are reported to have up to 200,000 C3 molecules when measured by other techniques. To resolve this discrepancy, EC43 were prepared by dialysis of blood samples against low ionic strength solution. About 97.5% of C3 remained in plasma after dialysis supporting the results of our tests. The new assay is an accurate and sensitive method of quantitating C3 fragments bound to RBC in vivo and in vitro.