Radioactive antiglobulin testing with 125I anti-C4 and anti-C3

The report describes methods for quantitating C4 and C3 coating of red blood cells using 125I anti-C4 and anti-C3. Two methods of preparing antisera were used; one involving absorption and elution from complement coated cells, and the other employing IgG fractions of antisera prepared on DEAE cellulose. White-cell-free red blood cells were used in the test procedure. Both the labeled antisera and the test cells were diluted in cold carrier proteins, initially bovine serum albumin, but more recently normal rabbit serum. We have found that normal rabbit serum is particularly effective in reducing the high uptake of nonspecific counts on normal cells, which had previously been a major technical problem for the radioactive antiglobulin technique. These methods are highly reproducible and relatively easy to perform once the labeled antisera are prepared. Furthermore, the use of labeled antisera permits analysis of specificity by the highly sensitive technique of radioimmunoelectrophoresis. One problem with the technique is that results indicate uptake of labeled antisera, rather than amounts of coating substances. Nonetheless, the ease of performance and the reproducibility of the method, make the procedure very useful in comparative studies of uptake of C4 and C3 by red blood cells.