FIG. 5.
Reactivities of MAbs used for measurement of C3 fragment deposition on RBCs. MAbs f1-7 and f1-23, raised for detection of C3 fragments, were coupled to CNBr-activated Sepharose columns and used for purification of C3 from cobra venom factor-activated normal serum. The eluted proteins were analyzed by SDS-PAGE (under reducing conditions) and Coomassie staining (left) or by immunoblotting with polyclonal anti-C3 antibodies as detection antibodies (right). Staining of the eluates from both columns by either method revealed bands with apparent molecular masses of approximately 75 kDa, 40 kDa, and 25 kDa, corresponding to the β-chain and the α2 and α1 portions of C3. Blotting of the f1-7 eluate also revealed an iC3b-specific α1-C3d portion (∼63 kDa), and the absence of a band corresponding to the intact α-chain (110 kDa) shows that f1-7 does not react with C3 or C3b. Conversely, the eluted fractions from the MAb f1-23 immunoaffinity purification produced a band of 110 kDa, corresponding to an intact α-chain, which suggests that this antibody also recognizes C3b.