Analysis of c3 deposition and degradation on bacterial surfaces after opsonization

C3b and iC3b, opsonic fragments of C3, interact with specific receptors on phagocytic cells. After bacterial opsonization, C3 fragments were analyzed by sodium dodecyl sul-fate-polyacrylamide gel electrophoresis, western blotting, and immunodetection. For bacteria opsonized in 50% pooled human serum (PHS), C3 deposition and cleavage to iC3b occurred rapidly. C3b, iC3b, and C3d made up 17%, 64%, and 19%, respectively, of the C3 on Staphylococcus aureus and 53%, 44%, and 2%, respectively, on Escherichia coli. Residual C3b was refractory to factor I cleavage, an occurrence enabling alternative pathway activation to continue. C3 deposited was quantitated by enzyme-linked immunosorbent assay; with 50% PHS, >50% and 90% of total C3 deposition occurred within 5 and 10 min, respectively. With a lower percentage of PHS, maximal deposition required up to 60 min and was not achieved in <10% PHS. Ester-bound fragments represented 34% and 82% of covalently bound C3 on S. aureus and E. coli, respectively.