Antibody-mediated complement activation on nucleated cells: A quantitative analysis of the individual reaction steps

Markus W. Ollert*, Joseph V. Kadlec, Kerstin David, Eugene C. Petrella, Reinhard Bredehorst, Carl Wilhelm Vogel

*Corresponding author for this work

Research output: Contribution to journalArticleResearchpeer-review

50 Citations (Scopus)


The sequential molecular events of the initiation, amplification, and membrane attack phases of classical C pathway activation on nucleated cells were investigated. As a model system, C-susceptible human melanoma cells (SK- MEL-93-2) expressing the disialoganglioside Ag G(D3) were studied. Activation of the classical C pathway was initiated by the anti-G(D3) mAb R24 (murine IgG3). The initiation phase is characterized by a very inefficient molar ratio of deposited C1q per Ab molecule. At an Ab density of 5.86 x 106 molecules/cell, only 3% of cell-bound R24 molecules form suitable pairs for C1q binding. During the amplification phase maximally 2.44 x 106 molecules of C4 and 0.67 x 106 molecules of C2/cell are being bound to form the C3 convertase. Despite the rather inefficient binding of C2, the C3 convertase is highly active in depositing high numbers of C3b molecules on the cell surface. Maximum binding of C3b occurred within 5 min of incubation with a total number of 2.1 x 107 molecules/cell. This indicates amplification factors at the level of C4 and C3 of 28 (C4/C1q) and 241 (C3/C1q), respectively. C3b was found to be rapidly cleaved into iC3b. As a result of this rapid C3b degradation, the membrane attack phase is initiated with a relatively inefficient C5 activation. The maximal number of 9.5 x 105 molecules C5b/cell corresponds to a molar ratio of C5:C3 of only 1:22. The deposition of C5b led to the subsequent maximum binding of the following numbers of molecules of terminal C components per cell: C6, 0.8 x 106; C7, 0.89 x 106; C8, 0.82 x 106; C9, 1.8 x 106. These numbers correspond to average molar ratios (calculated per C5b molecule) of C5b/C6/C7/C8/C9 of 1/0.85/0.94/0.86/1.88. In addition to the monomeric C9, dimeric and polymeric (12- to 16-mer) forms of the molecule could be demonstrated. Collectively, our data represent a first comprehensive quantitative analysis of classical pathway activation on a nucleated cell.

Original languageEnglish
Pages (from-to)2213-2221
Number of pages9
JournalJournal of Immunology
Issue number5
Publication statusPublished - 1 Sept 1994
Externally publishedYes


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