Reinvestigations into the formation and assay of C3bBbP complexes.
C3bBbP complex formation was studied by an enzyme linked immunosorbent assay (ELISA). Microtitre plates were coated with anti-P to trap the complexes and peroxidase labelled anti-C3 was used to detect them with the help of substrates of peroxidase. Incubation of normal serum pool (NSP) at 37 degrees C in the presence of high concentrations (greater than or equal to 0.5 mmol/l) of Mg2+, usually used in alternative pathway (AP) assay systems, caused the generation of C3bBbP complexes. This generation was not observed when NSP was incubated in the presence of low Mg2+ concentration (less than or equal to 0.2 mmol/l) or EDTA. The concentration of Mg2+ required for maximum complex formation was 2.0 mmol/l under the experimental conditions. Complexes could not be generated in B-depleted serum. Incubation of NSP with endotoxin or CoVF in the presence of 0.2 mmol/l Mg2+ caused the generation of the complexes. The generation was influenced by ionic strength in the incubation mixture. Endotoxin and Mg2+-dependent generation of complexes could not be detected when peroxidase-labelled anti-B was used instead of peroxidase-labelled anti-C3. Serum incubated with 0.2 mmol/l Mg2+ or EDTA apparently detected in vivo formed complexes whereas that incubated with 0.2 mmol/l Mg2+ and endotoxin reflected the complex forming capacity of the serum. The serum of a patient with Raynaud's phenomenon having 45% of normal AP activity did not show increased amounts of preformed complexes but had the ability to generate the complexes to a level of about 45% of that attainable by NSP. These observations suggest that the ELISA used here has the potential of detecting activation as well as the integrity of the AP under carefully controlled conditions.
D003176:Complement C3 / Q000032:analysis; D003179:Complement C3b / Q000096:biosynthesis*; D015933:Complement C3d; D004797:Enzyme-Linked Immunosorbent Assay; D006801:Humans; D007202:Indicators and Reagents
D003176:Complement C3; D007202:Indicators and Reagents; D003179:Complement C3b; D015933:Complement C3d