Reinvestigations into the formation and assay of C3bBbP complexes

Abstract

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.