Determination of complement activating circulating immune complexes and an example for antigen specific immune complex assays.

Abstract

For the screening of complement activating circulating immune complexes a new fluorescence linked immunosorbent assay was developed. Anti-human C3 or anti-human C4 F(ab')2 fragments were coupled to a nitrocellulose matrix. Nitrocellulose pieces of definite size covered with anti-C3 and anti-C4 were incubated with serum samples or standards for 15 min followed by a reaction with a fluorescence labelled anti-human IgG or anti-human-IgM. The nitrocellulose disks were then washed and the remaining fluorescence was read by a solid-phase fluorometer. The method was standardized by a WHO Tetanus toxoid anti-Tetanus immune complex reference substandard. (This standard and further controls were used to prove the reproducibility of the system.) Patients suffering from chronic inflammatory diseases were compared to healthy controls. The best discrimination between patients and controls was demonstrated by the determination of C3-IgG aggregates, followed by C3-IgM, C4-IgG, and C4-IgM aggregates. The easy performance, the stability of necessary chemical substances, the reliable standardization by a reference standard, and the clear-cut differences between patients and healthy control groups proved this test to be suitable for routine purposes. Furthermore, it could be demonstrated by means of an artificial model immune complex that this system can be expanded--by slight modifications--to antigen-specific CIC-assays. A CEA-anti-CEA CIC test is described as an example of an antigen specific CIC test not limited to complement activating CIC, and the data of 127 patients with colorectal carcinoma are given.