A C1-inhibitor-complex assay (INCA): a method to detect C1 activation in vitro and in vivo

Abstract

A radioimmunoassay (the C1-inhibitor-complex assay, INCA) is described for the detection of complexes that are composed of at least C1s and C1-inhibitor. This INCA is based on demonstrating that C1s and C1-inhibitor (C1-In) are linked: after an incubation with anti-C1s-Sepharose, bound C1sC1-In complexes are detected by 125I-anti-C1-In. C1sC1-In complexes were prepared by the addition of a slight excess of C1s to normal human serum (NHS). As little as 2 ng C1-In bound to C1s was detected. Additional free C1s in serum hardly influenced the detection of C1sC1-In complexes. Complexes presumably composed of C1rC1s(C1-In)2 were generated by the addition of aggregated IgG to NHS. This generation was inhibited by lowering the temperature to 0 degrees C, and by EDTA, and depended on the concentration of aggregated IgG. These complexes had a sedimentation value of approximately 9S. Complexes of C1s and C1-In were also generated in NHS by the addition of DNP-albumin and protein A, but not by zymosan. The INCA was applied to blood samples from normal donors and patients. Sixteen out of 19 samples from patients with acute glomerulonephritis contained increased amounts of C1rC1s(C1-In)2 complexes as compared with the amounts in blood samples from normal donors. The INCA provides a useful tool to assess the activation of C1 in the presence of C1-In, both in vitro and in vivo.