An analysis of the fluid phase C1q binding assay. The effect of endogenous C1q on the precipitation and detection of an immune complex model.
We examined the effect of endogenous C1q on the sensitivity of the fluid-phase C1q binding assay (C1qBA) in detecting an immune complex (IC) model, heat-aggregated IgG (HAIgG), at concentrations of 10-10,000 micrograms/ml sample. Results in normal human serum (NHS) or plasma (NHP) were compared with those in heat-inactivated NHS (NHS/56) in which most endogenous C1q was depleted by heat denaturation. Higher HAIgG concentrations were required in NHP and NHS to produce the same 125I-C1q precipitation seen in NHS/56. This decreased sensitivity varied from 70% at low HAIgG concentrations to 0% at high concentrations, as predicted for a large pool of endogenous C1q, in equilibrium with 125I-C1q, but in excess of that which could bind to all but the highest concentrations of IC model. In serum depleted of functional C1q on an immunoadsorbant of HAIgG, the precipitation of radiolabeled HAIgG under C1qBA conditions was concentration dependent and generated a saturation curve, showing that only a fraction of IC are usually precipitated in this assay. HAIgG precipitation was enhanced 1.4-fold in NHS/56 (8 micrograms C1q/ml) and three-fold in NHS (67 micrograms C1q/ml) suggesting that IC size is increased by endogenous C1q. In dual label experiments using 131I-HAIgG, the precipitation of 125I-C1q in NHS/56 was directly proportional to IC model precipitation, but markedly discordant in NHP, showing the measurement of IC in heat-inactivated sera superior to that in native serum. A comparison of the C1q:HAIgG ratio in PEG precipitates with that in samples, indicated that equilibrium was established between C1q and IC model. Thus the precipitation of 125I-C1q in the C1qBA represents (1) the fraction of total C1q bound to IC, and (2) the fraction of IC precipitated by PEG.
Jacobs
R J
RJ
Arthritis and Immunology Program, Oklahoma Medical Research Foundation, Oklahoma City 73104.
Mocharla
R
R
D000936:Antigen-Antibody Complex / Q000032:analysis; D011232:Chemical Precipitation; D003166:Complement Activating Enzymes / Q000378:metabolism*; D003172:Complement C1 / Q000378:metabolism*; D015922:Complement C1q; D003168:Complement Fixation Tests; D006358:Hot Temperature; D006801:Humans; D011485:Protein Binding
D000936:Antigen-Antibody Complex; D003172:Complement C1; D015922:Complement C1q; D003166:Complement Activating Enzymes