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Characterization of the soluble immune complexes that are detected by three different techniques
Abstract:
Three different tests which are based on different principles were used for the detection of soluble immune complexes (IC): (i) a PEG precipitation test, which is based on the solubility characteristics of IC; (ii) a solid-phase C1q binding assay, which is based on the complement binding property of IC, and uses peroxidase-linked anti-human IgG to detect the bound IC (C1q-ELISA); and (iii) the indirect granulocyte phagocytosis test (IGPT) which is based on the Fc R- and possibly the C3 R-binding of IC. Using heat-aggregated IgG (A-IgG) as a model for soluble IC all three tests showed a linear relation with the amount of A-IgG. The C1q-ELISA and the IGPT had a detection limit of less than 1 microgram/ml while the PEG test only detected quantities of more than 10 micrograms/ml. However, when using artificially produced soluble IC, which were prepared from human antibodies (ab) of different specificities and their respective antigens (ag) i.e., (i) tetanus toxoid, (ii) Helix pomatia hemocyanin (HPH), and (iii) dsDNA, and which consisted of the two components in a wide range of ag/ab ratios, distinct results were obtained with the three tests. Thus demonstrating that results obtained with A-IgG as a model for soluble IC can not simply be extrapolated to the behavior of real complexes in IC detection assays. No matter which ag was used, the composition of the IC, i.e., the ratio in which ag and ab were present, appeared to be the crucial factor for detectability in the different tests. The C1q-ELISA can detect IC over a wide range of ag/ab ratios, while it is particularly sensitive for IC formed in slight ag excess. The IGPT in contrast primarily detects, and is highly sensitive for, IC formed in ab excess. The PEG test appears to detect IC with freshly bound complement only. Another interesting finding has to be mentioned here: when increasing amounts of dsDNA were added to a SLE serum containing anti-DNA ab, the IC that had been detectable in the native serum with the IGPT completely disappeared, thus demonstrating that these complexes did consist of DNA and anti-DNA.