Detecting immune complexes in bodily fluids and tissues

Detection of immune complexes in clinical samples is of interest in a number of human and animal diseases. Furthermore, in countless test systems, immune complexes are the in vitro reaction product of laboratory diagnostic tests for detection of either specific antibodies or antigens. For demonstration of in vivo formed immune complexes in serum or other body fluids, where the antigen involved is unknown, two approaches may be pursued: 1) quantitative estimation of immune complexes with characterization of the complexed immunoglobulin isotype and bound complement component using conventional screening tests; 2) analysis of the antibody specificity and possibly of complexed antigen using more elaborate technology (Figures 1 and 2).

Rapid screening tests for the detection of immune complexes in a large number of samples are mostly based on the ability of immune complexes to interact with naturally occurring humoral and cell receptors. A classical technique is the Clq-binding assay that is based on the ability of immune complexes to interact with the globular head domains of the first component of human complement, Clq: purified, radiolabeled C1q binds to the immune complexes

PEG precipitable immune complexes

PEG precipitable immune complexes

Impurities

Protein A sorbent

Protein A sorbent

Immune complex remains bound to protein A

Impurities fall through

0 1 M Glycine buffer elufes purified immune complex

Figure 1 Isolation of immune complexes from the remainder of serum proteins. The figure depicts a possible protocol starting with coprecipitation of macromolecular proteins using 2-3% polyethylene glycol which precipitates proteins larger than approx 300 kDa. The precipitate is enriched in immune complexes but is still contaminated with other macromolecular proteins. In order to remove the latter proteins, the precipitate is passed over solid phase staphylococcal protein A which retains immune complexes; after thorough washing, the complexes are eluted from the column with glycine buffer.

0.1 m Glycine buffer elutes purified immune complex

Separation over acidified gel-filtration column

Effluent volume

Figure 2 Analysis of immune complex composition and specificity. The purified immune complexes are now ready for further analysis, e.g. for separation and examination of antigen and/or antibody. An easier procedure for purifying immune complexes is by cryoprecipitation but only a few immune complexes (e.g. a portion of complexes with hepatitis C infection) have the property of precipitating in the cold.

and the resulting Clq-immune complex assemblage is detected using size exclusion with polyethylene glycol (PEG). An even simpler technique mixes serum with low concentrations of PEG known to precipitate oversized macromolecules with subsequent analysis of the pellet. Monoclonal antibodies to Clq insolubilized on microtiter plates may be used to capture Clq in the analytical sample already bound to immune complexes in vivo, with subsequent demonstration of immunoglobulins in the captured material. Other solid-phase immune complex detection assays are possible using conglutinin (K) or rheumatoid factors as capture for immune complex bound complement. It is said that K preferentially binds small complexes and those having a high antigen: antibody ratio. On the other hand, Clq-binding assays identify immune complexes with a lower anti-gen:antibody ratio and a greater size range. In addition, K binds complexes that contain the small residual degradation fragment of C3, C3d, whereas Clq only binds those activating complement by the classical pathway of complement activation.

For detection of immune complexes in more elaborate assays, cellular receptors may be used, such as the Fc receptor (FcR) present on macrophages or platelets and, in the most successful of this type of assay, the Raji cell test, iC3b receptors (complement receptor CR3) present on in vitro transformed lym-phoblastoid cell lines. The erythrocyte-antibody-complement (EAC) rosette inhibition test measures the inhibition by immune complexes of rosette formation between a subpopulation of peripheral B lymphocytes and sensitized sheep erythrocytes carrying C3. The sensitivity of these techniques is generally good and is in the microgram per milliliter range, but the disadvantage is that cell preparations must be used, which makes them tedious and cumbersome for routine use. Standardization is difficult, but a fair comparison of results among different laboratories is noted with use of international reference preparations. Similar technology using cellular receptors may provide insights into the immune complex constituent that actually ligates to cell surface receptors. By blocking specific types of cell surface receptors with monoclonal antibodies, Fc binding can be distinguished from F(ab')2 binding or binding via immune complex fixed C3b and/or C4b.

The availability of preparative techniques providing a partially purified material allows further immunochemical and biochemical characterization of the complexes. Several techniques make the study of molecular composition of immune complexes for clinical research possible: direct electron microscopic examination of precipitates obtained by polyethylene glycol or cold precipitation; dissociation of complexes at acid pH (Figures 1 and 2), by urea or heat; in vitro manipulation of complexes by adding suspected antigen, with a possible shift of the antigen: antibody ratio towards antigen excess so that loss of their complement fixing ability can be ascertained; and absorption of immune complexes on congluti-nin-coated polystyrene beads allowing their further purification and study. Using this type of technology, it is possible to obtain information on the composition of immune complexes in disease (Table 1).

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