IMMUNOLOGY - Roitt I. - Mir 2000

Chapter 25. Hypersensitivity - Type III

DETECTION OF IMMUNE COMPLEXES

Immune complex deposition can be identified using immunofluorescence

Immune complex deposition is most likely to be detected in the affected organ. For this purpose, tissue sections are examined by immunofluorescence to check for the presence of IMMUNOGLOBULINS and Complement. Data on the composition and Nature of the complexes, as well as their localization site within the affected tissue, are crucial for assessing disease severity and prognosis. For instance, in membranous Glomerulonephritis, the presence of large granular IgG deposits in the subepithelial layer of the renal glomeruli indicates a poor prognosis, whereas localization within the mesangium suggests a favorable one. An inflammatory response is not always detectable in all Tissues that bind immune complexes. For example, in systemic lupus erythematosus (SLE), complexes are frequently found in biopsies of not only inflamed Skin but also outwardly normal skin.

Detection of immune complexes in the Blood

In the blood, complexes are not only bound to erythrocytes but also present in a free form within the plasma. Erythrocyte-bound complexes are less likely to cause tissue damage, making the determination of free complex levels of greater clinical interest. When collecting blood samples, certain precautions must be observed because bound complexes are readily released during clot formation under the action of factor I. To prevent this, erythrocytes should be separated from plasma promptly.

To identify large complexes containing IgG Antibodies, investigators frequently use polyethylene glycol (PEG) precipitation of immune complexes followed by the quantification of IgG in the precipitate. This principle underlies one of the commercial systems for immune complex determination (Fig. 25.23).

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Fig. 25.23. Polyethylene glycol (PEG) is added to a serum sample containing IgG complexes and monomeric IgG. At a 2% PEG concentration, only the complexes precipitate, while free antibodies remain in solution. The tubes are then centrifuged, forming a pellet of complexes at the bottom. The supernatant containing free antibodies is discarded. The pellet is washed and resuspended to determine The amount of IgG present within the complexes (e.g., using single radial immunodiffusion, nephelometry, or radioimmunoassay).

Circulating complexes are also frequently quantified based on their affinity for the C1q complement component, utilizing either radiolabeled or solid-phase (bound to a solid support) C1q (Fig. 25.24).

Fig. 25.24. Radioimmunoassay for the detection of immune complexes using C1q.

1. C1q is bound to an inert solid phase (support), typically a polystyrene tube or plate.

2. Complex-containing serum is added. The complexes bind to the solid-phase C1q via their Fc regions, which remain accessible for interaction with C1q.

3. Radiolabeled anti-IgG antibodies are added. Following washing, the radioactivity of the solid phase is measured using a gamma counter to calculate the amount of C1q-bound complexes.

Other receptors can also be employed to bind and quantify immune complexes, such as the C3 receptor of RAJI Cells (a B-Cell line tumor) or the platelet Fc receptor.

However, the detection of complexes in autoimmune diseases requires great caution. Such patients may harbor autoantibodies directed against Components of the test system itself. In SLE, for instance, anti-lymphocyte and anti-DNA antibodies are produced that bind to RAJI cells, leading to false-positive results in immune complex assays. Similarly, autoantibodies against C1q (whose Structure resembles Collagen) have been found in various Connective Tissue diseases, creating the potential for false-positive results when C1q-based assay systems are used.

Regardless of the method chosen, it is essential to verify that the detected compounds indeed possess a higher molecular weight than monomeric IgG. Finally, it should be noted that evaluating The Role of circulating complexes requires even greater caution than assessing The Significance of tissue deposits. Many circulating complexes are entirely harmless on their own; tissue damage only occurs when they become deposited in tissues.

Questions for thought

■ What factors may contribute to the persistence of immune complexes?

■ Why do immune complexes deposit in certain Organs and not others?

■ What novel therapeutic interventions targeting The Immune System could aid in treating immune complex diseases?

■ Is the complement activation triggered by immune complexes beneficial or harmful to the Organism?

■ How does the antibody isotype influence The Fate of immune complexes?

■ What difficulties arise in the laboratory determination of immune complexes?

Introduction/47.html">Further Reading

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