Detecting Immune Complexes

Tests Based on Physical Properties of Immune Complexes 

To perform the test it is best to allow a freshly drawn blood specimen to clot at 37°C. The serum is then held at 0-4°C for 48-72 hours (a Wintrobe tube is convenient). After centrifugation the volume of cryoprecipitate is measured (cryocrit). After washing the cryoprecipitate in the cold, it is dis- 

Tests Based on Reactivity with Serologic Receptors for Immunoglobulin 

With all the assays based on Clq binding, it is important to remember that the types of complexes detected are restricted by the specificity of Clq for IgM and the IgGl, IgC2, and IgC3 subclasses. The tests may also be affected by the Clq concentration in the test sample (Gl, H16, L21, M10, R8, Z3). More importantly, a number of nonimmunoglobulin substances that are Clq reactive may occur in sera and interfere with the results. Polyanions such as DNA, heparin, and bacterial endotoxins bind Clq (A7, Gl, L2, L21, S28, S39, T5, W26, Z3). C-Reactive protein reacts with Clq through a calcium-dependent bond, a problem usually circum- 

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Brorson SH, Andersen T, Haug S, Kristiansen I, Risstubben A, Tchou H, Ulstein J (1999) Antigen retrieval on epoxy sections based on tissue infiltration with a moderately increased amount of acceleratorto detect immune complex deposits in glomerular tissue. Histol Histopath 14 151 155... 

In the following review, we will discuss in some detail the principles and performance of the most commonly used tests for detecting immune complexes and summarize studies in those diseases for which considerable clinical experience with these tests has accumulated. Emphasis is on the clinical relevance of this technology. We consider a test clinically relevant if... 

Antigen-Nonspecific Methods fob Detecting Immune Complexes... 

Fig. 6. Immune complexes in sera from patients with disseminated gonococcal (GC) infection. Immune complexes were run in the staphylococci binding assay. Isotype-specific reagents and an anti-GC reagent were used to detect immune complex material. Standard deviations were determined on a panel of 27 normal sera.

T15. Theofilopoulos, A. N., Wilson, C. B., and Dixon, F. J., The Raji cell radioimmune assay for detecting immune complexes in human sera. J. Clin. Invest. 57, 169-182 (1976). 

A cascade of proteins of the immune response that can be triggered by antigen-antibody complexes and by the innate immune system (e.g. exposure to microbial polysaccharides) to raise the immune response. Complement proteins can detect and bind to foreign material or immune complexes and label them for phagocytosis. They can also cause inflammation by directly degranulating mast cells and releasing chemokines to recruit other immune cells into the affected area. 

Antibodies to very low density lipoprotein (VLDL) and LDL have been detected in the serum of patients with RA, but not control groups (Lazarevic et al., 1993). In these studies, 38% of patients with active RA tested positive for anti-VLDL/LDL antibodies whilst these autoantibodies were not detected in patients with psoriatic arthritis, osteoarthritis or healthy subjects. Lipoproteins were found in the dissociated components of circulating immune complexes in the serum of 30% of the RA patients. It was concluded that dyslipoproteinaemia in some RA patients may be due to an autoimmune component of the disease. 

Immunoassays are based on the ability of the immune system to produce a virtually unlimited variety of antibodies each with a high affinity for foreign compounds (immunogens like viruses, bacteria, proteins, and haptens). Analytically, this phenomenon can be exploited by detection of this immunoreaction using labeled antibodies or antigens (i.e., compounds that can be bound by antibodies). The equilibrium between antibody (Ab), antigen (Ag), and immune complex (Ab-Ag) may be expressed as ... 

In summary, these are the clinically relevant questions about the immunogenicity of rDNA species-specific proteins will antibody be induced in the recipient that will neutralize the therapeutic effect or lead to immune complex disease What is the class (e.g., IgG or IgE) and specificity (i.e., reactivity against specific protein or contaminant) of the antibody induced The former antibody type could potentially neutralize the product and produce immune complex disease, while the latter could result in an anaphylaxis response. It is possible that the antibody induced is of insignificant health consequence, and its presence is known only because of improvements made in the sensitivity of detection methods with the introduction of the enzyme-linked immunosorbent (ELISA) assay. 

Type III (immune complex related disease) reactions have been demonstrated by the presence of proteinuria and immune complex deposits in the kidneys of the Brown-Norway, Lewis, and PVG/C rat strains. However, susceptibility to the deposition and the subsequent lesions (glomerulamephritis) are often variable and dependent on the strain (Bigazzi, 1985). For example, despite the appearance of clinical signs and proteinuria, after two-months administration of mercuric chloride, detectable levels of circulating antinuclear autoantibodies can no longer be observed in the Brown-Norway strain (Bellon et al., 1982). By contrast, in PVG/C rats administered mercuric chloride, immune complex deposition and antinuclear autoantibodies are present for longer periods of time however, proteinurea is not observed (Weeping et al., 1978). 

Whereas antigen-retrieval technique serves to amplifying the immunocytochemical signal at the predetection phase, conventional methods of signal amplification, such as avidin biotin complex (ABC) and soluble enzyme-anti-enzyme immune complex techniques (peroxidase-anti-peroxidase complex and alkaline phosphatase-anti-alkaline phosphatase complex PAP and APAAP respectively), are applied in the phase of detection. For many years, the PAP and APAAP procedures represented the most sensitive and reliable and hence most popular techniques in many pathology laboratories. However, today these techniques are only rarely used, being substituted by modem more sensitive methods. 

Antigen can be detected in serum several weeks before seroconversion and is usually undetectable during the asymptomatic period of HIV disease. Serum antigen levels decrease as the level of antibody increases and immune complexes are formed. Production of detectable HIV antigen indicates both decreased antibody production and increased expression of viral genes and is an indication of progression of disease to a serious clinical state. 

The separated proteins were transferred to a polyvinylidene difluoride membrane, and nonspecific IgC binding sites were blocked by incubation with 5% nonfat dry milk for 1 h at room temperature. The membranes were then incubated overnight at 4°C with primary antibodies. Immune complexes were detected by enhanced chemiluminescence (Amersham Biosciences). 

GM Korf, JP Landers, DJ O Kane. Capillary electrophoresis with laser-induced fluorescence detection for the analysis of free and immune-complexed green fluorescent protein. Anal Biochem 251 210-218, 1997. 

Fig. 8. Morphological changes of apoptotic eosinophils induced by dexamethasone (Z2). After eosinophils were treated (a) without or (b) with dexamethasone (2 /u,M) for 12 h, cells were harvested and detected by TUNEL assay using the In Situ Cell Death Detection Kit (Boehringer Mannheim). Briefly, cells were fixed with 4% paraformaldehyde and permeabilized by proteinase K and incubated with the TUNEL reaction mixture containing terminal deoxynucleotidyl transferase (TdT). After washing to remove unbound enzyme conjugated antibody, the horseradish peroxidase retained in the immune complex was visualized by a substrate reaction with diaminobenzidine. The cell nucleus was counterstained with methanol green. Apoptotic eosinophils with nuclear DNA breaks were seen to stain dark brown using a Nikon Eclipse E800 microscope (Nikon Corporation, Tokyo, Japan) in Fig. 8b.

Fig. 4. Classification of reported noncompetitive immunoassays for haptens based on the assay principle. (A) Assays that include a chemical modification of hapten to allow sandwich-type detection. (B1) Improved single-antibody immunometric assays that separate immune complex and excess labeled antibody, either by using a hapten-immobilized affinity column or based on differences in their physical properties. (B2) A variation of single-antibody immunometric assays based on masking of unoccupied antibody by an immunoreactive macromolecule followed by selective capture and detection of the hapten-occupied antibody. (C) Assays employing a probe molecule specific to a hapten-antibody complex.

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