Immune Complex Assays

The ultimate validity of an immune complex assay depends on how it performs with clinical material, and there is a great deal of controversy regarding the relative merits of some immune complex assays. Some diseases may be associated with certain types of complexes which are detected by some tests but not by others. Since the tests differ in their complement dependence or independence and in the class or subclass of Ig that they detect, many of the discrepancies between assays can be directly attributed to this—a qualitative restraint imposed by the specificity and receptor principle used in a particular assay. Moreover, even with assays of similar principle, discordant results are not uncommon. Much of this variation is due to the performance characteristics of the assays themselves with respect to reproducibility, sensitivity, specificity, the size, quantity, and conformation of immune complexes detected, and the role of interfering substances. Per- 

In the WHO study, there were considerable differences between the tests in reproducibility, especially between-run variation. With the exception of the Raji assay, the bioassays which require viable, intact cells were much less reproducible than assays that use more stable and uniform reagents. Even the latter tests do not approach the precision one would demand from, say, a clinical chemistry test. 

The results with fractionated aggregates are also illustrated and confirmed for true immune complexes in Fig. 4. Declining assay readouts were obtained with increasing antigen excess, i.e., progressively smaller immune complexes. This figure also underscores why there is no truly relevant quan- 

Correlation and Concordance between IC Assays Tested on Patient Sera  

Two IC assays compared Receptor principle similar or different6 Correlation coefficient Concordance in results 7 Number of sera tested p value 

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Self, C. H., Dessi, J. L., and Winger, L. A., High-performance assays of small molecules Enhanced sensitivity, rapidity, and convenience demonstrated with a noncompetitive immunometric anti-immune complex assay system for digoxin. Clin. Chem. 40, 2035-2041 (1994). 

The preceding construct is a very useful concept for approaching immune complex assays. It allows a physicochemical appreciation of the manipulations by which immune complexes are preferentially detected. More importantly, it predicts that nonspecifically aggregated immunoglobulin may be detected by these assays and helps in understanding differences between the tests in sensitivity, specificity, types of complexes detected, and individual idiosyncrasies. 

In spite of the strong evidence for the role of DNA/anti-DNA complexes in causing the kidney lesions of systemic lupus (A6, K13, K15, W19), it is not certain that the material assayed in serum as immune complexes is actually composed of DNA linked to specific antibody. Several groups have demonstrated DNA (D2) or anti-DNA (H10) in immune complex material from sera, but careful studies by others have failed to repeat these findings (A7, H27, 18). DNA, by itself, does not persist in the circulation (G10, 17). Because of the multiplicity of autoantibodies found in lupus, any of a number of antigen-antibody systems may be involved (T4). Based on direct examination and the reactivity patterns with various immune complex assays, the immune complex material found in systemic lupus tends to be macromole-cular (>19 S) and complement fixing (A5, A7, C5, D2, El, F12, Gl, L2, M10, N6, T15). 

A3. Agnello, V., Immune complex assays in rheumatic diseases. Hum. Pathol. 14, 343-349 (1983). 

The feline CSF-1 receptor is a protein of apparent molecular weight of 165000 which can be pliosphorylated on tyrosine residues in an immune complex assay. Recently, the murine CSF-1 receptor has been purified and shown to possess intrinsic tyrosine kinase activity [31]. The receptor is expressed at relatively high lev-... 

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. 

Types II and III Hypersensitivity. No simple animal models are currently available to assess Type II (antibody-mediated cytotoxicity) hypersensitivity reactions. IgE antibodies and immune complexes in the sera of exposed animals can be assayed using ELISA or RIA techniques that require the use of specific antibodies to the drug. 

Assay Immune Complex Solid phase Solution phase... 

Figure 19.1 Dose-dependent response curve for thyroxine (T4) assay using E5-Ab and double-antibody immune complex. Solutions (20 juL) of the reference samples containing 0.0 to 25.0 /ig/dL T4 were mixed with 180 fiL of the releasing solution. Fractions (132 juL) of this mixture were combined with 38 //Lof an E5-Ab solution. After standard incubation, 76 A. of this mixture was spotted on a blank Stratus tab. Incubation times and reagent volumes used for the rest of the reagents, such as the conjugate and the substrate wash, were identical to those defined for an existing T4 assay on Stratus ...

In nearly all cases studied, the amount of primary antibody required in the E5-Ab complex to perform an assay has been found to be substantially less than that required for the double antibody immune complex format. This was found to be the case (Table 19.1) when the E5-Ab complex was either directly immobilized on the solid phase, to imitate the double-antibody immune complex format, or utilized in a solution phase format [12],... 

The general method utilized to prepare E5-Ab solutions obviates the need for stocking large numbers of reagents which would be necessary if different activation methods were used for each antibody. A number of specific antibodies immobilized by this process have shown response similar to that of the same antibodies when adsorbed as immune complexes in the Stratus system. In addition, the dendrimer-coupled antibodies have shown dramatic improvements in sensitivity, flexibility and precision for the enzyme immunoassay system. Feasibility demonstration of an assay for DNA probes is a prelude to what can possibly be achieved with these dendrimer-based reagents. 

Immune complexes are solubilised in 50 xl of 2X Laem mli buffer, boiled and subjected to SDS-polyacrylamide gel electrophoresis or washed once with the enzyme assay buffer and used for enzymatic assays. 

Immune complexes have been conjugated to 2, 7 -dichlorofluorescin and utilized as the phagocyte stimulus for a flow cytometric assay of oxidative metabolism (10). The reagents and a detailed protocol for this assay are available as the Fc-OxyBURST Kit from Molecular Probes. 

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.

T4. Tanaka, K., Kohno, T, Hashida, S., and Ishikawa, E., Novel and sensitive noncompetitive (two-site) enzyme immunoassay for h tens with amino groups. J. Clin. Lab. Anal. 4,208—212(1990). T5. Towbin, H., Motz, J., Oroszlan, R, and Zingel, O., Sandwich immunoassay for the hapten angiotensin II. A novel assay principle based on antibodies against immune complexes. J. Immunol. Methods 181, 167-176 (1995). 

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