1. Field of the invention:
This invention relates to methods of analyzing for circulating immune complexes in mammalian blood serum and more particularly to methods of analyzing for circulating immune complexes using an enzyme-linked immunoassay.
2. Description of the prior art:
Macromolecular circulating immune complexes (CIC) are formed in the blood serum of mammals by the interaction of endogenous or exogenous antigens and specific antibodies formed against these antigens by the host. Typical exogenous antigens which provoke the formation of antibodies include antigens asociated with infectious agents such as viruses, bacteria, parasites and fungi, as well as drugs, foods, and allergens. Typical endogenous antigens include cellular antigens such as ribosomes, nucleic acid, nucleoproteins, cell surface antigens, mitochondria and the like, and plasma factors such as rheumatoid factor, thyroglobulin, and tumor-associated antigens (e.g., carcinoembryonic antigen) and the like. These macromolecular complexes are removed from circulation under normal circumstances by a complement-dependent phagocytotic process or by deposition in specialized vasculature of kidneys, arteries, skin, lungs, joints and the choroid plexus. The glomerulus of the kidney is very prominent in removal of CIC from the blood. Often CIC can be demonstrated in normal healthy individuals immediately after eating or in normal as well as preeclamptic pregnancies.
The processing of immune complexes by the host can also result in immunopathology such as acute and chronic glomerulonephritis, vasculitis, pancreatitis, hepatitis, serum sickness, and the like, in both man and other mammals. Hence, it is desirable to have a method for determining the concentration of CIC in humans and other animals.
A number of methods of analyzing for CIC have been developed. Physical methods such as analytical ultracentrifugation, gel filtration, sucrose density gradient centrifugation, ultrafiltration, electrophoresis and nephelometry have been used. Chemical methods such as precipitation with polyethylene glycol and cryoprecipitation have also been used. Cellular methods depending on the interaction of CIC with Fc-receptors (FcR) in the cell membrane, such as platelet aggregation, Raji cell assay, rosette inhibition, release of soluble enzymes by eosinophils and basophils, and the bull sperm agglutination assay, have been used. Immunologic detection methods for CIC include competitive assays, complement dependent assays, antiglobulin methods and binding to staphylococcal protein-A. A review of prior assay methods for CIC is found in Ritzmann, S. E., and Daniels, J. C., Clinical Chemistry 28(6), 1259-1271 (1982).
The very existence of many methods of assaying CIC is an indication that a completely satisfactory method is not yet available. Problems involved in collecting, processing and storing the sera to be assayed, the stability of standards, ease of performance of the particular assay, and reproducibility of results are all factors to be considered in evaluating the suitability of a particular assay. A collaborative study conducted by the World Health Organization (WHO) has substantiated the importance of these problems and concluded that no single test is a valid CIC assay (Lambert, P. H., et al., J. Clin. Lab. Immunol. 1, 1-15 (1978).
A number of assays have been devised which depend on the precipitation of the CIC by polyethylene glycol (PEG), followed by reaction with staphylococcal protein-A (SPA).
Hallgren, R., et al., Ann. rheum. Dis. 35, 306-313 (1976) disclose assaying CIC by first separating CIC from the other proteins in the serum by exclusion chromatography on a polysaccharide gel and then precipitating with radiolabeled staphylococcal protein-A in the presence of polyethylene glycol. The excess reagent is separated and the radioactivity of the precipitated CIC is measured by counting in the usual way.
Stevens, W., et al., Immunology Letters 3, 1-4 (1981) disclose an assay for CIC wherein the complexes are first selectively precipitated by 5% polyethylene glycol and separated from the non-aggregated immunoglobulins. The CIC are then resuspended and reprecipitated with radiolabeled SPA, and quantitated in the usual way by counting.
Dobre, M., et al., Rev. roum. Biochim. 19 (2), 115-120) (1982) disclose an assay wherein CIC are first selectively precipitated with polyethylene glycol, then reprecipitated on Staphylococcus aureus. The precipitated CIC are then incubated with radiolabeled SPA, the excess reagent is removed, and the CIC are quantitated by counting.
Faiferman, I., et al., Arthritis and Rheumatism 25 (7), 799-801 (1982) disclose a fluoroimmunoassay for CIC wherein the complexes are first selectively precipitated with a 2.5% solution of polyethylene glycol, then resuspended and contacted with a solution of SPA linked to a fluorescent label, whereby a complex of CIC and fluorescent labeled SPA is formed. The complex is again precipitated with polyethylene glycol, the excess reagent is removed, and the fluorescence of the bound reagent is measured with a spectrofluorometer. Faiferman et al. disclose that the label used on the SPA could also be an enzyme label.
In all of these assays for CIC a preliminary separation of the CIC from the other proteins in the serum, particularly from the immunoglobulins, which also bind to SPA, is performed. Therefore, these assays involve at least two precipitation steps.
Hence a need has continued to exist for a simpler and more rapid assay for CIC, based on the binding of CIC to SPA, which avoids the disadvantages of the known assays, and in particular for an assay for CIC which does not require a preliminary separation of CIC from other serum proteins.