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Methods for measuring circulating immune complexes
Methods for Measuring Circulating Immune Complexes Stanley S. Levinson, Ph.D. Janice Goldman, Ph.D. Department of Laboratory Medicine Sinai Hospital Detroit, Michigan
The Nature of CIC Immune complexes are antibodies bound to antigens. Circulating immune complexes (CIC) are those found in serum or plasma. Endogenous CIC would be expected to bind complement. The size, structure, and composition of CIC are heterogeneous and depend on the binding characteristics of the components, which include antigens, immunoglobulins, complement, and other serum proteins. On first appearance, one might hypothesize that the complexity of such molecules would prevent effective measurement. Poor correlations between patient sera tested by different assay methods and the failure of CIC levels to correspond to the postulated pathological role in many patients have caused much controversy, and would seem to corroborate this hypothesis (14). A major reason for this controversy may be unreliable information obtained from ineffective assays. In addition to poor precision inherent in many assays, interference from monomeric immunoglobulins has been an important factor in causing misinformation. Failure to understand some pertinent aspects regarding the nature of CIC may be yet another factor. In this article, we critically examine the problems of interference and suggest alternatives to eliminate them. We also suggest a model of CIC that explains many observations. A number of distinct features regarding the nature of immune complexes is known: a) Endogenous immune complexes in serum and synovial fluid are composed mainly of immunoglobulins and other serum proteins (18, 19). Only small amounts of DNA that could not contribute significantly to the structure have been found (5, 26).
Clinical Immunology Newsletter 8:3,1987
b) Rheumatoid factors (RF) have been shown to be a significant component in CIC. c) In some infectious diseases, very sensitive methods indicate that organism-specific antigens are present in small amounts in CIC. Although these antigens may contribute only minimally to the overall CIC structure, they may be the seed that initiated the production of serum proteins, which comprise the bulk of the immune complex. In autoimmune disease, specific foreign antigens have not been identified in CIC. In autoimmune disease, the size of CIC has been shown to vary from 7S to greater than 19S (27). It is thought that smaller CIC do not contribute significantly to the disease process. Moreover, the range of sizes may be artifactually broad because methods such as gel filtration chromatography and sucrose gradient centrifugation used to analyze size may foster dissociation among many components of CIC, such that small complexes may increase (6, 13). Apparent size differences may actually represent differences in the internal binding forces of the aggregates. According to Jerne, the large majority of antibodies are idiotypic, forming networks with activity against other antibodies (9). If so, one might expect interactions among immunoglobulins to cause CIC in healthy persons. How, then, do we reconcile the finding that in healthy persons less than 0.5% of IgG is found in CIC? The binding forces between idiotypic antibodies and idiotopes may be weak; the networks may fragment easily with minor perturbations. In sick people, increases in RF, alterations in idiotypic antibody binding, and production of other proteins may facilitate adhesiveness like a glue and alter the intrinsic binding forces within these networks. Thus, CIC appear to be large aggregates with varying degrees of internal binding that depend on the reactivity of the components shown in Table 1.
Calibration of Methods for Measuring CIC Human aggregated T-globulin (HAG) is commonly used as a calibrator for CIC assays. This material, prepared by heating Cohn fraction 1I at 63°C, shows lot-to-lot inconsistency and is usually
© 1987 Elsevier Science Publishing Co., Inc.
Table 1 Nature of Immune Complexes Antigens Antibodies against antigens Antibodies against isotopes in antigens RF against the Fc portion of antibodies lmmunoglobulins that are low molecular weight precipitins of C l q Complement Other serum proteins
not stable for more than 2 - 3 months, even when stored at -70°C. Preparations of tetanus-toxoid antibody-antigen have been used for calibration material. The activity of these preparations is inconsistent because of varying antibody-antigen ratio, antibody affinity, and dilution ratio. Assays depending on the principle of binding complement components of CIC to solid-phase binders require that HAG or antibodyantigen calibrators be preincubated with normal serum to produce complement binding sites. In order to avoid problems of calibrator variability and inconsistency, some assays are standardized relative to a normal serum pool. In view of the inexactness of artificial preparations, this type of calibration has proven very useful even though it does not use absolute standards.
Methods for Measuring CIC Numerous methods for measuring CIC have been described. Many are based on the principle that complement or Fc receptors (FcR) on cells will preferentially bind CIC as compared to smaller immunoglobulins. Others use RF to preferentially bind CIC for measurement. Most use radioisotopes to identify and measure the bound components. Intrinsic difficulties in maintaining and standardizing RF preparations and cell cultures lead to poor reproducibility and tedium, as well as interference by high levels of lgG and autoantibodies. Because of its widespread use, Raji cell will be the only cell method discussed here. Other methods for discussion include the widely used C lq-liquid-phase assay; solid-phase assays, including Clqbinding, conglutinin, and antibody capture methods, which show promise for
0197-1859/87/$0.00 + 02.20
39
wide spread use because of adaptability into kit form; and extraction methods using polyethylene glycol (PEG) that precipitates CIC. An interesting en-. zyme immunoassay (EIA) that uses an enzyme-anti-enzyme probe, and is available as a kit, will also be mentioned.
Clq-Binding Assays The Clq component of complement reacts with immune complexes, but not with monomeric IgG from healthy persons, to form a precipitin. The Clq-binding liquid-phase assay uses radiolabeled Clq as a marker for precipitin formation after reaction with CIC. Precipitation of CIC from sera is assured by adding 3% PEG to the reaction mixture. For calibration, radioactivity in CIC precipitated by Clq is expressed as a ratio to radioactivity in total protein precipitated by trichloroacetic acid from normal pooled sera. Routine use is limited because of the need for radioiodination, and because human Clq is unstable and difficult to isolate in large yield. Recently, the use of animal Clq has reduced this latter problem. Clq has been attached to solid-phase materials for immunoradiometric assay. After reaction with serum, and washes to remove monomeric IgG, CIC bound to the Clq are identified by reaction with radiolabeled lgG; labeling of Clq is eliminated. A solid-phase Clq-binding kit (Cordis Laboratories, Inc., Miami, FL) that uses enzyme-labeled antibody for identifying immunoglobulins in CIC has been described (17). This kit uses goat Clq with freeze-dried preparations of HAG for calibration. Although the solid-phase assay has been shown to be more sensitive than the liquid for measuring HAG, it is less sensitive for identifying endogenous CIC (14). This would be expected if CIC were aggregates with varying degrees of internal binding, because washing should dissociate looser aggregates. Although it has been shown that Clq precipitates large aggregates, low molecular weight (7S) C lq-precipitins have also been isolated. These precipitins have been identified in lupus patients with elevated CIC levels but not in healthy people (20). These 7S-lgG
40
0197-1859/87/$0.00 + 02.20
molecules bind to Clq via the Fc portion. It seems reasonable to assume that this material acts like a glue, similar to RF and idiotypic antibodies, and increases the binding of complement to CIC.
are removed from consideration, Raji cell assay correlates better with C lqbinding in patient samples (15). In our opinion, Raji cell assay is ineffective for evaluating the clinical status of patients and should be discontinued.
Raji Cell
Conglutinin Assays
Raji cells are a line of cultured human lymphoblastoid cells derived from Burkitt's lymphoma; they contain receptors for C3 but show low avidity for the Fc portion of IgG. Cells are incubated with diluted serum, and CIC containing complement bind to cell receptors. Cells are washed to remove monomeric IgG, reacted with labeled antihuman IgG or protein A, and the concentration of CIC is estimated from the amount of isotope bound to the cells. HAG, preincubated with normal serum as a source of C3, is used to generate standard curves. Besides technical problems associated with poor reproducibility and sterile cell lines, there is abundant evidence that autoantibodies interfere with the Raji cell assay (1, 2, 7). The interference may be due to crossreaction between phosphoesters in the Raji cell membrane and autoantibodies with primary activity towards phosphoesters in DNA or RNA which are found in lupus patients (11, 24). Although Raji cells have been described as having a low avidity for the Fc portion of IgG, high levels of IgG in these patients may cause binding in excess of that seen in control sera that are used for assay blanking. This mechanism could also cause the observed interference. In any case, fractionation of sera by techniques that separate molecules on the basis of size consistently shows the major peak of Raji cell activity superimposed upon the 7S-IgG fraction. Discordance, in some lupus patients, between C lqbinding values that are normal and elevated Raji cell values most likely is due to one of these interfering mechanisms (15). We have consistently found Raji cell activity in such sera to coincide only with the 7S fraction upon gel filtration chromatography, in these patients, Raji cell provides a false and confusing impression of immune complex status. When snch discrepant data
Techniques that use different components of CIC for binding and identification (e.g., complement and IgG) promise greater specificity for CIC than methods such as the Clq assays that use a single component. Conglutinin is a protein (MW 750,000) found in ruminants; it has a high affinity for complement component C3bi. Bovine conglutinin has been used for solidphase immunometric assay. CIC are bound via complement, washed to remove monomeric lgG, and identified and measured by subsequent reaction with radio- or enzyme-labeled antilgG. A kit using conglutinin for measuring lgG, IgA, or IgM in CIC by means of an enzyme label is available (Shield Immunological Ltd., Dundee, UK). It uses HAG for calibration. Like the Clq solid-phase, the conglutinin assay shows greater sensitivity for identifying HAG than the Clq liquid-phase assay, but poorer sensitivity for endogenous CIC (3), consistent with dissociation during washing.
~ 1')87 Elsevier Science Publishing ('o . Inc.
Competitive Protein Binding Enzymatically Active Probe A novel approach for assaying CIC uses a probe formed by the reaction of [3-galactosidase with anti-[3-galactosidase (21). The immune complex probe competes with endogenous CIC in the sample for binding sites on solid-phase Clq or conglutinin. After a washing step, a colored product, which is inversely proportional to the amount of CIC in the sample, is generated from hydrolysis of o-nitrophenyl-13-D-galactopyranoside substrate by 13-galactosidase in the probe. Results are expressed as percent inhibition compared to a negative control. Donkey serum is added with the conglutinin assay as a source of complement for the probe. Unlike immunometric assays, binding of the label will he dependent on the stability of the probe rather than on the dissociation of endogenous CIC
Clinical Inamunology Newsletter 8:3.1987
200ul of sample
during washing. The method is available as a kit (Farmitalia Carlo Erba, Milano, Italy, and Kallestad Canada, Montreal, Quebec).
600ul of 3.3% PEG
stand overnight at 4°C centrifuge at 4°C and collect precipitate
Antibody Capture Assays Solid-phase anticomplement antibodies have been used to bind to complement components for measurement of CIC. After washing to remove monomeric IgG, CIC bound to the solid-phase are identified with labeled anti-IgG. The use of antibodies in reaction with two different components of CIC should provide high specificity. However, false positives due to endogenous antilgG antibodies that crossreact with animal antibodies used for capture have been reported (8, 22, 25). Preincubation of the sample with nonimmune animal sera has been shown to reduce this problem. An antibody capture kit for Clq that uses a mouse monoclonal antibody for capture is available (23) (Ortho Diagnostics, Raritan, N J, and Immunomedics, Newark, N J). Using this kit, we found, upon gel filtration chromatography, that the major activity from some elevated sera appeared in the monomeric IgG region. For this reason, we believe very careful evaluation of this kit is necessary to establish its reliability. PEG Extraction Methods CIC are precipitated with 2 - 3 % PEG (3). We have found that after precipitation, all measurable monomeric lgG is removed with two washes by a solution containing 2.5% PEG, and that lgG measured in the washed precipitate correlates well with the Clq-binding liquid-phase assay when patient sera are compared (15). Other investigators independently have obtained similar results (12, 27). We have shown that IgG in the precipitate can be measured using rate nephelometric (1CS, Beckman Instruments, Brea, CA) and fluorescent (FLAX, M.A. Bioproducts. Walkersville, MD) instrumentation that is available in many clinical laboratories, with minor modifications of kits from the manufacturer (4, 16). Stable IgG standards from the manufacturer can be used to calibrate the assays. Figure 1 illustrates this straightforward,
Clinical Immunology Newsletter 8:3.1987
/
800ul of Ice cold 2.5% PEG-BSA solution (shake vigorously)
Aspirate Supernatant
centrifuge at 4°C and collect precipitate 800ul of Ice cold 2.6% PEG-BSA solution (shake vigorously)
Aspirate / Supernatant
centrifuge at 4°C and collect precipitate Aspirate / Supernatant resuspend in 200ul of Trls-BSA buffer and assay Figure 1. Measurement of CIC by PEG extraction method. Samples are precipitated in 600 I~1 of 33 g/L PEG-saline solution, pH 7.4; washed with 800 ILl 25 g/L PEG-25 g/L bovine serum albumin (BSA )-saline solution, pH 7.4; reconstituted and assayed by a modification of the methods for assaying lgG in cerebrospinal fluid (4, 16).
reproducible approach for assaying CIC. PEG facilitates antibody-antigen binding, reducing the likelihood of dissociation during isolation. This may explain the high diagnostic sensitivity obtained by this technique (15). The complexes measured are predominantly those larger than 19S (28). We have found that some sera containing elevated CIC show only small CIC upon gel filtration chromatography, and that the eluate no longer shows PEG precipitable-lgG. The most likely explanation for the loss is that large immune complexes dissociated into smaller components (6, 13). A framework has been presented within which it is hoped CIC can be effectively measured. First and foremost is the elimination of methods, by alteration or discontinuation, that are susceptible to frequent interferences.
~ 1987 Elsevier Science Publishing Co.. Inc.
Cell assays, including Raji cell, should be discontinued. Antibody capture methods must be modified to eliminate interferences. Successful modification may lead to highly specific assays that lend themselves to production in kit form. At present, Clq and conglutinin methods seem effective. However, the stability of conglutinin, C lq, and calibrators may be difficult to control with widespread distribution in kits. We now recommend the PEG extraction method as a screen for CIC in laboratories with the appropriate instrumentation. It is reproducible, easy to perform, and can be standardized with stable monomeric lgG. It appears to measure large molecules only and correlates well with the C lq-binding liquid-phase assay. CIC may be bound together by a glue that includes RF, idiotypic antibodies, low molecular weight C lq-pre-
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41
cipitins, and other serum proteins, with varying degrees of adhesiveness. Normally, the network is loose and easily dissociable, but in disease, changes in the immune environment may lead to variations in composition that increase adhesiveness. Methods for measuring CIC may be affected by the degree of internal adhesiveness causing methods using more vigorous treatments to be clinically less sensitive. Clinically, CIC may be useful for following the progress of diseases, and for differentiating between conditions that overtly exhibit similar symptoms but that actually may have different underlying etiologies that require different classifications and treatments. The value of CIC measurement in clinical medicine needs to be reassessed.
We thank Dr. Stanley Levy for valuable discussion regarding the glue hypothesis.
References 1. Anderson, C. L. and W. S. Stillman (1980). Evidence for detection of Rajidirected immunoglobulin G antibody in sera from patients with systemic lupus erythematosus. J. Clin. Invest. 66:353-360. 2. Cooper, K. M. and M. Moore (1983). Reactivity of low molecular weight material in cellular immune complex assays. Clin. Exp. Immunol. 52:407- 416. 3. Digeon, M., M. Lauer, J. Riza, et al. (1977). Detection of circulating immmune complexes in human sera by simplified assays with polyethylene glycol. J. Immunol. Methods 16:165183. 4. Feldkamp, C. S., S. S. Levinson, M. Perry, and V. Amin (1985). Anti-lgG combined with rate nephelometry for measuring polyethylene glycol-precipitated circulating immune complexes. Clin. Chem. 31:2024-2027. 5. Harley, E. H. and P. Kiemp (1982). A specific DNA immune complex assay, and its application in SLE. Clin. Chem. Acta. 122:213-223. 6. Hansson, V. B., M. Vesson, and V. Alkner (1981 ). Isolation and characterization of intermediate complexes and other components with common antigenic determinants. Scand. J. Immunol. 13:57-66.
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7. Horsefall, C. A., P. J. W. Venables, P. A. Mumford, et al. (1981). Interpretation of the Raji cell assay in sera containing anti-nuclear antibodies and immune complexes. Clin Exp. lmmunol. 44:405 -415. 8. Jaebos, R. J. and M. Reiehlin (1985). Low molecular weight rheumatoid factor--a serological marker in systemic lupus erythematosis. Arthritis Rheum. 28:$65. 9. Jerne, N. K. (1985). The generative grammar of the immune system. Science 229:1057-1059. 10. Johnson, P. M., K. K. Phua, and H. B. Evans (1985). An idiotypic complementarity between rheumatoid factor and anti-peptidoglycan antibodies. Clin. Exp. lmmunol. 61:373378. 11. Koike, T., M. Sneishi, H. Funaki, et al. (1984). Anti-phospholipid antibodies and biological false positive serological test for syphilis in patients with systemic lupus erythematosus. Clin. Exp. lmmunol. 56:193-199. 12. Krapf, F., D. Renger, I. Sehedel, K. Leiendeeker, H. Leyssens, and H. Deieher (1982). A PEG-precipitation laser nephelometer technique for detection and characterization of circulating immune complexes in human sera. J. lmmunol. Methods 54:107- 117. 13. Kunkei, H. G., H. J. Eberhard, H. H. Fudenberg, and T. B. Tomas (1961). Gamma globulin complexes in rheumatoid arthritis and certain other conditions. J. Clin. Invest. 40:117128. 14. Lambert, P. H., F. J. Dixon, R. H. Znbler, et al. (1978). A WHO collaborative study for the evaluation of eighteen methods for detecting immune complexes in serum. J. Clin. Lab. lmmunol. 1:1-15. 15. Levinson, S. S. and J. O. Goldman (1984). Anti-lgG type test to assay circulating immune complexes from polyethylene glycol precipitates compared with Clq-binding and Raji cell tests. Clin. Biochem. 17:341-344. 16. Levinson, S. S. and J. O. Goldman (1986). Fluorescent anti-immunoglobulin G assay of circulating immune complexes extracted with polyethylene glycol. J. Clin. Microbiol. 23:29-32. 17. Lin, T. M., S. P. Halbert, and R. Cort (1983). An enzyme-like immunoassay for circulating immune complexes using solid-phase goat CIq. J. Immunol. Methods 63:187- 205. 18. Maire, M. A., M. Barret, N. Carpentier, P. A. Mieseher, and P. H. Lambert (1983). Identification of
,~ 1987 Elsevier Science Publishing Co.. Inc.
19.
20.
21.
22.
23.
24.
25.
26.
27.
28.
components of IC purified from human sera. Clin. Exp. Immunol. 51:215224. Male, D. K. and M. Roitt (1981). Molecular analysis of complement fixing rheumatoid synovial fluid immune complexes. Clin. Exp. lmmunol. 46:521-529, Marder, R. J., F. X. Burch, and F. R. Schmid (1978). Low molecular weight Clq-precipitins in hypocomplementemic vasculitis-urticaria syndrome: partial purification and characterization as immunoglobulin. J. Immunol. 121:613-618. Migliorini, P., G. Trovatello, S. Cantarella, and F. Manca (1983). An enzymatically active antigen-antibody probe to measure circulating immune complexes. II. E. Coli beta-galactosidase in the probe and CIq as the recognition unit. J. Immunol. Methods 59:245- 254. Olds, L. C. and J. J. Miller I l l (1984). Anti-F(ab~)2 antibodies that interfere with interpretation of the anti-C3 assay for immune complexes in children with rheumatic diseases. Arthritis Rheum. 27:330-336. Reckel, R. P., J. Harris, R. Botsko, R. Wellerson, and S. Varga (1984). The detection of circulating immune complexes containing C lq and IgG using a new rapid serum ELISA test system. Diagnostic Immunol. 2:228237. Shoenfeld, Y., J. Rauch, and H. Massicotte (1983). Polyspecificity of monoclonal lupus autoantibodies produced by human-human hybridomas. N. Engl. J. Med. 308:414-420. Takeuchi, T., O. Hosono, and J. Koide (1985). Suppression of rheumatoid factor synthesis by idiotypic antibody in rheumatoid arthritis patients with cross-reactive idiotypes. Arth. Rheum. 28:873- 881. Tron, F., J. Letarte, M. C. RoqueAntunes Barreira, and P. Lesavre (1982). Specific detection of circulating DNA: anti-DNA immune complexes in human SLE sera using murine monoclonal anti-DNA antibodies. Clin. Exp. Immunol. 49:481-487. Tung, K. S. K., R. J. DeHortatius, and R. C. Williams, Jr. (1981). Study of circulating immune complex size in systemic lupus erythematosis. Clin. Exp. Immunol. 43:615-625. Valentijn, R. M., L. A. Van Es, and M. R. Daha (1984). The specific detection of IgG, IgA, and the complement components C3 and C4 in circulating immune complexes. J. Clin. Lab. Immunol. 14:81-86.
Clinical Immunology Newsletter 8:3,1987
The Nature of CIC Immune complexes are antibodies bound to antigens. Circulating immune complexes (CIC) are those found in serum or plasma. Endogenous CIC would be expected to bind complement. The size, structure, and composition of CIC are heterogeneous and depend on the binding characteristics of the components, which include antigens, immunoglobulins, complement, and other serum proteins. On first appearance, one might hypothesize that the complexity of such molecules would prevent effective measurement. Poor correlations between patient sera tested by different assay methods and the failure of CIC levels to correspond to the postulated pathological role in many patients have caused much controversy, and would seem to corroborate this hypothesis (14). A major reason for this controversy may be unreliable information obtained from ineffective assays. In addition to poor precision inherent in many assays, interference from monomeric immunoglobulins has been an important factor in causing misinformation. Failure to understand some pertinent aspects regarding the nature of CIC may be yet another factor. In this article, we critically examine the problems of interference and suggest alternatives to eliminate them. We also suggest a model of CIC that explains many observations. A number of distinct features regarding the nature of immune complexes is known: a) Endogenous immune complexes in serum and synovial fluid are composed mainly of immunoglobulins and other serum proteins (18, 19). Only small amounts of DNA that could not contribute significantly to the structure have been found (5, 26).
Clinical Immunology Newsletter 8:3,1987
b) Rheumatoid factors (RF) have been shown to be a significant component in CIC. c) In some infectious diseases, very sensitive methods indicate that organism-specific antigens are present in small amounts in CIC. Although these antigens may contribute only minimally to the overall CIC structure, they may be the seed that initiated the production of serum proteins, which comprise the bulk of the immune complex. In autoimmune disease, specific foreign antigens have not been identified in CIC. In autoimmune disease, the size of CIC has been shown to vary from 7S to greater than 19S (27). It is thought that smaller CIC do not contribute significantly to the disease process. Moreover, the range of sizes may be artifactually broad because methods such as gel filtration chromatography and sucrose gradient centrifugation used to analyze size may foster dissociation among many components of CIC, such that small complexes may increase (6, 13). Apparent size differences may actually represent differences in the internal binding forces of the aggregates. According to Jerne, the large majority of antibodies are idiotypic, forming networks with activity against other antibodies (9). If so, one might expect interactions among immunoglobulins to cause CIC in healthy persons. How, then, do we reconcile the finding that in healthy persons less than 0.5% of IgG is found in CIC? The binding forces between idiotypic antibodies and idiotopes may be weak; the networks may fragment easily with minor perturbations. In sick people, increases in RF, alterations in idiotypic antibody binding, and production of other proteins may facilitate adhesiveness like a glue and alter the intrinsic binding forces within these networks. Thus, CIC appear to be large aggregates with varying degrees of internal binding that depend on the reactivity of the components shown in Table 1.
Calibration of Methods for Measuring CIC Human aggregated T-globulin (HAG) is commonly used as a calibrator for CIC assays. This material, prepared by heating Cohn fraction 1I at 63°C, shows lot-to-lot inconsistency and is usually
© 1987 Elsevier Science Publishing Co., Inc.
Table 1 Nature of Immune Complexes Antigens Antibodies against antigens Antibodies against isotopes in antigens RF against the Fc portion of antibodies lmmunoglobulins that are low molecular weight precipitins of C l q Complement Other serum proteins
not stable for more than 2 - 3 months, even when stored at -70°C. Preparations of tetanus-toxoid antibody-antigen have been used for calibration material. The activity of these preparations is inconsistent because of varying antibody-antigen ratio, antibody affinity, and dilution ratio. Assays depending on the principle of binding complement components of CIC to solid-phase binders require that HAG or antibodyantigen calibrators be preincubated with normal serum to produce complement binding sites. In order to avoid problems of calibrator variability and inconsistency, some assays are standardized relative to a normal serum pool. In view of the inexactness of artificial preparations, this type of calibration has proven very useful even though it does not use absolute standards.
Methods for Measuring CIC Numerous methods for measuring CIC have been described. Many are based on the principle that complement or Fc receptors (FcR) on cells will preferentially bind CIC as compared to smaller immunoglobulins. Others use RF to preferentially bind CIC for measurement. Most use radioisotopes to identify and measure the bound components. Intrinsic difficulties in maintaining and standardizing RF preparations and cell cultures lead to poor reproducibility and tedium, as well as interference by high levels of lgG and autoantibodies. Because of its widespread use, Raji cell will be the only cell method discussed here. Other methods for discussion include the widely used C lq-liquid-phase assay; solid-phase assays, including Clqbinding, conglutinin, and antibody capture methods, which show promise for
0197-1859/87/$0.00 + 02.20
39
wide spread use because of adaptability into kit form; and extraction methods using polyethylene glycol (PEG) that precipitates CIC. An interesting en-. zyme immunoassay (EIA) that uses an enzyme-anti-enzyme probe, and is available as a kit, will also be mentioned.
Clq-Binding Assays The Clq component of complement reacts with immune complexes, but not with monomeric IgG from healthy persons, to form a precipitin. The Clq-binding liquid-phase assay uses radiolabeled Clq as a marker for precipitin formation after reaction with CIC. Precipitation of CIC from sera is assured by adding 3% PEG to the reaction mixture. For calibration, radioactivity in CIC precipitated by Clq is expressed as a ratio to radioactivity in total protein precipitated by trichloroacetic acid from normal pooled sera. Routine use is limited because of the need for radioiodination, and because human Clq is unstable and difficult to isolate in large yield. Recently, the use of animal Clq has reduced this latter problem. Clq has been attached to solid-phase materials for immunoradiometric assay. After reaction with serum, and washes to remove monomeric IgG, CIC bound to the Clq are identified by reaction with radiolabeled lgG; labeling of Clq is eliminated. A solid-phase Clq-binding kit (Cordis Laboratories, Inc., Miami, FL) that uses enzyme-labeled antibody for identifying immunoglobulins in CIC has been described (17). This kit uses goat Clq with freeze-dried preparations of HAG for calibration. Although the solid-phase assay has been shown to be more sensitive than the liquid for measuring HAG, it is less sensitive for identifying endogenous CIC (14). This would be expected if CIC were aggregates with varying degrees of internal binding, because washing should dissociate looser aggregates. Although it has been shown that Clq precipitates large aggregates, low molecular weight (7S) C lq-precipitins have also been isolated. These precipitins have been identified in lupus patients with elevated CIC levels but not in healthy people (20). These 7S-lgG
40
0197-1859/87/$0.00 + 02.20
molecules bind to Clq via the Fc portion. It seems reasonable to assume that this material acts like a glue, similar to RF and idiotypic antibodies, and increases the binding of complement to CIC.
are removed from consideration, Raji cell assay correlates better with C lqbinding in patient samples (15). In our opinion, Raji cell assay is ineffective for evaluating the clinical status of patients and should be discontinued.
Raji Cell
Conglutinin Assays
Raji cells are a line of cultured human lymphoblastoid cells derived from Burkitt's lymphoma; they contain receptors for C3 but show low avidity for the Fc portion of IgG. Cells are incubated with diluted serum, and CIC containing complement bind to cell receptors. Cells are washed to remove monomeric IgG, reacted with labeled antihuman IgG or protein A, and the concentration of CIC is estimated from the amount of isotope bound to the cells. HAG, preincubated with normal serum as a source of C3, is used to generate standard curves. Besides technical problems associated with poor reproducibility and sterile cell lines, there is abundant evidence that autoantibodies interfere with the Raji cell assay (1, 2, 7). The interference may be due to crossreaction between phosphoesters in the Raji cell membrane and autoantibodies with primary activity towards phosphoesters in DNA or RNA which are found in lupus patients (11, 24). Although Raji cells have been described as having a low avidity for the Fc portion of IgG, high levels of IgG in these patients may cause binding in excess of that seen in control sera that are used for assay blanking. This mechanism could also cause the observed interference. In any case, fractionation of sera by techniques that separate molecules on the basis of size consistently shows the major peak of Raji cell activity superimposed upon the 7S-IgG fraction. Discordance, in some lupus patients, between C lqbinding values that are normal and elevated Raji cell values most likely is due to one of these interfering mechanisms (15). We have consistently found Raji cell activity in such sera to coincide only with the 7S fraction upon gel filtration chromatography, in these patients, Raji cell provides a false and confusing impression of immune complex status. When snch discrepant data
Techniques that use different components of CIC for binding and identification (e.g., complement and IgG) promise greater specificity for CIC than methods such as the Clq assays that use a single component. Conglutinin is a protein (MW 750,000) found in ruminants; it has a high affinity for complement component C3bi. Bovine conglutinin has been used for solidphase immunometric assay. CIC are bound via complement, washed to remove monomeric lgG, and identified and measured by subsequent reaction with radio- or enzyme-labeled antilgG. A kit using conglutinin for measuring lgG, IgA, or IgM in CIC by means of an enzyme label is available (Shield Immunological Ltd., Dundee, UK). It uses HAG for calibration. Like the Clq solid-phase, the conglutinin assay shows greater sensitivity for identifying HAG than the Clq liquid-phase assay, but poorer sensitivity for endogenous CIC (3), consistent with dissociation during washing.
~ 1')87 Elsevier Science Publishing ('o . Inc.
Competitive Protein Binding Enzymatically Active Probe A novel approach for assaying CIC uses a probe formed by the reaction of [3-galactosidase with anti-[3-galactosidase (21). The immune complex probe competes with endogenous CIC in the sample for binding sites on solid-phase Clq or conglutinin. After a washing step, a colored product, which is inversely proportional to the amount of CIC in the sample, is generated from hydrolysis of o-nitrophenyl-13-D-galactopyranoside substrate by 13-galactosidase in the probe. Results are expressed as percent inhibition compared to a negative control. Donkey serum is added with the conglutinin assay as a source of complement for the probe. Unlike immunometric assays, binding of the label will he dependent on the stability of the probe rather than on the dissociation of endogenous CIC
Clinical Inamunology Newsletter 8:3.1987
200ul of sample
during washing. The method is available as a kit (Farmitalia Carlo Erba, Milano, Italy, and Kallestad Canada, Montreal, Quebec).
600ul of 3.3% PEG
stand overnight at 4°C centrifuge at 4°C and collect precipitate
Antibody Capture Assays Solid-phase anticomplement antibodies have been used to bind to complement components for measurement of CIC. After washing to remove monomeric IgG, CIC bound to the solid-phase are identified with labeled anti-IgG. The use of antibodies in reaction with two different components of CIC should provide high specificity. However, false positives due to endogenous antilgG antibodies that crossreact with animal antibodies used for capture have been reported (8, 22, 25). Preincubation of the sample with nonimmune animal sera has been shown to reduce this problem. An antibody capture kit for Clq that uses a mouse monoclonal antibody for capture is available (23) (Ortho Diagnostics, Raritan, N J, and Immunomedics, Newark, N J). Using this kit, we found, upon gel filtration chromatography, that the major activity from some elevated sera appeared in the monomeric IgG region. For this reason, we believe very careful evaluation of this kit is necessary to establish its reliability. PEG Extraction Methods CIC are precipitated with 2 - 3 % PEG (3). We have found that after precipitation, all measurable monomeric lgG is removed with two washes by a solution containing 2.5% PEG, and that lgG measured in the washed precipitate correlates well with the Clq-binding liquid-phase assay when patient sera are compared (15). Other investigators independently have obtained similar results (12, 27). We have shown that IgG in the precipitate can be measured using rate nephelometric (1CS, Beckman Instruments, Brea, CA) and fluorescent (FLAX, M.A. Bioproducts. Walkersville, MD) instrumentation that is available in many clinical laboratories, with minor modifications of kits from the manufacturer (4, 16). Stable IgG standards from the manufacturer can be used to calibrate the assays. Figure 1 illustrates this straightforward,
Clinical Immunology Newsletter 8:3.1987
/
800ul of Ice cold 2.5% PEG-BSA solution (shake vigorously)
Aspirate Supernatant
centrifuge at 4°C and collect precipitate 800ul of Ice cold 2.6% PEG-BSA solution (shake vigorously)
Aspirate / Supernatant
centrifuge at 4°C and collect precipitate Aspirate / Supernatant resuspend in 200ul of Trls-BSA buffer and assay Figure 1. Measurement of CIC by PEG extraction method. Samples are precipitated in 600 I~1 of 33 g/L PEG-saline solution, pH 7.4; washed with 800 ILl 25 g/L PEG-25 g/L bovine serum albumin (BSA )-saline solution, pH 7.4; reconstituted and assayed by a modification of the methods for assaying lgG in cerebrospinal fluid (4, 16).
reproducible approach for assaying CIC. PEG facilitates antibody-antigen binding, reducing the likelihood of dissociation during isolation. This may explain the high diagnostic sensitivity obtained by this technique (15). The complexes measured are predominantly those larger than 19S (28). We have found that some sera containing elevated CIC show only small CIC upon gel filtration chromatography, and that the eluate no longer shows PEG precipitable-lgG. The most likely explanation for the loss is that large immune complexes dissociated into smaller components (6, 13). A framework has been presented within which it is hoped CIC can be effectively measured. First and foremost is the elimination of methods, by alteration or discontinuation, that are susceptible to frequent interferences.
~ 1987 Elsevier Science Publishing Co.. Inc.
Cell assays, including Raji cell, should be discontinued. Antibody capture methods must be modified to eliminate interferences. Successful modification may lead to highly specific assays that lend themselves to production in kit form. At present, Clq and conglutinin methods seem effective. However, the stability of conglutinin, C lq, and calibrators may be difficult to control with widespread distribution in kits. We now recommend the PEG extraction method as a screen for CIC in laboratories with the appropriate instrumentation. It is reproducible, easy to perform, and can be standardized with stable monomeric lgG. It appears to measure large molecules only and correlates well with the C lq-binding liquid-phase assay. CIC may be bound together by a glue that includes RF, idiotypic antibodies, low molecular weight C lq-pre-
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cipitins, and other serum proteins, with varying degrees of adhesiveness. Normally, the network is loose and easily dissociable, but in disease, changes in the immune environment may lead to variations in composition that increase adhesiveness. Methods for measuring CIC may be affected by the degree of internal adhesiveness causing methods using more vigorous treatments to be clinically less sensitive. Clinically, CIC may be useful for following the progress of diseases, and for differentiating between conditions that overtly exhibit similar symptoms but that actually may have different underlying etiologies that require different classifications and treatments. The value of CIC measurement in clinical medicine needs to be reassessed.
We thank Dr. Stanley Levy for valuable discussion regarding the glue hypothesis.
References 1. Anderson, C. L. and W. S. Stillman (1980). Evidence for detection of Rajidirected immunoglobulin G antibody in sera from patients with systemic lupus erythematosus. J. Clin. Invest. 66:353-360. 2. Cooper, K. M. and M. Moore (1983). Reactivity of low molecular weight material in cellular immune complex assays. Clin. Exp. Immunol. 52:407- 416. 3. Digeon, M., M. Lauer, J. Riza, et al. (1977). Detection of circulating immmune complexes in human sera by simplified assays with polyethylene glycol. J. Immunol. Methods 16:165183. 4. Feldkamp, C. S., S. S. Levinson, M. Perry, and V. Amin (1985). Anti-lgG combined with rate nephelometry for measuring polyethylene glycol-precipitated circulating immune complexes. Clin. Chem. 31:2024-2027. 5. Harley, E. H. and P. Kiemp (1982). A specific DNA immune complex assay, and its application in SLE. Clin. Chem. Acta. 122:213-223. 6. Hansson, V. B., M. Vesson, and V. Alkner (1981 ). Isolation and characterization of intermediate complexes and other components with common antigenic determinants. Scand. J. Immunol. 13:57-66.
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7. Horsefall, C. A., P. J. W. Venables, P. A. Mumford, et al. (1981). Interpretation of the Raji cell assay in sera containing anti-nuclear antibodies and immune complexes. Clin Exp. lmmunol. 44:405 -415. 8. Jaebos, R. J. and M. Reiehlin (1985). Low molecular weight rheumatoid factor--a serological marker in systemic lupus erythematosis. Arthritis Rheum. 28:$65. 9. Jerne, N. K. (1985). The generative grammar of the immune system. Science 229:1057-1059. 10. Johnson, P. M., K. K. Phua, and H. B. Evans (1985). An idiotypic complementarity between rheumatoid factor and anti-peptidoglycan antibodies. Clin. Exp. lmmunol. 61:373378. 11. Koike, T., M. Sneishi, H. Funaki, et al. (1984). Anti-phospholipid antibodies and biological false positive serological test for syphilis in patients with systemic lupus erythematosus. Clin. Exp. lmmunol. 56:193-199. 12. Krapf, F., D. Renger, I. Sehedel, K. Leiendeeker, H. Leyssens, and H. Deieher (1982). A PEG-precipitation laser nephelometer technique for detection and characterization of circulating immune complexes in human sera. J. lmmunol. Methods 54:107- 117. 13. Kunkei, H. G., H. J. Eberhard, H. H. Fudenberg, and T. B. Tomas (1961). Gamma globulin complexes in rheumatoid arthritis and certain other conditions. J. Clin. Invest. 40:117128. 14. Lambert, P. H., F. J. Dixon, R. H. Znbler, et al. (1978). A WHO collaborative study for the evaluation of eighteen methods for detecting immune complexes in serum. J. Clin. Lab. lmmunol. 1:1-15. 15. Levinson, S. S. and J. O. Goldman (1984). Anti-lgG type test to assay circulating immune complexes from polyethylene glycol precipitates compared with Clq-binding and Raji cell tests. Clin. Biochem. 17:341-344. 16. Levinson, S. S. and J. O. Goldman (1986). Fluorescent anti-immunoglobulin G assay of circulating immune complexes extracted with polyethylene glycol. J. Clin. Microbiol. 23:29-32. 17. Lin, T. M., S. P. Halbert, and R. Cort (1983). An enzyme-like immunoassay for circulating immune complexes using solid-phase goat CIq. J. Immunol. Methods 63:187- 205. 18. Maire, M. A., M. Barret, N. Carpentier, P. A. Mieseher, and P. H. Lambert (1983). Identification of
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19.
20.
21.
22.
23.
24.
25.
26.
27.
28.
components of IC purified from human sera. Clin. Exp. Immunol. 51:215224. Male, D. K. and M. Roitt (1981). Molecular analysis of complement fixing rheumatoid synovial fluid immune complexes. Clin. Exp. lmmunol. 46:521-529, Marder, R. J., F. X. Burch, and F. R. Schmid (1978). Low molecular weight Clq-precipitins in hypocomplementemic vasculitis-urticaria syndrome: partial purification and characterization as immunoglobulin. J. Immunol. 121:613-618. Migliorini, P., G. Trovatello, S. Cantarella, and F. Manca (1983). An enzymatically active antigen-antibody probe to measure circulating immune complexes. II. E. Coli beta-galactosidase in the probe and CIq as the recognition unit. J. Immunol. Methods 59:245- 254. Olds, L. C. and J. J. Miller I l l (1984). Anti-F(ab~)2 antibodies that interfere with interpretation of the anti-C3 assay for immune complexes in children with rheumatic diseases. Arthritis Rheum. 27:330-336. Reckel, R. P., J. Harris, R. Botsko, R. Wellerson, and S. Varga (1984). The detection of circulating immune complexes containing C lq and IgG using a new rapid serum ELISA test system. Diagnostic Immunol. 2:228237. Shoenfeld, Y., J. Rauch, and H. Massicotte (1983). Polyspecificity of monoclonal lupus autoantibodies produced by human-human hybridomas. N. Engl. J. Med. 308:414-420. Takeuchi, T., O. Hosono, and J. Koide (1985). Suppression of rheumatoid factor synthesis by idiotypic antibody in rheumatoid arthritis patients with cross-reactive idiotypes. Arth. Rheum. 28:873- 881. Tron, F., J. Letarte, M. C. RoqueAntunes Barreira, and P. Lesavre (1982). Specific detection of circulating DNA: anti-DNA immune complexes in human SLE sera using murine monoclonal anti-DNA antibodies. Clin. Exp. Immunol. 49:481-487. Tung, K. S. K., R. J. DeHortatius, and R. C. Williams, Jr. (1981). Study of circulating immune complex size in systemic lupus erythematosis. Clin. Exp. Immunol. 43:615-625. Valentijn, R. M., L. A. Van Es, and M. R. Daha (1984). The specific detection of IgG, IgA, and the complement components C3 and C4 in circulating immune complexes. J. Clin. Lab. Immunol. 14:81-86.
Clinical Immunology Newsletter 8:3,1987