Clin. exp. Immunol. (1977) 29, 428-435.
A test for antigen-antibody complexes in human sera using IgM of rabbit antisera to human immunoglobulins R. J. LEVINSKY & J. F. SOOTHILL Department of Immunology, Institute of Child Health, London
(Received 31 March 1977) SUMMARY
A simple semi-quantitative test for soluble antigen-antibody complexes using characterized nonhuman reagents and permitting analysis of their constituents is described. The agglutination of immunoglobulin-coated latex particles by rabbit IgM antibodies to the immunoglobulin is inhibited by complex-containing sera. No inhibition is obtained with monomer immunoglobulins. Semi-quantitative measurement of complexes may be made by electronically counting residual unagglutinated latex particles. The new method of linking proteins to latex by DNP coupling permits the technique to be applied to all constituents of complexes; immunoglobulins, complement and antigens. A new method of decomplementing sera by EDTA-Sigma cell-IgG absorption allows analysis of sera without the false positives and false negatives other methods give. The test gave positive results for IgG complexes in most of twenty-four patients with SLE nephritis. IgA complexes were identified in a patient with Henoch-Schonlein purpura nephritis.
INTRODUCTION Available methods for detecting immune complexes are not fully satisfactory and do not permit analysis of their constituents (Soothill, 1977). We have developed a simple semi-quantitative test for immune complexes, using characterized non-human reagents, which is capable of analysing the constituents. It is based on that of Lurhuma et al. (1976) in which immune complexes inhibit the agglutination of IgGcoated latex particles by rheumatoid factor or Clq. Human rheumatoid factors are low-affinity IgM antibodies to intrinsic IgG (Eisenberg, 1976; Dissanayake, Hay & Roitt, 1977) and, because of their multivalency, have a high avidity for aggregated IgG or IgG-containing complexes. We have prepared IgM antibodies in rabbits to the different complex constituents, which react with aggregated or complexed proteins but not with monomer. The new method of linking antigens to latex particles permits a far wider application of the method, and we have developed a method of removing complement which avoids the problems of the existing methods.
MATERIALS AND METHODS IgG. Purified human IgG was obtained by ion-exchange chromatography on Whatman DE 52 cellulose (Stanworth, 1960). The material was processed twice. Purity was assessed by immunoelectrophoresis using polyvalent anti-human serum. IgA. The IgA protein was purified from human myeloma serum by a three-stage procedure using Geon-Pevikon block electrophoresis, ion-exchange chromatography on a Whatman DE 52 cellulose column with a stepwise buffer gradient of 0-05-0-5 MTris-HCl, pH 8-0, and finally gel filtration on a Sephadex G-200 column. No impurities were found on immunoelectrophoresis and IgG, IgM and albumin were not detected by single radial immunodiffusion (Fahey & McKelvey, 1965). IgA and IgG were stored in a concentration of 10 mg/ml in saline at - 700C. Clq. Human Clq was prepared from fresh-frozen plasma by the method of Yonemasu & Stroud (1971). The protein was stored at a concentration of 500 pg/ml in saline at - 70'C. F(ab')2 fragment. This fragment was prepared from human IgG (Kabi) by pepsin digestion for 48 hr using an enzyme: substrate ratio of 1:50 (Stanworth & Turner, 1973). The fragment was isolated by gel filtration of the digest on Sephadex G-200. Correspondence: Dr R. J. Levinsky, Department of Immunology, Institute of Child Health, London W.C. 1.
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Immunoabsorbents. Insoluble immunoabsorbents of IgG and F(ab')2 were made by linking these proteins to Sigma cell (Sigma Chemicals, 50 p size) by cyanogen-bromide activation. 5 g Sigma cell was suspended in 100 ml water. 5 g cyanogen bromide (Fluka) was dissolved in 100 ml water in a fume cupboard. The two reagents were mixed together for 10 min at room temperature, maintaining the pH between 10-11 by the addi tion of 1 M sodium hydroxide. The activated Sigma cell was washed with 5 litres of cold 0- 1 M sodium bicarbonate solution in a Buchner funnel and then resuspended in 20 ml coupling buffer (76-8 ml M NaHCO3 mixed with 7*7 ml M Na2CO3 and made up to 1 litre with water). 150 mg of the protein to be linked was dissolved in 7 ml of coupling buffer and added to the activated Sigma cell. Coupling occurred overnight at 40C. The washed F(ab')2-Sigma cell was stored at 4VC in Tris-HCl, pH 8-2. The IgG-Sigma cell was stored as a 1:5 suspension (1 vol packed Sigma cell:4 vol buffer) in 0-27 M glycine saline adjusted to pH 8-2 with 1 M sodium hydroxide at 4VC, ready for use in the decomplementing stage of the immune-complex test. Preparation of rabbit JgM reagent. 10 mg of purified protein (IgG or IgA) in Freund's complete adjuvant was injected i.m. in four sites into rabbits. The rabbits were bled out at 10 days and the serum obtained was separated on a Sephadex G-200 column. The exclusion peak containing IgM was concentrated to starting volume by ultrafiltration. Light-chain specificity was removed by shaking with Sigma cell F(ab')2 immunoabsorbent for 1 hr at room temperature. The supernatant following centrifugation was stored in aliquots at - 700C. Latex coupling. Polyvinyl toluene latex particles 1l15 pm in diameter (s.d.+ 0-0055 pm),), supplied by Coulter Electronics as a 10%I w/v solution, were used. 800 pl latex was washed twice in 0-054 M glycine saline, pH 8-2 (1 vol. 0-27 M glycine saline, pH 8-2, +4 vol. water). The latex was centrifuged at 12,500 g and reconstituted in 20 ml 0 054 M glycine saline, pH 8-2. 302 pul of a 10 mg/ml solution of IgG (40 ,pg IgG/mg dry weight latex) was added and the suspension mixed for 30 min at room temperature. The latex was then washed twice in 0 054 M glycine saline, pH 8-2, and reconstituted to 20 ml in 0-27 M glycine saline, pH 8-2, containing 0- 1% human serum albumin to block remaining latex protein binding sites. All buffers were millipore filtered (0-22 ,pm) prior to use. This method results in the latex binding IgG, but not IgA or many other proteins. However, if DNP is substituted into the protein molecule latex coupling may be effected. DNP-IgA preparation. 10 mg IgA in 1 ml saline was dialysed for 3 hr at 370C against 10%4 w/v sodium bicarbonate. 3 ,ul DNFB (2-4-dinitrofluorobenzene, Sigma Chemicals) was added with constant mixing at 37°C, and when a bright yellow colour was obtained, the reaction was stopped by the addition of an equal vol. of benzene to extract excess DNFB. The resultant DNP-IgA solution was dialysed for 24 hr against 0 054 M glycine saline, pH 8-2. DNP-IgA may be coupled to latex using the same method as for IgG. Coated latex preparations were stored at 40C and were stable for at least 3 months. The latex suspension was sonicated before use each time. Slide agglutination test. This was used initially for testing agglutinating titres of antisera. Doubling dilutions of the sera were prepared. 25,pl of antisera dilution was mixed with 25 ,pl latex suspension and agglutination read visually at 3 min. When the test for soluble complex is done on a slide, 25 ,ul of suitably diluted IgM reagent, 25 p1 of IgG-coated latex and 25 ,pl of 1:10 dilutions of decomplemented patient's serum are mixed and read as above. Further dilution of patient's sera may be made. Immune complex test. Sera (50 pl) were decomplemented by incubation with 50,u1 02 M EDTA, pH 7-6, for 10 min at room temperature to release Clq. To remove this, 500 ,pl of freshly washed and resuspended (1 vol. packed immunoabsorbent: 4 vol. 0-27 M glycine saline, pH 8-2) IgG-coated Sigma cell was added to the EDTA serum and shaken for 20 min at room temperature. Any rheumatoid factor in the serum is also removed by this procedure. The supernatant after centrifugation (750 g for 3 min) is a 1:10 dilution. The Coulter counter model ZB, using the 30 ,pm orifice tube, was set to count latex of 1-15 ,um size by adjusting the upper and lower thresholds to obtain an electronic window which included the monomer peak but excluded smaller particles downwards and aggregates of dimer upwards. The latex suspension was adjusted to give a count of 2-2-5 x 104 particles/50 ,ul of final dilution, so that errors due to coincidence were < 1%/ and could be disregarded (other counters may be used). Detection ofIgG-containing complexes. Each batch of rabbit IgM and IgG was titrated; the dilution giving 70-80%, agglutination of IgG-coated latex was used in the test. 100 ul of decomplemented serum (1:10 dilution) was mixed with 100 p1I rabbit IgM anti-human IgG at appropriate dilution and 100,1l of IgG-coated latex particles in a plastic tube. This was incubated on a rotating mixer for 30 min at room temperature. The latex particles were diluted 1:500 in Isoton buffer (Coulter Electronics) and the number of unagglutinated particles counted in the Coulter counter. Samples were tested in duplicate and known positive and negative sera included in each batch. Results are expressed as percentage inhibition of agglutination (X%) and calculated thus: X=
(b-c) Ox 100%; (a-c)
where (a) is the number of particles added to each tube; (b) is the number of particles remaining after inhibition of agglutination by immune complex; and (c) is the number of particles remaining after maximum agglutination. Inhibition of IgG-coated latex agglutination by Clq may be used to detect Clq-binding immune complexes. Similarly, inhibition of DNP-IgA-coated latex agglutination by rabbit IgM anti-human IgA will detect IgA-containing complexes. Size calibration of complexes. A 90 x 3-2 cm Sepharose CL 6B (Pharmacia) column was calibrated using monomer IgM, dimer IgG, monomer IgG, F(ab')2 and pFc'. The column was run at 60 ml/hr using Tris-HCl pH 7-6 buffer. 2 ml samples of patient's sera were fractionated and all the fractions were tested for immune complexes by inhibition of latex agglutination.
R. J. Levinsky L J. F. Soothill
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Serum samples. Serum samples were obtained from patients with active systemic lupus erythematosus and nephritis (Levinsky, Cameron & Soothill, 1977) and one patient with Henoch-Schonlein purpura nephritis. Samples were separated within 3 hr of collection and stored in aliquots at - 70'C until analysed. Control serum samples were obtained from healthy adults and similarly handled.
RESULTS IgM antibody production Fig. 1 shows the results of sequential bleeds following an injection of 50 ug IgG into a rabbit. Serum was separated on Sephadex G-200 and the first and second peaks concentrated to starting volume by ultrafiltration. Agglutinating activity was titrated by slide latex agglutination, and precipitating activity by double gel diffusion. In the first peak, agglutination occurred maximally at day 15, whereas no precipitating activity was detected at any time. In the second peak, agglutination occurred at 12 days and precipitation at 15 days. Following immunization with 50 ig, 5 mg and 10 mg IgG in saline or Freund's complete adjuvant, the highest titre of agglutinating antibody in the first peak material was obtained using 10 mg IgG in FCA after bleeding at 10 days, and no precipitating antibody was obtained. This regimen has been used for all subsequent immunizations. After separating the first peak on a Sephadex G-200 column, it was absorbed with F(ab')2-Sigma cell immunoabsorbent to remove anti-light-chain
specificity. Quantitative latex agglutination Fig. 2 demonstrates the agglutination of IgG-coated latex particles by the rabbit IgM anti-human IgG reagent. As agglutination increases to a maximum, the number of single unagglutinated particles diminishes, and the subsequent rise is due to the prozone phenomenon of antibody excess. The reaction is 85% complete by 30 min and dilution in Isoton stops further agglutination. The antibody dilution giving 80-90% agglutination is used in the immune-complex test (1:100 in Fig. 2). This reaction may be inhibited by increasing concentrations of heat-aggregated IgG (10 mg/ml IgG heated to 630C for 15 min) (Fig. 3). 1 pg/ml of this preparation in buffer may easily be detected and as this is only 33% aggregated, actual sensitivity is less than 350 ng/ml of aggregated IgG. 1 yg/ml of column-separated aggregated IgG was also detected in 1:10 dilutions of normal sera. Monomeric IgG up to a concentration of 3 mg/ml (1 mg/ml in Fig. 3) gave less than 5% inhibition.
Decomplementing sera C1 inhibits agglutination of IgG-coated latex particles (Schubart, 1959) and Clq agglutinates them
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Days FIG. 1. Serum from sequential bleeds following injection of 50 ,ug human IgG in a rabbit were separated on Sephadex G-200. Titres (-log2) of agglutination of human IgG-coated latex are plotted for the IgMcontaining peak (o) and the IgG-containing peak (o). Presence of precipitating IgG antibody to human IgG on double gel diffusion is also shown (top line; (-) no precipitating antibody; (+) presence of precipitating antibody). No precipitating IgM antibody was obtained at any time.
A testfor antigen-antibody complexes
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Antibody dilution FIG. 2. Quantitative determination of agglutination of IgG-coated latex by sequential dilutions of rabbit IgM anti-human IgG reagent are shown. Agglutination increases until a prozone is reached (arrow). The antibody dilution giving 80-90% agglutination (1:100 in this case; shown by x on dotted line) is used in the immunecomplex test.
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FIG. 3. Inhibition of agglutination of IgG-latex by rabbit 1gM anti-human IgG reagent with increasing concentration of heat-aggregated (10 mg/ml heated to 630C for 15 mmn) IgG (.), and by monomeric IgG (0) (log probit scale). FIG. 4. Inhibition of agglutination of IgG-coated latex by either the rabbit 1gM anti-human IgG reagent, or by Clq, with sera from patients with SLE nephritis and sera from healthy adults (P< 0005, rank sum test).
R. J. Levinsky L J. F. Soothill
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TABLE 1. Normal sera incubated for 10 min at room temperature with 0-2 M EDTA, pH 7-6, strongly agglutinates IgG-coated latex to varying titres, but not after IgGSigma cell absorption
IgG-latex agglutination titre Sera no. EDTA only
EDTA-IgG-Sigma cell
1:512 1:256 1:128 1:256 1:64 1:256
0 0 0 0 0 0
1 2 3 4 5 6
(Ewald & Schubart, 1966). The Cl of fresh normal sera inhibits IgG-coated latex agglutination by rheumatoid factor, etc. (Lurhuma et aL, 1976). Clq released after incubating serum with EDTA agglutinates such particles, but after absorption with IgG-coated Sigma cell, no agglutination occurs (Table 1). The inhibitory effect of fresh sera, or serum decomplemented by our method or by heating on the agglutination of IgG-coated latex particles by the anti-IgG reagent or Clq are shown in Table 2. Four sera were from healthy subjects and two sera contained immune complexes. Our method greatly reduces the inhibition by normal serum, but the effect of heating is much less, presumably due to aggregation of IgG. After decomplementation, the abnormal sera were still inhibited because of immune complexes, but in the SLE serum, there was much less inhibition after heating; presumably because such complexes may be heat-labile (Zubler et al., 1976). Our method also removes intrinsic rheumatoid factor which would give a false negative in the test (e.g. heated rheumatoid serum gave a slide IgG-latex agglutination titre of 1:128 before absorption; 1:1 after absorption with IgG-Sigma cell). Immune complexes in SLE patients Fig. 4 shows the results of the tests for Clq binding and IgG-containing complexes applied to sera from twelve healthy adults and twenty-four patients with SLE nephritis (Levinsky, Cameron & Soothill, 1977). Correlation between the two tests was good (r = 0-91, P 3%/ inhibition are plotted. Arrow marks 9% inhibition of IgG-latex agglutination.
Reproducibility Preliminary data on nineteen samples run in duplicate show that the mean difference between duplicates was 2.8% inhibition of agglutination (range 0 1%-8.4%). Five samples run for a second time showed a mean difference between tests of 3.8% inhibition of agglutination (range 1.1%-6.4%). DISCUSSION The ideal tests for soluble immune complexes in serum-would permit easy sensitive measurement of complexes, with analysis for size and constituents (immunoglobulin class, complement components, antigen, etc.), without false positives. None of the many existing tests satisfy these criteria (Soothill, 1977) and strict quantification is clearly impossible in view of the heterogeneous nature of complexes. Our new test probably satisfies most of the other criteria, and is usefully semi-quantitative. Most existing tests depend on a combination of size, or physical precipitability with antigenic identification of the immunological reagent involved, or on the specificity of human antibodies or cell receptors for activated complement or denatured IgG (Soothill, 1977). The former are complex, insensitive and prone to false positives, and the latter are dependent on human reagents which are largely uncharacterized. Our method is derived from that of Lurhuma et al. (1976), in which complex-containing sera inhibit the agglutination of IgG-coated latex particles by human Clq or human serum containing rheumatoid factor. Both these reagents detect IgG-containing complexes only. Cl gives false positives which they overcame by storing sera for 1 week at 4VC before study, or heating sera to 560C which produces false positives and false negatives (Table 2). Apart from immune complexes, Clq combines with free DNA bacterial endotoxins and other proteins (Sobel, Bokisch & Muller-Eberhard, 1975), giving rise to false positives in the Clq test, whilst intrinsic rheumatoid factor results in false negatives in both tests. Many of these objections apply to most of the available tests for soluble complex. The automation of the latexagglutination-inhibition method (Cambiaso, Riccomi & Masson, 1977) is impressive, but open to the criticism that the latex used (0.79 p) is comparable in size to chylomicra in lipaemic sera. Rheumatoid factor specificity depends on low-affinityIgM anti-IgG antibody obtaining stable binding to denatured (aggregated) IgG by multivalent binding (Eisenberg, 1976; Dissanayake et al., 1977). It is likely that most IgM antibody is of low affinity and we have shown that reagents of this type can be prepared in rabbits by appropriate immunization and early bleeding. This should permit extension of the method to the study of most complex constituents and we have shown it for IgA as well as IgG. It should be possible to raise similar antibodies to viral, bacterial or food antigens. For identification of a complex constituent, the purified antigen used to raise the rabbit IgM antibody must be linked to the latex particles. It is not known how IgG attaches to latex, but most proteins do not. Our chance obser-
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vation of the effect of DNP permits a far wider use of latex agglutination in this and other tests. When latex is used for quantitative agglutination procedures, whether by DNP bonding or spontaneous IgG bonding, it is important to use low ionic strength buffers and appropriately low concentrations of coating protein, or spontaneous aggregation of latex particles occurs. Existing methods of decomplementing sera for analysis of immune complexes are unsatisfactory. Heating sera to 560C for 1 hr (Johnson, Mowbray & Porter, 1975), even at serum dilutions of 1:10, results in false positives in normal sera due to aggregation of intrinsic IgG, and false negatives due to destruction of immune complexes in some pathological sera (Zubler et al., 1976; Table 2). Our method of decomplementing does not cause aggregation of intrinsic IgG or loss of immune complexes. Intrinsic rheumatoid factor is also removed. The method would be applicable to any immune complex test which requires decomplementation as a preliminary procedure. The use of 1-15 pm latex particles prevents false positives from another cause. The very narrow meansize distribution of those particles (s.d.: + 0*0055 pm) allows the electronic window on the Coulter counter to be set to count that size particles only. As chylomicra are mainly less than 1 pm in size (Coulter Electronics, personal communication), lipaemic sera or post-prandial samples do not give false positives in this method. Fourteen out of twenty-four patients with SLE nephritis had IgG-containing complexes greater than the highest level obtained in the normal sera. The positive values obtained in some normal sera may have been detecting complexes, since it is probable that healthy people have low levels of them, and the sensitivity of our method may allow the variation expected within a normal population to be studied. Preliminary data on reproducibility of duplicates and repeated samples is good and could be improved further by the use of automated techniques. The technique does not require the sophisticated electronic particle counter. Slide latex agglutination inhibition would provide a useful simple diagnostic test. However, electronic particle counting gives a highly sensitive system which may be automated for rapid large-scale screening of sera. We would like to thank Professor J. S. Cameron for SLE sera, Dr M. W. Turner for help with immunochemical techniques and for the pFc', Dr D. R. Stanworth for IgM and dimer-rich IgG preparations, Dr P. L. Masson for helpful advice, and the National Fund for Research into Crippling Diseases for Fellowship support. Aspects of this technique are covered by U.K. Patent Application No. 48995/76.
REFERENCES CAMBIASO, C.L., RIccoMI, H. & MASSON, P.L. (1977) Automated determination of immune complexes by their inhibitory effect on the agglutination of IgG-coated particles by rheumatoid factor or Clq. Ann. Rheum Dis., 36, Suppl. 1, 40. DISSANAYAKE, S., HAY, F.C. & RoITT, I.M. (1977) The binding constants of IgM rheumatoid factors and their univalent fragments for native and aggregated human IgG. Immunology, 32, 309. EISENBERG, R. (1976) The specificity and polyvalency of binding of a monoclinal rheumatoid factor. Immunochemistry, 13, 355. EWALD, R.W. & SCHUBART, A.R. (1966) Agglutinating activity ofthecomplementcomponentClq in the F-Il latexfixation test. A. Immunol. 97, 100. FAHEY, J.L. & McKELvEY, E.M. (1965) Quantitative determination of serum immunoglobulins in antibodyagar plates. A. Immunol. 94, 84. JOHNSON, A.H., MOWBRAY, J.F. & PORTER, K.A. (1975) Detection of circulating immune complexes in pathological human sera. Lancet, i, 762. LEVINSKY, R.J., CAMERON, J.S. & SOOTHILL, J.F. (1977) Serum immune complexes and disease activity in lupus nephritis. Lancet, i, 564. LURHUMA, A.Z., CAMBIASO, C.L., MASSON, P.L. & HEREMANS, J.F. (1976) Detection of circulating antigen-
antibody complexes by their inhibitory effect on the agglutination of IgG-coated particles by rheumatoid factor or Clq. Clin. exp. Immunol. 25, 212. SCHUBART, A.F. (1959) Latex fixation test in rheumatoid arthritis. II. Characterization of the thermolabile inhibitor by a serologic study. New Engl. J. Med. 261, 579. SOBEL, A.J., BOKISCH, V.A. & MULLER-EBERHARD, M.J. (1975) Clq deviation test for the detection of immune complexes, aggregates of IgG, and bacterial products in human serum. I. exp. Med. 142, 139. SOOTHILL, J.F. (1977) Tests for soluble antigen-antibody complex in blood. Ann. rheum. Dis. 36, Suppl. I, 64. STANWORTH, D.R. (1960) A rapid method of preparing pure serum gamma-globulins. Nature (Lond.), 188, 156. STANWORTH, D.R. & TURNER, M.W. (1973) Immunochemical analysis of immunoglobulins and their subunits. Handbook of Experimental Immunology (ed. D. M. Weir), 2nd edn, Chap. 10. Blackwell Scientific Publications. YONEMASU, K. & STRouD, R.M. (1971) Clq: rapid purification method for preparation of monospecific antisera and for biochemical studies. ]. Immunol. 106, 304. ZUBLER, R.H., LANGE, G., LAMBERT, P.H. & MIESCEIER, P.A. (1976) Detection of immune complexes in unheated sera by a modified "2'I-labelled Clq-binding test. J. Immunol. 116, 232.
A test for antigen-antibody complexes in human sera using IgM of rabbit antisera to human immunoglobulins R. J. LEVINSKY & J. F. SOOTHILL Department of Immunology, Institute of Child Health, London
(Received 31 March 1977) SUMMARY
A simple semi-quantitative test for soluble antigen-antibody complexes using characterized nonhuman reagents and permitting analysis of their constituents is described. The agglutination of immunoglobulin-coated latex particles by rabbit IgM antibodies to the immunoglobulin is inhibited by complex-containing sera. No inhibition is obtained with monomer immunoglobulins. Semi-quantitative measurement of complexes may be made by electronically counting residual unagglutinated latex particles. The new method of linking proteins to latex by DNP coupling permits the technique to be applied to all constituents of complexes; immunoglobulins, complement and antigens. A new method of decomplementing sera by EDTA-Sigma cell-IgG absorption allows analysis of sera without the false positives and false negatives other methods give. The test gave positive results for IgG complexes in most of twenty-four patients with SLE nephritis. IgA complexes were identified in a patient with Henoch-Schonlein purpura nephritis.
INTRODUCTION Available methods for detecting immune complexes are not fully satisfactory and do not permit analysis of their constituents (Soothill, 1977). We have developed a simple semi-quantitative test for immune complexes, using characterized non-human reagents, which is capable of analysing the constituents. It is based on that of Lurhuma et al. (1976) in which immune complexes inhibit the agglutination of IgGcoated latex particles by rheumatoid factor or Clq. Human rheumatoid factors are low-affinity IgM antibodies to intrinsic IgG (Eisenberg, 1976; Dissanayake, Hay & Roitt, 1977) and, because of their multivalency, have a high avidity for aggregated IgG or IgG-containing complexes. We have prepared IgM antibodies in rabbits to the different complex constituents, which react with aggregated or complexed proteins but not with monomer. The new method of linking antigens to latex particles permits a far wider application of the method, and we have developed a method of removing complement which avoids the problems of the existing methods.
MATERIALS AND METHODS IgG. Purified human IgG was obtained by ion-exchange chromatography on Whatman DE 52 cellulose (Stanworth, 1960). The material was processed twice. Purity was assessed by immunoelectrophoresis using polyvalent anti-human serum. IgA. The IgA protein was purified from human myeloma serum by a three-stage procedure using Geon-Pevikon block electrophoresis, ion-exchange chromatography on a Whatman DE 52 cellulose column with a stepwise buffer gradient of 0-05-0-5 MTris-HCl, pH 8-0, and finally gel filtration on a Sephadex G-200 column. No impurities were found on immunoelectrophoresis and IgG, IgM and albumin were not detected by single radial immunodiffusion (Fahey & McKelvey, 1965). IgA and IgG were stored in a concentration of 10 mg/ml in saline at - 700C. Clq. Human Clq was prepared from fresh-frozen plasma by the method of Yonemasu & Stroud (1971). The protein was stored at a concentration of 500 pg/ml in saline at - 70'C. F(ab')2 fragment. This fragment was prepared from human IgG (Kabi) by pepsin digestion for 48 hr using an enzyme: substrate ratio of 1:50 (Stanworth & Turner, 1973). The fragment was isolated by gel filtration of the digest on Sephadex G-200. Correspondence: Dr R. J. Levinsky, Department of Immunology, Institute of Child Health, London W.C. 1.
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Immunoabsorbents. Insoluble immunoabsorbents of IgG and F(ab')2 were made by linking these proteins to Sigma cell (Sigma Chemicals, 50 p size) by cyanogen-bromide activation. 5 g Sigma cell was suspended in 100 ml water. 5 g cyanogen bromide (Fluka) was dissolved in 100 ml water in a fume cupboard. The two reagents were mixed together for 10 min at room temperature, maintaining the pH between 10-11 by the addi tion of 1 M sodium hydroxide. The activated Sigma cell was washed with 5 litres of cold 0- 1 M sodium bicarbonate solution in a Buchner funnel and then resuspended in 20 ml coupling buffer (76-8 ml M NaHCO3 mixed with 7*7 ml M Na2CO3 and made up to 1 litre with water). 150 mg of the protein to be linked was dissolved in 7 ml of coupling buffer and added to the activated Sigma cell. Coupling occurred overnight at 40C. The washed F(ab')2-Sigma cell was stored at 4VC in Tris-HCl, pH 8-2. The IgG-Sigma cell was stored as a 1:5 suspension (1 vol packed Sigma cell:4 vol buffer) in 0-27 M glycine saline adjusted to pH 8-2 with 1 M sodium hydroxide at 4VC, ready for use in the decomplementing stage of the immune-complex test. Preparation of rabbit JgM reagent. 10 mg of purified protein (IgG or IgA) in Freund's complete adjuvant was injected i.m. in four sites into rabbits. The rabbits were bled out at 10 days and the serum obtained was separated on a Sephadex G-200 column. The exclusion peak containing IgM was concentrated to starting volume by ultrafiltration. Light-chain specificity was removed by shaking with Sigma cell F(ab')2 immunoabsorbent for 1 hr at room temperature. The supernatant following centrifugation was stored in aliquots at - 700C. Latex coupling. Polyvinyl toluene latex particles 1l15 pm in diameter (s.d.+ 0-0055 pm),), supplied by Coulter Electronics as a 10%I w/v solution, were used. 800 pl latex was washed twice in 0-054 M glycine saline, pH 8-2 (1 vol. 0-27 M glycine saline, pH 8-2, +4 vol. water). The latex was centrifuged at 12,500 g and reconstituted in 20 ml 0 054 M glycine saline, pH 8-2. 302 pul of a 10 mg/ml solution of IgG (40 ,pg IgG/mg dry weight latex) was added and the suspension mixed for 30 min at room temperature. The latex was then washed twice in 0 054 M glycine saline, pH 8-2, and reconstituted to 20 ml in 0-27 M glycine saline, pH 8-2, containing 0- 1% human serum albumin to block remaining latex protein binding sites. All buffers were millipore filtered (0-22 ,pm) prior to use. This method results in the latex binding IgG, but not IgA or many other proteins. However, if DNP is substituted into the protein molecule latex coupling may be effected. DNP-IgA preparation. 10 mg IgA in 1 ml saline was dialysed for 3 hr at 370C against 10%4 w/v sodium bicarbonate. 3 ,ul DNFB (2-4-dinitrofluorobenzene, Sigma Chemicals) was added with constant mixing at 37°C, and when a bright yellow colour was obtained, the reaction was stopped by the addition of an equal vol. of benzene to extract excess DNFB. The resultant DNP-IgA solution was dialysed for 24 hr against 0 054 M glycine saline, pH 8-2. DNP-IgA may be coupled to latex using the same method as for IgG. Coated latex preparations were stored at 40C and were stable for at least 3 months. The latex suspension was sonicated before use each time. Slide agglutination test. This was used initially for testing agglutinating titres of antisera. Doubling dilutions of the sera were prepared. 25,pl of antisera dilution was mixed with 25 ,pl latex suspension and agglutination read visually at 3 min. When the test for soluble complex is done on a slide, 25 ,ul of suitably diluted IgM reagent, 25 p1 of IgG-coated latex and 25 ,pl of 1:10 dilutions of decomplemented patient's serum are mixed and read as above. Further dilution of patient's sera may be made. Immune complex test. Sera (50 pl) were decomplemented by incubation with 50,u1 02 M EDTA, pH 7-6, for 10 min at room temperature to release Clq. To remove this, 500 ,pl of freshly washed and resuspended (1 vol. packed immunoabsorbent: 4 vol. 0-27 M glycine saline, pH 8-2) IgG-coated Sigma cell was added to the EDTA serum and shaken for 20 min at room temperature. Any rheumatoid factor in the serum is also removed by this procedure. The supernatant after centrifugation (750 g for 3 min) is a 1:10 dilution. The Coulter counter model ZB, using the 30 ,pm orifice tube, was set to count latex of 1-15 ,um size by adjusting the upper and lower thresholds to obtain an electronic window which included the monomer peak but excluded smaller particles downwards and aggregates of dimer upwards. The latex suspension was adjusted to give a count of 2-2-5 x 104 particles/50 ,ul of final dilution, so that errors due to coincidence were < 1%/ and could be disregarded (other counters may be used). Detection ofIgG-containing complexes. Each batch of rabbit IgM and IgG was titrated; the dilution giving 70-80%, agglutination of IgG-coated latex was used in the test. 100 ul of decomplemented serum (1:10 dilution) was mixed with 100 p1I rabbit IgM anti-human IgG at appropriate dilution and 100,1l of IgG-coated latex particles in a plastic tube. This was incubated on a rotating mixer for 30 min at room temperature. The latex particles were diluted 1:500 in Isoton buffer (Coulter Electronics) and the number of unagglutinated particles counted in the Coulter counter. Samples were tested in duplicate and known positive and negative sera included in each batch. Results are expressed as percentage inhibition of agglutination (X%) and calculated thus: X=
(b-c) Ox 100%; (a-c)
where (a) is the number of particles added to each tube; (b) is the number of particles remaining after inhibition of agglutination by immune complex; and (c) is the number of particles remaining after maximum agglutination. Inhibition of IgG-coated latex agglutination by Clq may be used to detect Clq-binding immune complexes. Similarly, inhibition of DNP-IgA-coated latex agglutination by rabbit IgM anti-human IgA will detect IgA-containing complexes. Size calibration of complexes. A 90 x 3-2 cm Sepharose CL 6B (Pharmacia) column was calibrated using monomer IgM, dimer IgG, monomer IgG, F(ab')2 and pFc'. The column was run at 60 ml/hr using Tris-HCl pH 7-6 buffer. 2 ml samples of patient's sera were fractionated and all the fractions were tested for immune complexes by inhibition of latex agglutination.
R. J. Levinsky L J. F. Soothill
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Serum samples. Serum samples were obtained from patients with active systemic lupus erythematosus and nephritis (Levinsky, Cameron & Soothill, 1977) and one patient with Henoch-Schonlein purpura nephritis. Samples were separated within 3 hr of collection and stored in aliquots at - 70'C until analysed. Control serum samples were obtained from healthy adults and similarly handled.
RESULTS IgM antibody production Fig. 1 shows the results of sequential bleeds following an injection of 50 ug IgG into a rabbit. Serum was separated on Sephadex G-200 and the first and second peaks concentrated to starting volume by ultrafiltration. Agglutinating activity was titrated by slide latex agglutination, and precipitating activity by double gel diffusion. In the first peak, agglutination occurred maximally at day 15, whereas no precipitating activity was detected at any time. In the second peak, agglutination occurred at 12 days and precipitation at 15 days. Following immunization with 50 ig, 5 mg and 10 mg IgG in saline or Freund's complete adjuvant, the highest titre of agglutinating antibody in the first peak material was obtained using 10 mg IgG in FCA after bleeding at 10 days, and no precipitating antibody was obtained. This regimen has been used for all subsequent immunizations. After separating the first peak on a Sephadex G-200 column, it was absorbed with F(ab')2-Sigma cell immunoabsorbent to remove anti-light-chain
specificity. Quantitative latex agglutination Fig. 2 demonstrates the agglutination of IgG-coated latex particles by the rabbit IgM anti-human IgG reagent. As agglutination increases to a maximum, the number of single unagglutinated particles diminishes, and the subsequent rise is due to the prozone phenomenon of antibody excess. The reaction is 85% complete by 30 min and dilution in Isoton stops further agglutination. The antibody dilution giving 80-90% agglutination is used in the immune-complex test (1:100 in Fig. 2). This reaction may be inhibited by increasing concentrations of heat-aggregated IgG (10 mg/ml IgG heated to 630C for 15 min) (Fig. 3). 1 pg/ml of this preparation in buffer may easily be detected and as this is only 33% aggregated, actual sensitivity is less than 350 ng/ml of aggregated IgG. 1 yg/ml of column-separated aggregated IgG was also detected in 1:10 dilutions of normal sera. Monomeric IgG up to a concentration of 3 mg/ml (1 mg/ml in Fig. 3) gave less than 5% inhibition.
Decomplementing sera C1 inhibits agglutination of IgG-coated latex particles (Schubart, 1959) and Clq agglutinates them
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Days FIG. 1. Serum from sequential bleeds following injection of 50 ,ug human IgG in a rabbit were separated on Sephadex G-200. Titres (-log2) of agglutination of human IgG-coated latex are plotted for the IgMcontaining peak (o) and the IgG-containing peak (o). Presence of precipitating IgG antibody to human IgG on double gel diffusion is also shown (top line; (-) no precipitating antibody; (+) presence of precipitating antibody). No precipitating IgM antibody was obtained at any time.
A testfor antigen-antibody complexes
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Antibody dilution FIG. 2. Quantitative determination of agglutination of IgG-coated latex by sequential dilutions of rabbit IgM anti-human IgG reagent are shown. Agglutination increases until a prozone is reached (arrow). The antibody dilution giving 80-90% agglutination (1:100 in this case; shown by x on dotted line) is used in the immunecomplex test.
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FIG. 3. Inhibition of agglutination of IgG-latex by rabbit 1gM anti-human IgG reagent with increasing concentration of heat-aggregated (10 mg/ml heated to 630C for 15 mmn) IgG (.), and by monomeric IgG (0) (log probit scale). FIG. 4. Inhibition of agglutination of IgG-coated latex by either the rabbit 1gM anti-human IgG reagent, or by Clq, with sera from patients with SLE nephritis and sera from healthy adults (P< 0005, rank sum test).
R. J. Levinsky L J. F. Soothill
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TABLE 1. Normal sera incubated for 10 min at room temperature with 0-2 M EDTA, pH 7-6, strongly agglutinates IgG-coated latex to varying titres, but not after IgGSigma cell absorption
IgG-latex agglutination titre Sera no. EDTA only
EDTA-IgG-Sigma cell
1:512 1:256 1:128 1:256 1:64 1:256
0 0 0 0 0 0
1 2 3 4 5 6
(Ewald & Schubart, 1966). The Cl of fresh normal sera inhibits IgG-coated latex agglutination by rheumatoid factor, etc. (Lurhuma et aL, 1976). Clq released after incubating serum with EDTA agglutinates such particles, but after absorption with IgG-coated Sigma cell, no agglutination occurs (Table 1). The inhibitory effect of fresh sera, or serum decomplemented by our method or by heating on the agglutination of IgG-coated latex particles by the anti-IgG reagent or Clq are shown in Table 2. Four sera were from healthy subjects and two sera contained immune complexes. Our method greatly reduces the inhibition by normal serum, but the effect of heating is much less, presumably due to aggregation of IgG. After decomplementation, the abnormal sera were still inhibited because of immune complexes, but in the SLE serum, there was much less inhibition after heating; presumably because such complexes may be heat-labile (Zubler et al., 1976). Our method also removes intrinsic rheumatoid factor which would give a false negative in the test (e.g. heated rheumatoid serum gave a slide IgG-latex agglutination titre of 1:128 before absorption; 1:1 after absorption with IgG-Sigma cell). Immune complexes in SLE patients Fig. 4 shows the results of the tests for Clq binding and IgG-containing complexes applied to sera from twelve healthy adults and twenty-four patients with SLE nephritis (Levinsky, Cameron & Soothill, 1977). Correlation between the two tests was good (r = 0-91, P 3%/ inhibition are plotted. Arrow marks 9% inhibition of IgG-latex agglutination.
Reproducibility Preliminary data on nineteen samples run in duplicate show that the mean difference between duplicates was 2.8% inhibition of agglutination (range 0 1%-8.4%). Five samples run for a second time showed a mean difference between tests of 3.8% inhibition of agglutination (range 1.1%-6.4%). DISCUSSION The ideal tests for soluble immune complexes in serum-would permit easy sensitive measurement of complexes, with analysis for size and constituents (immunoglobulin class, complement components, antigen, etc.), without false positives. None of the many existing tests satisfy these criteria (Soothill, 1977) and strict quantification is clearly impossible in view of the heterogeneous nature of complexes. Our new test probably satisfies most of the other criteria, and is usefully semi-quantitative. Most existing tests depend on a combination of size, or physical precipitability with antigenic identification of the immunological reagent involved, or on the specificity of human antibodies or cell receptors for activated complement or denatured IgG (Soothill, 1977). The former are complex, insensitive and prone to false positives, and the latter are dependent on human reagents which are largely uncharacterized. Our method is derived from that of Lurhuma et al. (1976), in which complex-containing sera inhibit the agglutination of IgG-coated latex particles by human Clq or human serum containing rheumatoid factor. Both these reagents detect IgG-containing complexes only. Cl gives false positives which they overcame by storing sera for 1 week at 4VC before study, or heating sera to 560C which produces false positives and false negatives (Table 2). Apart from immune complexes, Clq combines with free DNA bacterial endotoxins and other proteins (Sobel, Bokisch & Muller-Eberhard, 1975), giving rise to false positives in the Clq test, whilst intrinsic rheumatoid factor results in false negatives in both tests. Many of these objections apply to most of the available tests for soluble complex. The automation of the latexagglutination-inhibition method (Cambiaso, Riccomi & Masson, 1977) is impressive, but open to the criticism that the latex used (0.79 p) is comparable in size to chylomicra in lipaemic sera. Rheumatoid factor specificity depends on low-affinityIgM anti-IgG antibody obtaining stable binding to denatured (aggregated) IgG by multivalent binding (Eisenberg, 1976; Dissanayake et al., 1977). It is likely that most IgM antibody is of low affinity and we have shown that reagents of this type can be prepared in rabbits by appropriate immunization and early bleeding. This should permit extension of the method to the study of most complex constituents and we have shown it for IgA as well as IgG. It should be possible to raise similar antibodies to viral, bacterial or food antigens. For identification of a complex constituent, the purified antigen used to raise the rabbit IgM antibody must be linked to the latex particles. It is not known how IgG attaches to latex, but most proteins do not. Our chance obser-
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vation of the effect of DNP permits a far wider use of latex agglutination in this and other tests. When latex is used for quantitative agglutination procedures, whether by DNP bonding or spontaneous IgG bonding, it is important to use low ionic strength buffers and appropriately low concentrations of coating protein, or spontaneous aggregation of latex particles occurs. Existing methods of decomplementing sera for analysis of immune complexes are unsatisfactory. Heating sera to 560C for 1 hr (Johnson, Mowbray & Porter, 1975), even at serum dilutions of 1:10, results in false positives in normal sera due to aggregation of intrinsic IgG, and false negatives due to destruction of immune complexes in some pathological sera (Zubler et al., 1976; Table 2). Our method of decomplementing does not cause aggregation of intrinsic IgG or loss of immune complexes. Intrinsic rheumatoid factor is also removed. The method would be applicable to any immune complex test which requires decomplementation as a preliminary procedure. The use of 1-15 pm latex particles prevents false positives from another cause. The very narrow meansize distribution of those particles (s.d.: + 0*0055 pm) allows the electronic window on the Coulter counter to be set to count that size particles only. As chylomicra are mainly less than 1 pm in size (Coulter Electronics, personal communication), lipaemic sera or post-prandial samples do not give false positives in this method. Fourteen out of twenty-four patients with SLE nephritis had IgG-containing complexes greater than the highest level obtained in the normal sera. The positive values obtained in some normal sera may have been detecting complexes, since it is probable that healthy people have low levels of them, and the sensitivity of our method may allow the variation expected within a normal population to be studied. Preliminary data on reproducibility of duplicates and repeated samples is good and could be improved further by the use of automated techniques. The technique does not require the sophisticated electronic particle counter. Slide latex agglutination inhibition would provide a useful simple diagnostic test. However, electronic particle counting gives a highly sensitive system which may be automated for rapid large-scale screening of sera. We would like to thank Professor J. S. Cameron for SLE sera, Dr M. W. Turner for help with immunochemical techniques and for the pFc', Dr D. R. Stanworth for IgM and dimer-rich IgG preparations, Dr P. L. Masson for helpful advice, and the National Fund for Research into Crippling Diseases for Fellowship support. Aspects of this technique are covered by U.K. Patent Application No. 48995/76.
REFERENCES CAMBIASO, C.L., RIccoMI, H. & MASSON, P.L. (1977) Automated determination of immune complexes by their inhibitory effect on the agglutination of IgG-coated particles by rheumatoid factor or Clq. Ann. Rheum Dis., 36, Suppl. 1, 40. DISSANAYAKE, S., HAY, F.C. & RoITT, I.M. (1977) The binding constants of IgM rheumatoid factors and their univalent fragments for native and aggregated human IgG. Immunology, 32, 309. EISENBERG, R. (1976) The specificity and polyvalency of binding of a monoclinal rheumatoid factor. Immunochemistry, 13, 355. EWALD, R.W. & SCHUBART, A.R. (1966) Agglutinating activity ofthecomplementcomponentClq in the F-Il latexfixation test. A. Immunol. 97, 100. FAHEY, J.L. & McKELvEY, E.M. (1965) Quantitative determination of serum immunoglobulins in antibodyagar plates. A. Immunol. 94, 84. JOHNSON, A.H., MOWBRAY, J.F. & PORTER, K.A. (1975) Detection of circulating immune complexes in pathological human sera. Lancet, i, 762. LEVINSKY, R.J., CAMERON, J.S. & SOOTHILL, J.F. (1977) Serum immune complexes and disease activity in lupus nephritis. Lancet, i, 564. LURHUMA, A.Z., CAMBIASO, C.L., MASSON, P.L. & HEREMANS, J.F. (1976) Detection of circulating antigen-
antibody complexes by their inhibitory effect on the agglutination of IgG-coated particles by rheumatoid factor or Clq. Clin. exp. Immunol. 25, 212. SCHUBART, A.F. (1959) Latex fixation test in rheumatoid arthritis. II. Characterization of the thermolabile inhibitor by a serologic study. New Engl. J. Med. 261, 579. SOBEL, A.J., BOKISCH, V.A. & MULLER-EBERHARD, M.J. (1975) Clq deviation test for the detection of immune complexes, aggregates of IgG, and bacterial products in human serum. I. exp. Med. 142, 139. SOOTHILL, J.F. (1977) Tests for soluble antigen-antibody complex in blood. Ann. rheum. Dis. 36, Suppl. I, 64. STANWORTH, D.R. (1960) A rapid method of preparing pure serum gamma-globulins. Nature (Lond.), 188, 156. STANWORTH, D.R. & TURNER, M.W. (1973) Immunochemical analysis of immunoglobulins and their subunits. Handbook of Experimental Immunology (ed. D. M. Weir), 2nd edn, Chap. 10. Blackwell Scientific Publications. YONEMASU, K. & STRouD, R.M. (1971) Clq: rapid purification method for preparation of monospecific antisera and for biochemical studies. ]. Immunol. 106, 304. ZUBLER, R.H., LANGE, G., LAMBERT, P.H. & MIESCEIER, P.A. (1976) Detection of immune complexes in unheated sera by a modified "2'I-labelled Clq-binding test. J. Immunol. 116, 232.
