A new radioimmunoassay for IgM and IgG rheumatoid factors, based on a double antibody method

Journal oflmmunological Methods, 47 (1981) 87--97

87

Elsevier/North-Holland Biomedical Press

A NEW RADIOIMMUNOASSAY FOR IgM AND IgG RHEUMATOID FACTORS, BASED ON A DOUBLE ANTIBODY METHOD

OLE N O R D F A N G 1, MIMI HOIER-MADSEN 2, POUL HALBERG 3 and J O H A N N A LIEBERKIND 2

I Department o f Nuclear Medicine, Rigshospitalet, Blegdamsvej 9, DK-21 O0 Copenhagen, 2 Bloodbank/Bloodgrouping Department, Statens Seruminstitut, Amager Boulevard 80, DK-2300 Copenhagen, and 3 Medical Department Division o f Rheumatology, Hvidovre Hospital, Kettegaard Alle 30, DK-2650 Hvidovre, Denmark (Received 20 February 1981, accepted 30 June 1981)

A new radioimmunoassay has been developed for measuring IgM and IgG rheumatoid factors. Diluted sera from donors and patients were incubated with immunoprecipitates prepared from sheep serum and rabbit anti-sheep IgG antiserum. The precipitates were washed, and radioiodinated rabbit F(ab')2 fragments specific for human IgM or IgG were added. The precipitates were isolated by filtration and measured in a gamma counter. With this assay IgM rheumatoid factors were detected in sera from all patients with seropositive rheumatoid arthritis and in sera from 40% of patients with seronegative rheumatoid arthritis. IgG rheumatoid factors were found in sera from 68% of the seropositive and 40% of the seronegative patients. Gel filtration experiments demonstrated that it is possible to detect monomeric IgG rheumatoid factors and IgM rheumatoid factors of molecular weight smaller than pentameric IgM. Furthermore it has been shown that IgG rheumatoid factor activity is still present after reduction of IgM rheumatoid factors with dithiothreitol.

INTRODUCTION

Rheumatoid factors (RF), i.e., antibodies with specificity for the Fc region of IgG (Johnson and Faulk, 1976), are demonstrable by tests which have been used for many years designed by Waaler (1940), Rose et al. (1948), and Singer and Plotz (1956). The classical tests for RF detect IgM but not IgG RF, since high avidity antibodies are needed for agglutination of sensitized cells and latex particles (Yamagata et al., 1979). IgG RF has been demonstrated by binding assays. Torrigiani and Roitt (1967, 1970) utilized binding of RF to aggregated insoluble IgG, but the technique is cumbersome and difficult to standardize (Turner, 1976). Hay et al. (1975) and Carson et al. (1977) substituted plastic tubes coated with IgG. Using a similar technique, which measures IgM RF reliably (Vejtorp et al., 1979), we failed to detect IgG RF in sera of patients with rheumatoid arthritis (unpublished observation). We have developed a new assay, based on the principle of double anti0022-1759/81/0000--0000/$02.75 © 1981 Elsevier/North-Holland Biomedical Press

88

ShlgO ( SHEEP SERUM )

R ~ S h l g O ( RABBIT ANTISERUM ) ABSORBED WITH HUMAN IgG

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COLLECT PRECIPITATE AND COUNT

Fig. 1. Procedure for precipitation RIA for rheumatoid factors.

b o d y radioimmunoassay, which has high reproducibility (Nordfang, 1981). The principle of the radioimmunoassay is shown in Fig. 1. MATERIALS AND METHODS

Antisera Rabbits were immunized with 1 mg of DEAE purified sheep IgG in complete Freund's adjuvant followed b y booster injections o f 100 pg at intervals o f 5 weeks. The rabbits were bled 1 and 2 weeks after each booster injection. Rabbit anti-human IgM and rabbit anti-human IgG were obtained from DAKO (Copenhagen). Protein chemistry Highly purified human IgG was obtained from ammonium sulfate precipitated normal human immunoglobulin. After DEAE chromatography, the IgG fraction was further purified b y gel filtration on Ultrogel AcA 34 (LKB, France). Fractions were tested b y rocket electrophoresis, as described b y Weeke (1973), with anti-IgM and anti-IgG, and the IgG peak contained no detectable IgM. Highly purified human IgM was obtained in a similar way, from ammonium sulfate precipitated immunoglobulin from the serum of a patient with WaldenstrSm's macroglobulinemia.

s9 For gel filtration experiments, IgG and IgM from normal human serum were used as markers. The main IgG and IgM peaks, determined by rocket electrophoresis, were taken to represent monomeric IgG and pentameric IgM respectively. F(ab')2 fragments were prepared from rabbit immunoglobulin by a modification of the method of Nisonoff (1964). The immunoglobulin was digested for 18 h with 2% (w/w) pepsin at pH 4. After neutralization and dialysis the F(ab'): fragments were obtained by gel filtration and absorption with protein-A Sepharose (Pharmacia, Sweden) at pH 8 (Endersen, 1978; Nordfang, 1981).

Absorption o f rabbit anti-sheep IgG antiserum 1.8 g human immunoglobulin (ammonium sulfate precipitated) was coupled to 125 ml CNBr-activated Sepharose according to the directions of the manufacturer (Pharmacia, Sweden). This was used for absorption of 40 ml rabbit anti-sheep IgG antiserum. Non-reacted antiserum was eluted with PBS (phosphate-buffered saline, pH 7.4), and concentrated to the original volume. The immunosorbent was regenerated with 5 M guanidine hydrochloride/1 M NaC1. Preparation o f monospecific anti-human IgM and anti-human IgG F(ab'): fragments from 100 mg rabbit anti-human IgG were absorbed with 100 mg ammonium sulfate precipitated sheep immunoglobulin coupled to 25 ml Sepharose. The absorbed F(ab'): fragments were passed through a column containing 20 mg highly purified human IgG coupled to 10 ml Sepharose. Specific antibodies were eluted with 0.1 M glycine-HC1 buffer, pH 2.5, and collected in 1 M Tris buffer, pH 8. The F(ab')~ fragments were filtered through Ultrogel AcA 44 to remove possible aggregates formed during purification. The amount of monospecific F(ab')2 fragments against human IgG obtained in this way was 1.5 mg. Monospecific F(ab')2 fragments against human IgM were obtained in a similar way, with a yield of 2.0 mg. Radioiodination This was carried out using the chloramine-T method of Hunter and Greenwood (1962). 50 gg protein was labeled with 0.5 mCi ~:sI, with resulting specific activity of 6 ~Ci/~g. Radioimm u noassay The principle of the radioimmunoassay is shown in Fig. 1.20 pl absorbed rabbit anti-sheep IgG antiserum and 50 gl 1.5% (v/v) normal sheep serum in buffer A were incubated for 1 h at 37°C to form an immune precipitate. Buffer A was 1% (w/v) BSA in PBS containing 0.02% sodium azide. The rabbit antiserum and the sheep serum were heat inactivated. If these reagents were not inactivated, donor sera stored for about 3 years at --20°C were positive in the subsequent test for IgG RF.

90 The precipitate was incubated with 50/~l human serum, diluted in buffer A, for 1 h at 37°C and 1 h at 4°C. A 400-fold dilution was used, since this resulted in the best discrimination between 10 d o n o r sera and 10 Waaler-Rose positive sera. The precipitate was washed 3 times with 2 ml 0.1% (w/v) BSA. After centrifugation (5 min at r o o m temperature, 1700 X g), the supernatant was drained o f f b y turning the tubes upside down. 10 ng radioiodinated F(ab')2 anti-human IgM or IgG in 50/~l buffer A was then added to each tube. After overnight incubation at r o o m temperature, 1.5 ml 4% PEG (polyethylene glycol 6000) was added and the precipitate recovered in a Scatron cell harvester with Whatman GF/B glass-fiber filters as described by Jensenius (1977). Rheumatoid factors present were measured as a percentage of the amount of radioiodinated anti-human immunoglobulin added. 100% was equal to the a m o u n t precipitated with trichloroacetic acid (TCA, 10%), and 0% was the non-specific binding. Non-specific binding was determined in samples witho u t human serum, and was a b o u t 1% of the counts found in the TCA precipitates. Dithiothreitol treatment Ten pl serum was incubated for 1 h at 37°C with 100 pl 0.005 M dithiothreitol (DL-DTT) in PBS, and a final dilution o f 1 : 400 was prepared in buffer A. Following this procedure, no gel formation in the serum was observed, and the final dilution was used directly in the radioimmunoassay. Clinical material D o n o r sera were obtained from 200 healthy subjects. 10 sera from patients with seronegative rheumatoid arthritis were a gift from Dr. P. Helin, King Christian X's Rheumatic Hospital, Graasten. Sera from patients with psoriatic arthritis were supplied b y Dr. O. Clemmensen, Dermatology Clinic, the Finsen Hospital, Copenhagen. All other sera were from patients in the Dept. of Medicine, Division of R h e u m a t o l o g y , Hvidovre Hospital, Copenhagen. All patients with seropositive rheumatoid arthritis had Waaler-Rose titers o f 40 or more as determined b y the routine test for IgM R F p e r f o r m e d by the State Serum Institute, Copenhagen. The patients with seropositive rheumatoid arthritis included 11 with active synovitis, 2 with Felty's syndrome and 9 with vasculitis w i t h o u t active synovitis. For comparison of paired samples the Mann-Whitney U-test was used. RESULTS IgM rheumatoid factors determined by R I A in various diseases An upper normal limit o f 8.75% binding of anti-IgM was calculated from results on 200 d o n o r sera after excluding the 5% with the highest binding values. Fig. 2 shows that all patients with seropositive arthritis were positive

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Fig. 2. IgM rheumatoid factor levels in different diseases. The vertical line sets the limit between positive and negative sera.

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Fig. 3. IgG rheumatoid factor in different diseases. The vertical line sets the limit between positive and negative sera.

92

in the RIA test for IgM RF. The patients with Felty's syndrome and vasculitis had higher values than the patients with active synovitis (P < 0.05). 40% of the patients with seronegative rheumatoid arthritis were positive for IgM RF by the RIA test. The IgM RF levels in sera from patients with seronegative rheumatoid arthritis were significantly higher than in the donor sera

(P < 0.01). IgG rheumatoid factors determined by RIA in various diseases IgG RF was found in 5% of the donor sera, if 4% binding or more of antiIgG was taken as abnormal. According to the normal limit thus established, 68% o f sera from patients with seropositive arthritis were positive for IgG RF, including both patients with Felty's syndrome and 7. of 9 patients with vasculitis. There was no significant differences in level between the sera of the Felty's syndrome patients, and those with vasculitis or synovitis in the binding o f anti-IgG in the assay. IgG RF was found in 10 o f 24 sera from patients with seronegative rheumatoid arthritis, and 50% of the routinely seronegative sera positive for IgM RF by RIA were also positive for IgG RF. IgG RF levels were significantly higher in patients with seronegative rheumatoid arthritis than in the donors (P < 0.01). All the patients with psoriatic arthritis were negative for IgG RF and levels did not differ from those in the donors.

TABLE 1

Effect of D T T treatment on the a m o u n t s of IgG and IgM rheumatoid factor detected. Sera from 6 donors and sera form 6 RIA IgG RF positive patients with seropositive rheumatoid arthritis were tested in the usual way (--DTT), and after reduction with DTT (+DTT).

% binding of anti-IgG

% binding o f anti-IgM

--DTT

+DTT

--DTT

+DTT

15.1 9.2 7.3 17.7 9.2 15.0

14.5 5.3 7.6 14.7 3.8 15.2

33.1 29.8 37.8 34.2 32.1 38.8

3.6 3.2 6.7 3.8 2.4 7.3

2.7 2.3 1.4 1.8 0.4 0.5

1.5 0.7 1.2 1.8 0.0 1.2

3.7 2.3 5.6 6.1 2.0 3.1

1.1 0.6 0,4 0.6 0.3 0.7

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Fig. 4. Ultrogel A c A - 3 4 f r a c t i o n s o f a p o o l o f sera having Waaler-Rose titers >/ 5120. E a c h

fraction was tested undiluted in the RIA. Arrows indicate fractions containing the highest a m o u n t o f IgM or IgG b y r o c k e t e l e c t r o p h o r e s i s , e, e x t i n c t i o n at 280 n m ; ~, IgM R F c o n -

tent; ~, IgG R F content.

TABLE 2 S p e c i f i c i t y in t h e R I A . T w o s e r u m p o o l s w e r e i n c u b a t e d w i t h d i f f e r e n t c o m p o n e n t s o f the p r e c i p i t a t e . Per c e n t b i n d i n g is given as t h e m e a n o f t w o i n d e p e n d e n t assay ± 1 S.D. NShS (+/--) d e n o t e s i n c u b a t i o n w i t h or w i t h o u t n o r m a l s h e e p s e r u m . R--> ShIg (+/--) d e n o t e s i n c u b a t i o n with o r w i t h o u t r a b b i t a n t i - s h e e p IgG a n t i s e r u m . R ~ ShIg

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8.6±1.1 24.1±1.8

8.2±2.8 36.6±3.1

3.1±0.2 2.3±0.4

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Fig. 5. U]troge] A c A - 3 4 f r a c t i o n s o f a single serum. Each f r a c t i o n was tested u n d i l u t e d in

the RIA. Arrows indicate the fractions containing the highest a m o u n t of IgM or IgG

according to rocket electrophoresis, e, extinction at 280 nm; D, IgM RF content; a, IgG RF content.

D T T treatment and gel filtration o f sera containing rheumatoid factors According to Slobin and Singer (1968) and Olsop et al. (1976) DTT is a reducing agent which under certain conditions (Capel et al., 1980) destroys the a n t i b o d y activity of IgM and spares that of IgG. Table 1 shows that DTT treatment abolishes most of the IgM RF activity, but hardly any of the IgG RF activity. Fig. 4 shows the result of Ultrogel AcA 34 fractionation of a pool of sera with very high titers in the Waaler-Rose test. The peak IgM RF activity was f o u n d in fractions containing pentameric IgM, but some of the activity was also f o u n d in fractions with lower molecular weight. IgG RF activity peaked in the same fractions as monomeric IgG. Fig. 5 shows the result of fractionation of a serum with strong IgG RF activity, the peak of which was f o u n d in the same fractions as monomeric IgG. It will be seen t h a t some activity was also present in fractions with higher molecular weight, indicating complex formation. Specificity o f the rheumatoid factors in the assay Table 2 shows t h a t the IgM RF RIA detects some binding of IgM R F

95

which is n o t specific for the immune precipitate. With a pool of 20 seropositive sera, 8% of the anti-IgM was b o u n d in the absence o f b o t h c o m p o n e n t s o f the precipitate, and 24% was b o u n d in the presence o f only one component, e.g., rabbit anti-sheep IgG. This IgM R F activity in the absence of either c o m p o n e n t o f the precipitate could n o t be abolished by thorough centrifugation of the sera before testing. DISCUSSION

In this new RIA for IgM R F and IgG RF, the RFs are b o u n d to immune precipitates (sheep IgG/rabbit anti-sheep IgG) and detected with radioiodinated rabbit F(ab')2 fragments with specificity for human immunoglobulin. To avoid cross-reactions, the reagents are absorbed as shown in Fig. 1. Rabbit anti,sheep IgG is absorbed with human immunoglobulin to avoid binding o f normal human immunoglobulin. Anti-human immunoglobulin is absorbed with sheep IgG to avoid non-specific binding of the tracer to the precipitate. F(ab')2 fragments are used to avoid binding of anti-IgG if IgM R F is present, and vice versa (Carson et al., 1977). Monospecific anti-human immunoglobulin is used to reduce non-specific binding of radioiodinated rabbit immunoglobulin. Despite these absorptions some non-specific binding of IgM b u t not of IgG R F persisted. The nature of this binding in the absence o f either c o m p o n e n t o f the precipitate remains unexplained. IgM, b u t n o t IgG R F , m a y also be measured with reduced efficiency with rabbit anti-sheep IgG alone. The same effects was seen with another hyperimmune rabbit antiserum (rabbit anti-mouse IgG), b u t not with normal rabbit serum. This suggests that complexes in hyperimmune sera are sufficient to bind IgM RF in the assay. The advantage o f this assay as compared with the coated t u b e m e t h o d of Hay et al. (1975, 1979) is that reproducible immune precipitates may be made each day, whereas it m a y be difficult to achieve constant coating of tubes (Hunter, 1978). Furthermore, b o u n d rheumatoid factors are separated from the human immunoglobulin which sticks to plastic tubes (Koopman, 1980). The fractionation experiments described with the new RIA indicate that IgM R F exists as pentamers but also as molecules with a smaller degree of polymerization. The distribution of the IgM R F activity appears to vary from patient to patient. The assay also indicates that IgG R F may exist both as m o n o m e r and in complexes. These findings are in agreement with those of Carson et al. (1977) and Sato et al. (1977). The demonstration of considerable R F activity in fractions almost free of IgM RF, and after reduction of IgM RF with DTT, suggest that IgG RF is n o t a misinterpretation of complexes consisting o f IgG and IgM RF. Some o f the sera showed a slight decrease in IgG R F activity after DTT treatment. The reason for this may be that a small a m o u n t of normal IgG is b o u n d b y IgM R F in these sera. This is likely to occur also in other assays for IgG R F , and treatment with DTT

96

might be included in all tests for IgG RF. However, Yamagata et al. (1979) f o u n d that the concentration of IgG R F rises after DTT treatment, resulting in false positive values for IgG RF. When DTT treatment was performed on a 10-fold serum dilution, we did not observe this effect. According to Theofilopoulos et al. (1974) IgG RF is found in particularly high concentrations in patients with vasculitis. IgG RF has also been reported in the sera of patients with psoriatic arthritis (Howell et al., 1972). The present work does not confirm these findings. We found that IgG RF values were higher in patients with Felty's syndrome or with vasculitis than in patients with synovitis, b u t that the difference was not significant. These results with RIA demonstrate that the classical tests for IgM R F have low sensitivity, since several patients with seronegative rheumatoid arthritis were positive for IgM R F in the RIA test. This may be due to the fact that IgM RF in these patients is of low avidity. Four of the patients who were routinely seronegative and negative for IgM R F by the RIA were positive for IgG RF. ACKNOWLEDGEMENTS

The skilful technical assistance o f B. Rumler is gratefully acknowledged. We thank dr. J. Andersen, Statens Seruminstitut and our colleagues in Dept. o f Nuclear Medicine, Rigshospitalet for valuable comments on the manuscript. The animal department o f Statens Seruminstitut t o o k care of the rabbits. REFERENCES

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97 Singer, J.M. and C.M. Plotz, 1956, Am. J. Med. 21,888. Slobin, L.I. and S.J. Singer, 1968, J. Biol. Chem. 243, 1777. Theofilopoulos, A.N., G. Burtonboy, J.L LoSpalluto and M. Ziff, 1974, Arthr. Rheum. 17,272. Torrigiani, G. and I.M. Roitt, 1967, Ann. Rheum. Dis. 26, 334. Torrigiani, G. and I.M. Roitt, 1970, Lancet i, 14. Turner, M.W., 1976, in: Infection and Immunology of the Rheumatic Diseases, ed. C.D. Dumonde (Blackwell, London) p. 41. Vejtorp, M., M. H~bier-Madsen and P. Halberg, 1979, Scand. J. Rheumatol. 8, 65. Waaler, E., 1940, Acta Pathol. Microbiol. Scand. 17,172. Weeke, B., 1973, A Manual for Quantitative Immunoelectrophoresis, Methods and Applications (Universitetsforlaget, Oslo). Yagamata, J., E.V. Barnett, D.W. Knutson, H. Nasu and D. Chia, 1979, J. Immunol. Methods 29, 43.