Journal o f l mmu n o l o g ic a l Methods, 31 (1979) 109--118

109

© Elsevier/North-Holland Biomedical Press

A SIMPLE ENZYME IMMUNOASSAY FOR THE DEMONSTRATION OF RHEUMATOID FACTOR

MARIANNE GRIPENBERG l, FUAT WAFIN 2, HEIKKI ISOMAKI LINDER I

2

and EWERT

1 Department o f Bacteriology and Immunology, University o f Helsinki, Haartmaninkatu 3, SF-00290 Helsinki 29 and 2 Rheumatism Foundation Hospital, SF-18120 Heinola 12, Finland

(Received 20 March 1979, accepted 1 July 1979)

An enzyme-linked immunosorbent assay (ELISA) for the demonstration of rheumatoid factor (RF) is described. Human IgG is used as antigen in the assay and the presence of RF is demonstrated by anti-human IgM conjugated to alkaline phosphatase. The assay is simple to perform and can be completed in one day. The recording of results is objective by means of a spectrophotometer, or alternatively the results can be read by the naked eye. There is good agreement between the RF-ELISA results and those obtained by the classical latex fixation and Waaler--Rose tests. All sera with a latex fixation titre />128 and 70% (14/20) of sera with a titre of 64 were positive in the RF-ELISA, whereas 17/107 (16%) sera with titres between 4 and 32 were positive in the assay. 3 out of 70 (4%) Waaler--Rose positive sera gave negative results in the RF-ELISA. A positive RF-ELISA result was obtained in one out of 26 patients with non-specific arthritis studied. In 43 patients with a diagnosis of probable or definite rheumatoid arthritis, RF was detected in 27 cases (63%) by the RF-ELISA, whereas the classical tests detected RF in 21 cases (49%).

INTRODUCTION A n t i g l o b u l i n s , or r h e u m a t o i d f a c t o r s ( R F ) , d i r e c t e d a g a i n s t a n t i g e n i c sites s i t u a t e d p r i m a r i l y on t h e F c p o r t i o n o f t h e I g G m o l e c u l e ( J o h n s o n a n d F a u l k , 1 9 7 6 ) , are a f e a t u r e o f r h e u m a t o i d a r t h r i t i s ( R A ) , b u t m a y also o c c u r in o t h e r c o n d i t i o n s c h a r a c t e r i z e d b y c h r o n i c i n f l a m m a t i o n . R F are p r i n c i p a l l y o f IgM class, a l t h o u g h R F o f I g G o r I g A class also e x i s t ( S t a g e a n d Mann i k , 1 9 7 2 - - 7 3 ) ; t h e m a j o r i t y o f t h e I g M - R F has s p e c i f i c i t y f o r b o t h h u m a n a n d r a b b i t I g G ( N o r m a n s e l l , 1 9 7 2 ) . T h e i n c i d e n c e o f s e r o p o s i t i v i t y in R A is b e t w e e n 60 a n d 80%, d e p e n d i n g o n t h e d e t e c t i o n s y s t e m f o r R F . I f c o m p l e x e s b e t w e e n R F a n d n a t i v e I g G f o r m in vivo, t h e R F a c t i v i t y m a y be ' h i d d e n ' ( A l l e n a n d K u n k e l , 1 9 6 6 ) a n d a f t e r s p e c i a l t r e a t m e n t o f sera, R F m a y be d e t e c t e d in a b o u t 50% o f ' s e r o n e g a t i v e ' cases o f R A ( H a n s s o n a n d Winblad, 1978). T h e classical m e t h o d s o f d e t e c t i n g R F are t h e s h e e p cell a g g l u t i n a t i o n t e s t (Waaler, 1 9 4 0 ; R o s e et al., 1 9 4 8 ) a n d t h e l a t e x f i x a t i o n t e s t ( S i n g e r a n d

110 Plotz, 1956). For both clinical and research purposes there is a need for sensitive and accurate tests which detect RF, a fact reflected by the numerous reported methods for radioimmunoassay (Nineham et al., 1976; Carson et al., 1977; Knez and Reimer, 1977; Ziola et al., 1978) and enzyme-immunoassay (Maiolini et al., 1978; Willems and Klaassen de Kort, 1978). A reduction of the number of different methods now in use for the demonstration of antibodies would be of considerable advantage for the clinical laboratory. Various antibodies are readily demonstrated by the enzyme-linked immunosorbent assay (ELISA) (Voller et al., 1978) and the a u t o m a t i o n of this assay is also possible. In this report we describe an application of ELISA to detection of RF. This m e t h o d is simple and allows a large number of specimens to be tested simultaneously. The results are objectively recorded by a spectrophotometer or may be read by the naked eye. MATERIALS AND METHODS Standard methods for the demonstration o f R F The presence of RF was demonstrated by the standard latex fixation (Singer and Plotz, 1956) and Waaler--Rose tests (Waaler, 1940; Rose et al., 1948). The latex test was performed on slides (Latex-RF reagent, Behringwerke AG, Marburg, F.R.G.), a titre of ~>64 being regarded as positive, or by the single tube procedure according to Singer and Plotz (1958). In the Waaler--Rose test, heat-inactivated (56°C, 30 m i n ) s e r a were tested with rabbit anti-sheep cell IgG-coated sheep red blood cells and an agglutination titre of ~>30 was regarded as positive. Se ra 330 serum samples sent for investigation of the presence of RF were used in the studies. From these, latex-positive and latex-negative sera were pooled and used as positive and negative reference sera, respectively. The normal controls consisted of 105 sera from healthy blood donors. Sera from 43 patients (12 men, 31 w o m e n ) with probable (23 patients) or definite (20 patients) RA and from 26 patients (11 men, 15 women) with non-specific or post-infective arthritis or Reiter's disease were obtained from the Rheumatism F o u n d a t i o n Hospital, Heinola, Finland. Antigens The IgG fraction of human gamma globulin (Ab Kabi, Stockholm, Sweden) was purified by chromatography on DEAE cellulose. The purity of this preparation was checked by immunoelectrophoresis. Soluble aggregated IgG was produced by heating the IgG preparation for 20 min at 63 ° C. Rabbit IgG was prepared by chromatography of normal rabbit serum on DEAE cellulose.

111

RF-ELISA The ELISA pr ocedur e was p e r f o r m e d essentially as described previously (Gripenberg et al., 1978). Disposable 1-ml p o l y s t y r e n e microcuvettes, 9 in a block (Finnpipette-Labsystems, Helsinki, Finland), and flat b o t t o m microtitre plates {Cooke Engineering Co., Alexandria, VA, U.S.A.) were used as solid phase in the assay. Th e cuvettes were coated with 200 pl o f solutions containing 1--500 pg IgG/1 ml phosphate-buffered saline (PBS), pH 7.2. After incubation for 3 h at 37°C, the cuvettes were washed 3 times with distilled water. When n o t used immediately t h e y were stored dry at 4°C for up to 8 weeks. Sera were diluted in PBS containing 0.05% Tween 20 (PBS-T), and 150 pl of each dilution was added t o the cuvettes. All samples were run in duplicate and incubated for 2 h at 37 ° C. After t h o r o u g h washing with distilled water, 150 pl of an anti-human IgM-alkaline phosphatase conjugate (Orion Diagnostica, Helsinki, Finland), diluted 1 : 500 in PBS-T was added and left overnight at r o o m temp er atur e . After the usual washing procedure, 150 gl of the substrate solution (1 mg Sigma 104 R phosphatase substrate, Sigma Chem. Corp., St. Louis, MO, U.S.A./1 ml of a 1 M diethanolamine buffer, pH 9.7, containing 0.5 mM MgCl2) was added. The e nzym at i c reaction was inhibited after incubation at 37°C f or 1 h by adding 250 pl of 0.16 M NaOH. The absorbance was measured vertically through the cuvettes with a FP-9 phot o m e t e r (Labsystems, Helsinki, Finland) at a wavelength of 404.7 nm. Sera in non-coated cuvettes and PBS-T in coated cuvettes were run as technical controls. The assay on microtitre plates was p e r f o r m e d as described above or alternatively with shorter incubation times, i.e. 1 h at 37°C for t he serum dilutions, 1 h at r o o m t e m p e r a t u r e and 1 h at 37°C for the conjugate and 1 h was allowed for the e n z y m a t i c reaction to take place. The absorbance values obtained were r e cor de d with a Titertek Multiskan p h o t o m e t e r (Flow Laboratories, Eflab Oy, Helsinki, Finland) at a wavelength of 405 nm, or alternatively the results were read as positive or negative by the naked eye. Inhibition experiments Equal volumes of sera diluted 1 : 50 and dilutions of IgG or aggregated IgG were mixed and incubated overnight at 4 ° C. The sera were then tested in the ELISA at a final dilution of 1 : 500. RESULTS The optimal conditions for the assay were determined using the pooled positive and negative reference sera. An IgG c o n c e n t r a t i o n of 5 pg/ml for the coating p r o ced u r e was f o u n d suitable and was used in the furt her studies. Representative titration curves obtained in the RF-ELISA are shown in Fig. 1. According to these results, sera were subsequently tested at dilutions o f 1 : 50, 1 : 500 and 1 : 5000. The reproducibility of the assay was evalu-

112

2.o_

I 1.5

1.0

0.5

I~o

,,~)o

,,'o

lUlCIPIIOCAL MIIlUM I~LU~ION

Fig. 1. R e p r e s e n t a t i v e titration curves o b t a i n e d in the R F - E L I S A . • p o o l e d positive r e f e r e n c e s e r u m ; • p o o l e d negative r e f e r e n c e serum; i , ©, [] individual R F - p o s i t i v e sera.

TABLE 1 R E S U L T S O B T A I N E D IN T H E R F - E L I S A A F T E R I N H I B I T I O N O F S E R A WITH HUMAN IgG IN N A T I V E O R A G G R E G A T E D F O R M R F activity is e x p r e s s e d as a b s o r b a n c e at 405 nm. Sera

P o o l e d positive r e f e r e n c e s e r u m P o o l e d negative r e f e r e n c e s e r u m A B C D E F

Not inhibited

0.530 0.120 1.880 1.015 0.565 2.860 2.608 0.595

R F activity o f sera i n h i b i t e d w i t h a aggregated IgG (mg/ml)

native IgG (mg/ml)

0.1

1

0.1

1

0.205 0.110 0.585 0.290 0.320 1.995 1.645 0.275

0.140 0.105 0.260 0.140 0.170 0.895 1.010 0.180

0.490 0.120 1.435 0.885 0.455 2.640 2.308 0.420

0.350 0.120 0.935 0.445 0.265 2.600 2.440 0.220

a Equal v o l u m e s o f sera d i l u t e d 1:50 and s o l u t i o n s o f IgG were m i x e d and i n c u b a t e d overnight at +4°C, the sera were t h e n t e s t e d in the R F - E L I S A in a final dilution o f 1:500.

113

ated b y the results o b t a i n e d with the p o o l e d reference sera and one Waaler-Rose positive serum tested o n 9 d i f f e r e n t d a y s u n d e r s t a n d a r d c o n d i t i o n s . T h e average d a y to d a y c o e f f i c i e n t o f variation in a b s o r b a n c e values at a s e r u m dilution o f 1 : 500 was 20%, whereas the w i t h i n - d a y c o e f f i c i e n t o f variation was 11%.

Specificity o f the assay T h e technical c o n t r o l s consisting o f sera in n o n - c o a t e d cuvettes and the c o n j u g a t e c o n t r o l s gave a b s o r b a n c e values o f less than 0.150. The results o f the i n h i b i t i o n e x p e r i m e n t s are s h o w n in Table 1. The activity was m o r e readily a b s o r b e d b y aggregated IgG t h a n b y native IgG. As an a d d i t i o n a l c o n t r o l a s e r u m s p e c i m e n f r o m a p a t i e n t with W a l d e n s t r 6 m ' s m a c r o g l o b ulinemia was tested and f o u n d n o t to react in the assay.

Expression o f results and results obtained with normal sera The results o f the R F - E L I S A were expressed as units, i.e. the a b s o r b a n c e value o b t a i n e d at a s e r u m dilution o f I : 500 ( m e a n o f t w o d e t e r m i n a t i o n s ) was calculated as per c e n t o f the a b s o r b a n c e value o f the p o o l e d positive r e f e r e n c e s e r u m at the same dilution run on the same occasion. The 105 n o r m a l sera tested gave a m e a n value o f 24-+ 16 (mean_+ S.D.). 65 sera negative in the latex f i x a t i o n test gave a c o r r e s p o n d i n g result o f 27-+ 12. A c c o r d i n g l y , values o f > 50 units in the R F - E L I S A were regarded as positive. The r e p r o d u c i b i l i t y o f the assay was also evaluated using the u n i t s y s t e m f o r expression o f the results. The c o e f f i c i e n t o f variation calculated b y the results o b t a i n e d with o n e W a a l e r - - R o s e positive and o n e n o r m a l h u m a n s e r u m , tested o n 8 d i f f e r e n t occasions was 15%.

Comparison o f the RF-ELISA results with the standard methods T h e results o f the R F - E L I S A c o m p a r e d to the results o b t a i n e d in the latex f i x a t i o n and the Waaler--Rose test are s h o w n in Tables 2 and 3. There was g o o d a g r e e m e n t b e t w e e n the classical m e t h o d s and the R F - E L I S A ( P
/30 regarded as positive.

0.001). T h e m e a n value o f the 5 0 / 3 3 0 (15%) sera negative in the Waaler-Rose test b u t positive in the R F - E L I S A was 127 + 72. The results o b t a i n e d in t h e R F - E L I S A were the same w h e t h e r testing fresh or inactivated sera. The R F - E L I S A units p l o t t e d against the titre o f the latex fixation test and t h e W a a l e r - - R o s e test are d e p i c t e d in Figs. 2 and 3. There was a significant c o r r e l a t i o n b e t w e e n the titres a n d the R F - E L I S A units (P < 0 . 0 0 1 , calculated b y variance analysis). T h r e e o u t o f 70 (4%) Waaler--Rose positive sera

400_

ee

300_

go 200_

~00_ ee e~,Q

~

~

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eele

ee

ee

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~ 8

, 1

:

/ 2

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4

, 128

I 266

I 512

llLIDI T|ST

Fig. 2. Comparison between titres obtained in the latex fixation slide test and RF-ELISA units.

115

400_

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*

*

*~

• :

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:" ":"

.

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120

240

WULiR-IOil

480

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Fig. 3. C o m p a r i s o n b e t w e e n titres o b t a i n e d in t h e W a a l e r - - R o s e test a n d R F - E L I S A units.

were negative in the R F - E L I S A . All sera with a latex f i x a t i o n titre ~>128 were positive in the R F - E L I S A , whereas 1 4 / 2 0 (70%) ()f the sera with a titre o f 64 and 1 7 / 1 0 7 (16%) o f t h e sera positive in dilutions ranging f r o m 1 : 4-1 : 32 were positive in the assay. Results obtained in R A and non-specific arthritis Positive results in the R F - E L I S A were o b t a i n e d in 27 o f 43 (63%) p a t i e n t s with p r o b a b l e or definite R A and in o n e o u t o f 26 (4%) patients with posti n f e c t i o u s or non-specific arthritis or in Reiter's disease (Table 4). T h e m e a n

TABLE 4 C O M P A R I S O N B E T W E E N THE R F - E L I S A A N D THE L A T E X F I X A T I O N A N D W A A L E R - - R O S E T E S T S IN R H E U M A T O I D A R T H R I T I S A N D N O N - S P E C I F I C ARTHRITIS Diagnosis

Probable R A Definite R A Non-specific arthritis

Positive results o b t a i n e d in t h e R F - E L I S A a L a t e x < 64 WaRo 64 WaRo /64 WaRo 930

1/13 6/9 1/26

4/5 2/2 0/0

5/5 9/9 0/0

a Results a b o v e 50 R F - E L I S A u n i t s r e g a r d e d as positive.

Total

%

10/23 17/20 1/26

43 85 4

116 TABLE 5 COMPARISON BETWEEN RESULTS EXPRESSED AS RF-ELISA UNITS AND RESULTS RECORDED AS TITRES IN THE ONE-DAY RF-ELISA Serum dilution giving a positive result in the one-day microtitre RF-ELISA 1:100

RF-ELISA units -50

50--150

150-250

250-350

350-

21 1

9 11

-8

-4

4

-

6

3

8

3

R F - E L I S A unit value was 88 +- 98 in the g r o u p consisting o f p r o b a b l e R A a n d 136 -+ 101 in definite R A , whereas t h e valuc was 28 ± 12 in non-specific arthritis.

R a b b i t IgG used as antigen in the assay F o r t y - e i g h t sera positive and 16 sera negative in the R F - E L I S A were tested in parallel in cuvettes c o a t e d with IgG o f h u m a n and r a b b i t origin (5 p g / m l ) . All the sera negative in the h u m a n I g G - R F assay were also negative in t h e r a b b i t assay. Five o u t o f t h e 48 positive sera r e a c t e d preferentially with r a b b i t IgG, while 2 r e a c t e d with h u m a n IgG b u t did n o t react with rabbit IgG.

The R F - E L I S A performed on microtitre plates T h e r e was n o d i f f e r e n c e in a b s o r b a n c e values w h e t h e r using m i c r o c u v e t t e s or m i c r o p l a t e s a n d the same i n c u b a t i o n time. When the assay on the m i c r o titre plates was c o m p l e t e d in o n e day, the a b s o r b a n c e values were approxim a t e l y 60% lower. T h e unit values r e m a i n e d , h o w e v e r , r o u g h l y the same w h e n using a dilution o f 1 : 50 for the calculation. A c o m p a r i s o n b e t w e e n t h e results r e c o r d e d as titres b y the n a k e d eye in the o n e - d a y m i c r o t i t r e assay and the c o r r e s p o n d i n g R F - E L I S A units are s h o w n in Table 5. DISCUSSION The classical m e t h o d s for the d e t e c t i o n o f R F , the Waaler--Rose test a n d the latex f i x a t i o n test, are widely used b u t suffer f r o m s o m e disadvantages. T h e reagents f o r the Waaler--Rose test are labile and a b s o r p t i o n and inactivation o f sera p r i o r t o testing is necessary. In a d d i t i o n b o t h tests suffer f r o m difficulties in s t a n d a r d i z a t i o n and r e p r o d u c i b i l i t y . Lately, there has been great interest in e n z y m e i m m u n o a s s a y s (Voller et al., 1 9 7 8 ) , and various m o d i f i c a t i o n s o f this new m e t h o d have also been a d o p t e d for the d e m o n s t r a -

117 tion of RF. Maiolini et al. (1978) have developed a sandwich m e t h o d using glucose oxidase-labeled aggregated IgG; Ziola et al. (1978) have described a m e t h o d employing the binding of RF to IgG in immune complexes on a solid phase, and Willems and Klaassen de Kort (1978) have used rabbit IgG as antigen and investigated the occurrence of RF with anti-human IgM coupled to peroxidase. We have developed a simple enzyme-linked immunosorbent assay, using human IgG as antigen, and find good agreement between the results of this assay and those obtained by the classical methods. The reproducibility of the assay is good and the m e t h o d is suitable for routine use. The recording of the results is objective by means of a spectrophotometer, but the assay may be performed without any complicated equipment. The antiglobulins are a heterogeneous group of antibodies and several sites on the IgG molecule have been claimed to be the antigenic site for RF (Johnson and Faulk, 1976). We have used native human IgG as antigen in our assay. As the m e t h o d is a solid phase assay, conformational changes may, however, occur in the antigen molecule as it adheres to the surface (Kochwa et al., 1967). In the RF-ELISA the antibody activity was more readily inhibited by aggregated than by native IgG, which apparently is due to polyvalent interactions between RF and aggregated IgG (Normansell, 1971; Eisenberg, 1976). The majority of RFs react with both human and rabbit IgG (Normansell, 1972). In our assay most of the sera tested showed a stronger reaction with human than with rabbit IgG and only 2/48 sera reacted solely with IgG of human origin. There was a statistically significant correlation between the RF-titres obtained with the classical methods and the results of the RF-ELISA. Some discrepancies were, however, seen. These findings could at least partly be explained by differences in affinity of the RF. The ELISA is known to be influenced by the antibody affinity (Butler et al., 1978) and favors the detection of high affinity antibodies. The RF-ELISA gave positive results in a frequency of about 15% of sera which were negative in the Waaler--Rose test. In the patients with rheumatoid arthritis studied, the ELISA detected RF in 63% of the cases, whereas RF was demonstrated in 49% by the classical methods in the same material. After treatment of rheumatoid sera in various ways, so called 'hidden' RF are revealed in some cases (Allen and Kunkel, 1966; Hansson and Winblad, 1978). As there is no need to inactivate sera before testing and as the sera are tested in great dilutions in the RF-ELISA, it is possible that this assay detects some hidden RF. The RF-ELISA may also detect RF devoid of agglutinating activity. The role of RF in the pathogenesis of rheumatoid arthritis has n o t been settled (Johnson and Faulk, 1976). Lately, there has been a great interest in RF of IgG class, and different methods for the demonstration of IgG-RF have been described (Torrigiani et al., 1970; Nineham et al., 1976; Carson et al., 1977). Our RF-ELISA was primarily designed for the detection of RF of IgM class, but it is possible to m o d i f y the m e t h o d for the demonstration of RF of IgG class according to the m e t h o d described by Carson et al. (1977).

118 ACKNOWLEDGEMENTS

We thank the Pharmaceutical Company Medica for financial support. The technical assistance of Ms. Merja Swanljung and Ms. Anna-Liisa Ruuska is gratefully acknowledged. REFERENCES Allen, J.C. and H.G. Kunkel, 1966, Arthritis Rheum. 9 , 7 5 8 . Butler, J.E., T.L. Feldbush, P.L. McGivern and N. Stewart, 1978, Immunochemistry 15, 131. Carson, D.A., S. Lawrance, M.A. Catalano, J.H. Vaughan and G. Abraham, 1977, J. Immunol. 119,295. Eisenberg, R., 1976, Immunochemistry 13,355. Gripenberg, M., E. Linder, P. Kurki and E. Engvall, 1978, Scand. J. Immunol. 7,151. Hansson, U.-B. and S. Winblad, 1978, J. Immunol. Methods 22, 155. Johnson, P.M. and W.P. Faulk, 1976, Clin. Immunol. Immunopath. 6, 414. Knez, V. and C.B. Reimer, 1977, J. Immunol. Methods 18, 105. Kochwa, S., M. Brownell, R.E. Rosenfield and L.R. Wasserman, 1967, J. Immunol. 99, 981. Maiolini, R., B. Ferrua, J.F. Quaranta, A. Pinoteau, L. Euller, G. Ziegler and R. Massayeff, 1978, J. Immunol. Methods 20, 25. Nineham, L.J., F.C. Hay and I.M. Roitt, 1976, J. Clin. Path. 29, 1121. Normansell, D.E., 1971, Immunochemistry 8 , 5 9 3 . Normansell, D.E., 1972, Immunochemistry 9, 725. Rose, H.M., C. Ragan, E. Pearce and M.O. Lipman, 1948, Proc. Soc. Exp. Biol. Med. 68, 1. Singer, J.M. and C.M. Plotz, 1956, Am. J. Med. 2 1 , 8 8 8 . Singer, J.M. and C.M. Plotz, 1958, Arthritis Rheum. 1,142. Stage, D.E. and M. Mannik, 1972-73, Bull. Rheum. Dis. 23,720. Torrigiani, G., I.M. Roitt, K.N. Lloyd and M. Corbett, 1970, Lancet i, 14. Voller, A . , A . Bartlett and D.E. Bidwell, 1978, J. Clin. Path. 31,507. Waaler, E., 1940, Acta Path. Microbiol. Scand. 17,172. Willems, F.Th.C. and C.C.M. Klaassen de Kort, 1978, Lancet i, 994. Ziola, B., O. Meurman, M.-T. Matikainen, A. Salmi and J.L. Kalliomiiki, 1978, J. Clin. Microbiol. 8 , 1 3 4 .