Enzyme-linked immunosorbent assay for anti-histone antibodies and their presence in systemic lupus erythematosus sera

Journal of Immunological Methods, 44 (1981) 311--322

311

Elsevier/North-Holland Biomedical Press

ENZYME-LINKED IMMUNOSORBENT ASSAY F O R ANTI-HISTONE ANTIBODIES AND T H E I R PRESENCE IN SYSTEMIC LUPUS E R Y T H E M A T O S U S SERA

A. AITKACI, J.C. MONIER and N. MAMELLE

Laboratoire de Mddecine Prdventive, Facultd A. Carrel, Rue Guillaume Paradin, 69008 Lyon, France (Received 3 December 1980, accepted 9 April 1981)

We describe an enzyme-linked immunosorbent assay (ELISA) with adsorption of histones (total and fractions) on glass beads and saturation of excess sites with sheep serum. The anti-histone antibodies are detected with peroxidase conjugate and developed with Trinder's reagent which has great stability. This very sensitive method detects anti-histone antibodies in 53% of SLE patients and in virtually no other diseases. Positive reactions are observed only with total histones and fractions H1 and H2b.

INTRODUCTION

Antinuclear antibodies commonest in systemic lupus erythematosus (SLE) have specificities for DNA-histone, native or denatured DNA, or Sm antigens. Other antibodies reacting with histones have been detected in SLE sera by complement fixation (Kunkel et al., 1960; Stollar, 1971} and b y immunofluorescence (Tan et al., 1976; Fishbein et al., 1979). We have devised a simple, more sensitive and more reproducible test for these antibodies which employs enzyme-linked immunosorbent assay (ELISA) with the advantage of being readily automated. The technique consists of adsorption of histories (total or fractions} to the surface of glass beads and saturation of excess sites with sheep serum. The beads react with test sera, and any attached antibodies are detected by means of a second reaction with peroxidase-conjugated anti-human Ig serum. A sensitive specific colour detection system permits measurement of peroxidase b o u n d to the mixed antibody-antigen complexes attached to the glass bead. Tests were performed on 382 serum samples from normal controls or patients with SLE, rheumatoid arthritis, other autoimmune diseases and other illnesses. These indicate that some 50% of SLE patients produce antihistone antibodies, b u t that they are very rare in other circumstances. The method described is sensitive, but some sera from both control and diseased subjects give false positive reactions. These are easily distinguished from true positive reactions since they give equally strong reactions with beads sensitized with sheep serum without histone antigens. 0022-1759/81/0000--0000/$02.50 © Elsevier/North-Holland Biomedical Press

312 MATERIALS

AND METHODS

(Fig. 1 )

Glass beads 5 mm diameter (Laboratoire Chamagne) were washed 3 times in detergent (Neo Disher, Alkalisher reiniger A8, Chemische Fabrik), then rinsed first with tap water and then with distilled water before being stirred in sodium hydroxide (0.1 M) for 30 min, and then washed 10 times in distilled water. At this stage, the beads may either be used immediately, or dried and stored. Total calf t h y m u s histones type II-s or fractions H1, H2a-H4, H2b and H3, prepared by Johns' m e t h o d (1964, 1967) were purchased from Sigma Chemicals. These antigens were adsorbed to the beads by placing 10 beads in a stoppered haemolysis tube with 3 ml of antigen solution (50 gg/ml) on a rocker for 1 h at room temperature, followed by overnight incubation at 4°C w i t h o u t rocking. The sensitized beads were then washed 3 times with phosphate-buffered saline pH 7,2, 0.1 M phosphate (PBS) and unoccupied sites were saturated with normal sheep serum diluted 1 : 5 in PBS. Control beads were treated with the same dilution of normal sheep serum w i t h o u t prior contact with histone. The beads were washed a further 4 times in PBS before use.

Sera were from normal subjects of different ages, from patients with autoimmune disease with antinuclear antibodies (ANAb) detected by immunofluorescence on smears of mouse blood, and from patients with other diseases. The majority of patients with a u t o i m m u n e disease had either confirmed SLE (at least 4 positive diagnostic criteria) or probable SLE (3 positive diagnostic criteria). For screening, sera were diluted 1 : 8 in PBS containing 0.05% Tween 20 (PBS-Tween 20) supplemented with 5% normal sheep serum. Sheep anti-human Ig serum conjugated with peroxidase was from the Institut Pasteur, Paris and diluted (1 : 200) in PBS-Tween 20 immediately before use. Y2

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313

For testing, single antigen~oated and control beads were placed in the wells of 20- or 60-well A b b o t t plates with 0.2 ml of diluted serum. After incubation at r o o m temperature for 1 h the beads were washed 3 times with PBS-Tween 20 b y the A b b o t t 'pentawash' automatic system, and 0.2 ml o f freshly diluted peroxidase-conjugated sheep anti-human Ig was added to each well and allowed to react for 30 min at room temperature with rocking. The beads were then washed a further 4 times with PBS-Tween 20 with the pentawash system and transferred to clean haemolysis tubes for detection of b o u n d enzyme activity. Each serum was tested on antigen coated beads and on control beads. Detection of enzyme activity was by a colorimetric reaction with Trinder's reagent (Gallati, 1977}. Each tube containing a single bead was incubated at 37°C for 80 min with 2 ml of freshly prepared reagent (Phenol 25 mM, amino 4-phenazone 2 mM and hydrogen peroxide 0.4 mM), and the intensity of the colour reaction was read in a Pye Unicam model SP6-500 spectrophotometer at 492 nm. The optical density (OD) multiplied b y 10 -3 was expressed as OD units. RESULTS AND DISCUSSION

Glass beads were chosen as antigen support rather than the more usual polystyrene tubes or wells for the ELISA reaction for the following reasons (1) they permit the use of the A b b o t t automatic washing system which is efficient, reproducible and rapid; (2) the composition of the glass is uniform from one batch to another whereas polystyrene may show considerable variation between batches (Masseyeff, 1978); and (3) the bead may be transferred to a clean tube before the final colour reaction is read thereby avoiding non-specific reactions due to adsorption of the conjugate to the wall of the containing tube. The reaction conditions described were established b y a series of preliminary experiments. The concentration of sensitizing antigen was varied between 10 and 500 pg/ml, and uptake by the beads was estimated b y meaTABLE 1 Adsorption of histone to glass beads. Histone concentrations (pg/ml)

Uptake of histone by one bead (Pg)

10 25 50 100 250 500

0.8 2.2 3 6 7 7

314 suring the residual histone in the supernatant at the end of incubation by OD at 289 nm. The results in Table 1 show that m a x i m u m uptake of histone occurs at a concentration of 250 pg/ml. Results with beads sensitized with various concentrations of histone and tested with selected human sera positive and negative for anti-histone antibodies, and with sera which gave strong false po.sitive reactions through non-specific binding are shown in Fig. 2. Reproducible results were obtained when the beads were sensitized with 7> 25 /~g/ml histone. We therefore chose a concentration of 50 /~g/ml for routine sensitization. Absorption of histone to the beads is rapid, satisfactory antigenic activity being achieved after only 1 h contact at room temperature. However, reproducibility was improved if contact was prolonged overnight at 4°C. Such sensitized beads could be stored immersed in antigen solution for up to 10 days w i t h o u t detectable loss of activity. Since antigen is attached to the beads by physical adsorption, other proteins such as human immunoglobulin or peroxidase conjugate might also be non-specifically taken up by unsaturated sites leading to false positive reactions. To avoid this, we saturated the beads by further incubation with normal serum from the species (sheep) used to produce the conjugated anti-human Ig serum (Masseyeff, 1978). It was found that rocking antigen-sensitized beads with normal sheep serum diluted 1 : 5

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in PBS for 1 h at r o o m temperature, followed by overnight incubation at 4°C eliminated non-specific uptake of protein from the majority o f normal sera (Figs. 3 and 4). To avoid possible false positive reactions due to the presence of antibodies to sheep serum protein in the test sera, we routinely diluted the serum specimens in PBS-Tween 20 containing 5% normal sheep serum, incubated them at 37°C for 1 h and left them overnight at 4°C before testing (Fig. 4). Despite these precautions some normal sera gave strong positive reactions, in some cases comparable with those given b y SLE sera with histone-sensitized beads. Such false positive reactions are easily identified since t h e y give equally strong or stronger reactions with beads treated with normal sheep serum alone. Similar reactions are occasionally observed with SLE sera. It is therefore necessary to test all sera with both antigen sensitized and control beads, the difference in intensity o f the colour reaction measuring the amount of anti-histone antibody. This procedure may result in some loss of sensitivity, b u t this is compensated for by a corresponding gain in specificity.

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Fig. 4. E f f e c t o f saturating e x c e s s sites with normal sheep serum on d e t e c t i o n o f antih i s t o n e a n t i b o d i e s with histone-sensitized beads. A: beads n o t treated with sheep serum. B: beads treated w i t h sheep serum. 1, sera diluted in P B S - T w e e n 20; 2, sera diluted in P B S - T w e e n 20 c o n t a i n i n g 5% normal s h e e p serum; C, c o n j u g a t e control in absence o f test serum; S - , negative serum; S ÷, positive serum. Filled bars represent reactions w i t h beads s e n s i t i z e d with normal sheep serum in the absence o f histones.

We attempted to further diminish such non-specific reactions which are probably due to adsorption on the bead of serum protein(s) recognised by the conjugate. Variation of the ionic strength and pH of the PBS, dilution of the conjugate in PBS-Tween 20 containing 5% normal sheep serum, saturation of excess absorption sites on the sensitized beads with materials other than sheep serum (e.g. BSA, SDS, heparin or dithionite), ultracentrifugation of the diluted test sera at 110,000 X g for 45 min to remove possible immune complexes, or the use of specific conjugate reacting with human IgG all gave no significant reduction in non-specific reactions. The serum components responsible have not yet been identified. Masseyeff (1978) has incriminated rheumatoid factor or anti-sheep antibodies. These are unlikely to be the cause of the reactions seen in the present study since they are not affected by substitution of specific anti-IgG conjugate for anti-

317

total Ig conjugate, or by pre-incubation of the test sera with sheep serum. Another possible cause is a-2M which binds to histones (Stollar and Rezuke, 1978), and may be recognised by antibodies present in some batches of conjugate (Aitkaci and Monier, 1979). In the conditions described, however, we found that a-2M is only weakly adsorbed by the glass beads, and its uptake becomes undetectable after saturation of excess sites with sheep serum. Finally, the false positive reactions do not appear to be due to affinity for the peroxidase moiety of the conjugate, since incubation o f serum-treated beads in a solution of peroxidase (100 pg/ml) instead of conjugate produces no positive colour reaction. One hour's contact between the test serum and the sensitized beads at room temperature suffices for maximum uptake of antibody, and many OD 3

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318 positive sera attain m a x i m u m fixation after 30 min. At 4°C between 3 and 6 h are required to give the same values. When the intensity o f the final colour reaction on the sensitized bead is corrected by subtraction of that obtained with the cont r ol bead for the same serum, the values obtained vary very little with time of c ont a c t with the test serum, up to 18 h. The dilution o f the peroxidase-anti-human Ig conjugate is det erm i ned for each batch b y titration with k n o w n positive and negative test sera. Various batches supplied by the Institut Pasteur (Paris) gave o p t i m u m results diluted 1 : 200. As shown in Fig. 5, no increase in the uptake o f conjugate was observed when the c ont a c t time with a n t i b o d y positive beads was increased fr o m 30 min to 2 h. Overnight incubation resulted in uni form increase in u p t a k e for all samples w i t h o u t improving the difference between positive and negative sera. We f o u n d th at the substrate usually e m p l o y e d for peroxidase in ELISA, 5-amino salicylic acid, apart from the hazards of its manipulation, has the disadvantage o f being seriously unstable. Bullock and Walls (1978) were unsuccessful in attem pt s to use sodium h y d r o x i d e to com bat this instability. The optical density (OD) of solutions of this substrate increases rapidly even in the absence o f enzym e . For this reason we chose to use Trinder's reagent as substrate for peroxidase as r e c o m m e n d e d by Gallati (1977). This reagent gives no spontaneous increase in OD in the absence of e n z y m e so that the colour reactions observed in the test are strictly representative of the a m o u n t o f peroxidase conjugate present on the bead. The optimal time for the enzyme-substrate reaction was de t e r m i ne d with several test sera o f different degrees o f positivity for anti-histone antibodies. The OD increases rapidly over the first 40 min of cont act and more slowly thereafter. Under the reaction conditions described in Materials and Methods and expressing the results a s (ODtest---ODcontrol) , we found that duplicate measurements varied b y less than 5%. Tested on different occasions, the corrected OD measurements for the same serum were reproducible to within 10 OD units as long as t he dilution o f conjugate was freshly prepared for each test series. With patients' sera, where duplicate measurements did not agree to within 5%, the test was repeated in duplicate. The intensity of the colour reaction obtained at 1 : 8 dilution o f test sera correlated with t he titres o f t h e same sera and could t h e r e f o r e be used as an estimate o f the a m o u n t o f a n t i b o d y present (Fig. 6). The anti-histone activity o f sera o f 109 healthy individuals was measured using the above technique, t he uppe r limit of normal activity being defined as t h at including 95% of these specimens. This corresponds to a corrected OD value o f 20 for beads sensitized with total histones, and of 30 for beads sensitized with histone fractions. The f r e q u e n c y of anti-histone antibodies in serum samples f r o m patients with confirmed or probable SLE, with o t h e r a u t o i m m u n e manifestations or with unrelated diseases was tested on beads sensitized with to t al histones. Positive reactions were observed in 56 of 129 samples f r o m clinically confirmed cases o f SLE (43.5%) and 24 o f the 45

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patients furnishing these samples had at least one positive test. For cases of probable SLE (3 positive clinical signs) with antinuclear antibody (ANA), titres/> 4 0 9 6 , 16 of 52 samples (30.8%) representing 11 of 31 patients (35.5%) gave positive reactions. For similar cases with ANA titres ~ 4 096, 2 of 8 samples (25%) had significant levels of anti-histone antibodies. Positive reactions were also observed in 4 o f 6 samples from one patient with medullary aplasia, in a single sample from a patient with malignant lympho~pithelial t h y m o m a and in one of 20 serum samples from 18 cases of scleroderma. No positive reactions were observed in serum samples from patients with various forms of autoimmune diseases: 30 samples from 29 cases of rheumatoid arthritis, 8 samples from 7 cases of drug-induced LED, or 2 samples from 2 cases of dermatomyositis. Similarly, 20 samples from different cases of various other diseases (active chronic hepatitis, dermatomyositis, cancer, cirrhosis, etc.) gave uniformly negative results (Fig. 7). It would appear that anti-histone antibodies are associated essentially with cases of spontaneous SLE and two of the patients with other diseases whose sera contained similar antibodies presented several biological criteria reminiscent of SLE. All serum samples positive for anti-histone antibodies also containes ANA at titres o f at least 256 and usually of 4 096. Studies o f sequential serum samples from some patients showed that the anti-histone antibodies fre-

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ANA

anti-I~NA Ab (crithidia)

H

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June

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321

quently appear when the ANA titre reaches about 1 024, and tend to regress before any diminution in ANA titre is observed (Table 2). Fritzler and Tan (1977) measured anti-histone antibodies by immunofluorescence on eluted liver nuclei which had been reconstituted by incubation in a solution of histones. By this m e t h o d , t h e y showed that 35% of patients with spontaneous SLE produced anti-histones antibodies, but t h e y also found that 96% of cases of drug-induced lupus produced such antibodies. In the present study we only examined a small number o f samples from cases of iatrogenic lupus, but none of the 8 samples from 7 patients gave positive reactions with our antigen-coated beads although 6 had ANA titres ~ 4 0 9 6 . Two differences in the antigen preparations used might account for this lack of concordance. First, histones associated with nuclear DNA may present different antigenic sites from those directly adsorbed to glass and second, Fritzler and Tan (1977) used histones prepared by saline extraction, which have been shown to differ antigenically from those prepared by extraction in acid (Mihalakis et al., 1976). The histones and fractions used in the present study were prepared in acid medium (Johns, 1964, 1967). The sera giving positive reactions with total histones were re-tested on beads sensitized with fractions H1, H2a-H4, H2b and H3. Positive reactions were observed only with fractions H1 and H2b; even the samples with the strongest reactivity towards total histone gave no reaction with either H2aH4 or with H3 of greater intensity than that given by normal sera. Of 79 sera with antibody activity towards total histone we found that 22 reacted with H1 only, 14 with H2b only, and 43 with both fractions. In all cases, the colour reaction was more intense than with the same serum tested on a bead sensitized with total histone. The coefficient of correlation between the titre of ANA or of DNA Ab and the strength of reaction with beads coated with total histone or with the different fractions was determined for the above sera (Table 3). Significant correlations were found between ANA or antiDNA titres and reactions with total histone and with fraction H1 and fraction H2b as antigen. No significant correlation was found with fractions H2a or F3. The comparison o f our results for the presence of antibodies to individual histone fractions in cases of spontaneous SLE with those described by Fritzler and Tan (1977) is complicated by the fact that only those with the TABLE 3 C o r r e l a t i o n s b e t w e e n A N A , a n t i - D N A a n d a n t i - h i s t o n e A b . N u m b e r s s h o w n are r values.

ANA Anti-DNA Ab ap~< 0.05.

H

H1

H2a-H4

H2b

H3

0.33 a 0.24 a

0.23 a 0.26 a

0.01 --0.04

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0.13 0.12

322

H1 fraction are directly c o m p a r a b l e . Their findings that, o f 5 positive sera, 2 reacted with H1 and 2 with a H 2 a - H 2 b c o m p l e x are c o m p a t i b l e with our o b s e r v a t i o n o f reactions with H1 and H2b; b u t their fifth serum reacted with a H3-H4 c o m p l e x , and we f o u n d no serum o u t o f 79 tested which reacted either with H3 or H2a-H4 c o m p l e x . An E L I S A test for anti-histone antibodies such as t h a t described here m i g h t be o f considerable value for the diagnosis and surveillance of SLE, comp l e m e n t i n g the m e a s u r e m e n t o f ANA. Our findings suggest t h a t such antibodies are i n f r e q u e n t in o t h e r disease states w h e t h e r t h e y axe o f a u t o i m m u n e origin or not. REFERENCES Aitkaci, A., and J.C. Monier, 1979, R@union Soc. Franq. hnmunol. 1, 1. Bullock, S.L. and K.W. Walls, 1978, J. Infect. Dis. 136 (Suppl.), $279. Fishbein, E., D. Alarcon Segovia and J.M. Vega, 1979, Clin. Exp. Immunol. 36,145. Fritzler, M.J. and E.M. Tan, 1977, Clin. Res. 25,483a. Gallati, H., 1977, J. Clin. Chim. Biochim. 15,699. Johns, E.W., 1964, Biochem. J. 92, 55. Johns, E.W., 1967, Biochem. J. 105,611. Kunkel, H.G., H.R. Holman and H.R.G. Deicher, 1960, CIBA Found. Symp. 429. Masseyeff, R., 1978, Ann. Biol. Clin. 36,375. Mihalakis, N., O.J. Miller and B.F. Erlanger, 1976, Science 192,469. Stollar, B.D., 1971, Arthr. Rheum. 14,485. Stollar B.D. and W. Rezuke, 1978, Arch. Biochem. Biophys. 190,398. Tan, E.M., J. Robbinson and P. Robitaille, 1976, J. Immunol. 5,811.