A recombinant 70K protein ELISA

Journal of Immunological Methods, 143 (1991) 11-24 © 1991 Elsevier Science Publishers B.V. All rights reserved 0022-1759/91/$03.50 ADONIS 0022175991002854

11

JIM06060

A recombinant 70K protein E L I S A Screening for antibodies against UlsnRNP proteins in human sera H.P. Seelig, H. Ehrfeld, H. Schroeter, Claudia H e i m and M. Renz Institute of lmrnunology and Molecular Genetics, Kriegsstrasse 99, 7500Karlsruhe, F.R.G. (Received 28 March 1991, revised received 24 May 1991, accepted 24 May 1991)

Antibodies to uridylic acid rich small nuclear ribonucleoprotein particles (UsnRNP) are mainly detected in patients with systemic lupus erythematosus (SLE) or mixed connective tissue disease (MCTD). Particularly those directed against epitopes of the 70K protein of UlsnRNP serve as important markers for the diagnosis of MCTD. To establish an ELISA for determination of anti-70K protein antibodies in patients' sera a 1239 bp long cDNA insert coding for the epitopes of the 70K protein was ligated into a fusion expression vector. The bacterially expressed fusion protein was purified by chromatography on DEAE cellulose. Microtiter plates were coated with the fusion protein as well as with partially purified calf thymus extract (CTE) containing all natural UsnRNP antigens and RNase digested calf thymus extract (CTERNase) in which the natural 70K antigen was destroyed by the nuclease treatment. 10,888 sera of patients with suspected or overt rheumatic diseases were analyzed for antibodies against these antigens simultaneously. Antibodies against CTE or CTERNase were not detected in 9123 sera, none of these showed reactivity with the 70K protein indicating a high degree of specificity of the assay. Positive results in each the 70K protein, CTE as well as the CTERNas e ELISAs were obtained with 474 sera. 319 sera were only positive with CTE and 70K protein. Of these 793 anti-70K protein ELISA positive sera, 79% could be confirmed by immunoblot. Of 967 sera reacting with CTE and CTERNas~ but not with the recombinant 70K protein, 31% contained antibodies against various other UsnRNP proteins as shown by immunoblotting. 2.4% of these sera revealed also antibodies against the 70K protein. The use of the recombinant 70K protein as antigen meets the criterion for a simple and specific assay to detect anti-UlsnRNP antibodies. Nevertheless, the sole use of this recombinant protein for anti-UlsnRNP antibody screening may not be appropriate, because antibodies against other frequently occurring UlsnRNP proteins (A, C) cannot be detected with this test. Therefore it should be used together with a natural UsnRNP antigen until further studies in patients with well established diagnoses will show whether natural antigens may be omitted.

Correspondence to: H.P. Seelig, Institute of Immunology and Molecular Genetics, Kriegsstrasse 99, 7500 Karlsruhe, F.R.G. Abbreviatons: ELISA, enzyme-linked immunosorbent assay; EDTA, ethylenediaminetetraacetic acid; LB-broth, Luria-Bertani medium; SDS-PAGE, sodium dodecyl sulfate polyacrylamide gel electrophoresis; PMSF, phenylmethylsulfonylfluoride; CTE, calf thymus extract; CTERNase, calf thymus extract digested by RNase; Tris, tris[hydroxy-methyl]aminomethane;anti-Ig-POD, anti-immunoglobulin-peroxidase conjugate; MES, 2-(N-morpholino)ethane sulfonic acid; CMC, cyclohexyl-3-(2-morpholinoethyl)-carbodiimide-methyl-4-toluolsulfate; OPD, o-phenylenediamine; OD, optical density; BSA, bovine serum albumin; BGG, bovine gamma globulin.

12

Key words: Anti-UlsnRNP; Autoimmunity;U1-70K; Systemic lupus erythematosus (SLE); Mixed connective tissue disease;

autoantibody

Introduction

Antinuclear antibodies are important diagnostic markers in patients with rheumatic diseases. Information concerning the antibody specificity may allow valuable considerations in differential diagnosis (for review: Seelig, 1983; Tan 1989). Patients suffering from systemic lupus erythematosus (SLE) may develop antibodies against the soluble nuclear antigen Sm (Tan et al., 1966). Patients with mixed connective tissue disease (MCTD) exhibit antibodies against a different nuclear antigen which in contrast to Sm is destroyed by RNase (Sharp et al., 1972). Because of its solubility it was originally named extractable nuclear antigen (ENA) and later, according to its ribonucleoprotein nature, RNP or UlsnRNP. Both antigens belong to a group of small nuclear ribonucleoprotein particles (snRNP) which are composed of a low molecular weight RNA rich in uridine (URNA) and several proteins with molecular weights from 9 to 52 kDa (Lerner et al., 1979; Zieve et al., 1990). The particles which are involved in mRNA splicing (Mattaj et al., 1989) differ from each other by their RNA (U1, U2, U4/6, U5 RNA) contributing to the particles name (i.e., UlsnRNP) and by their associated proteins carrying antigenic epitopes. UsnRNPs contain common core proteins (B'B, D, E, F, G) as well as proteins unique for particular particles, e.g., 70K, A and C protein in UlsnRNP. Antibodies against the 70K protein are found in most patients with MCTD, less frequently in SLE or other rheumatic diseases (Habets et al., 1983, 1985; Pettersson et al., 1984, 1986; Combe et al., 1989; Kallenberg et al., 1990). On the other hand, SLE sera with anti-Sm activity react with B'B and D core proteins (Guldner et al., 1983, 1986). The UlsnRNP particle carries the 70K protein as well as the Sm epitopes, therefore a discrimination and reliable quantification of anti-Sm and anti70K protein antibodies which frequently occur

simultaneously becomes difficult with natural antigens and methods like agar gel double diffusion (Kumar et al., 1984), counter current electrophoresis (Kurata et el., 1976), passive hemagglutination (Sharp et al., 1972), ELISA (Struckmann et al., 1981; Warlow et al., 1984) or RIA (Seelig, 1983; Whittingham, 1983). Only immunoblot analyses (Guldner et al., 1983; Pettersson et al., 1984) allow the proper detection of antibody specificities but are difficult to quantify and may sometimes give rise to false negative results especially at low antibody concentrations. Following the first reports concerning a few patients only (Theissen et al., 1986; Spritz et al., 1987; Guldner e t al., 1988; Netter et al. 1988) several groups are now developing ELISAs with recombinant proteins for detection of anti-70K protein antibodies. 70K fusion proteins containing/3-galactosidase (Habets et al., 1989a; Clair et al., 1990) or MS-2 polymerase (Renz et al., 1989a; Seelig et al., 1990) were used, the latter providing higher specificity because natural antibodies against MS-2 polymerase do not occur in humans. Assays with recombinant 70K proteins may be regarded as a step forward in definition of antibody specificity. Therefore, we made a comparative study of more than 10,000 sera with ELISAs based on recombinant and natural antigens. As it was fomerly suggested from tests using natural antigens a negative anti-UlsnRNP assay may rule out the diagnosis of MCTD (Sharp et al., 1976; Alarc6n-Segovia et al., 1987; Kasukawa et al., 1987). The term anti-UlsnRNP (anti-RNP, antiENA) also includes antibodies to the individual A and C proteins of this particle which cannot be detected with the 70K protein antigen. Therefore, another aim of this study was to evaluate the proportion of the latter antibodies with regard to anti-70K protein antibodies in order to define the statistical predictions which can be drawn from a positive or negative anti-70K protein test.

13

Materials and methods SeFa

10,888 sera of patients with suspected or manifest rheumatic diseases were tested simultaneously by ELISA for autoantibodies to (a) recombinant 70K protein, (b) nuclear extract from calf thymus containing UsnRNP (CTE), and (c) CTE after RNase digestion (CTEnNase). All sera positive in one of these assays (n = 1760) were subjected to immunoblot analyses with UsnRNP proteins of HeLa cell nuclear extract. All sera were submitted for routine ANA testing. Controls Control sera were obtained from the Center of Disease Control (CDC, Atlanta, U.S.A.). Own reference sera were characterized by agar gel double diffusion (Kumar et al., 1984), URNA precipitation with Wil-2 cell nuclear extract (McNeilage et al., 1984; Forman et al., 1985), immunoblot and ELISA techniques with natural antigens, recombinant proteins (A, C, D, B'B, SS-A, SS-B, Scl-70) or synthetic epitopes of peptides (B'B, D), respectively (Renz et al., 1989b). Sera with monospecific antibodies against 70K, A, C, B'B and D proteins of UlsnRNP, SS-A (60 kDa protein), SS-A (48 kDa protein), SS-B or topoisomerase I (Scl-70), respectively, were available for specificity testing as there were polyspecific sera containing antibodies against several nuclear antigens (ds-DNA, ss-DNA, histones, centromeres, Jo-1, Ku-1, PM-Scl) and sera from healthy donors. Recombinant 70K protein The 1239 bp insert of the cDNA clone pEMBL70.1 (Theissen et al., 1986) containing the coding sequences for 380 N terminal amino acids (87%) of the human 70K protein of UlsnRNP was isolated after digestion with EcoRI and HindIII, its restriction sites were deleted by filling in with the Klenow fragment of E. coli polymerase I and blunt-ligated into the similarly treated, EcoRI opened bacterial expression vector pEx34b (Strebel et al., 1986). After transfection into competent E. coli 537 cells (Remaut et aI., 1983) and plating, colonies were first screened for the presence of 70.1 DNA by colony hy-

bridization. Positive colonies were transferred to new agar plates and checked, after induction of protein synthesis, for immunoreactivity with human anti-RNP sera. One of several immunopositive colonies was selected, characterized by DNA sequencing to confirm the correct reading frame, and used for preparative synthesis of the 70K fusion protein. Cells in 80 ml LB broth containing 30 /.~g/ml kanamycin and 100 /~g/ml ampicillin were grown under selective pressure at 30°C to high density for 15 h. To induce synthesis of fusion protein, cells were diluted with 320 ml of prewarmed (42°C) culture medium without antibiotics and incubated for 3 h at 42°C under good aeration. Bacteria were pelleted, washed once with 50 ml 0.1 M NaC1, 50 mM Tris-HC1 (pH 8.0). The cell pellets were frozen at -20°C for at least 30 min, thawed at room temperature, resuspended in 3.2 ml 10% sucrose, 50 mM TrisHC1 (pH 8.0) and, after the addition of 0.8 ml of lysozyme (10 mg/ml) and 0.4 ml 0.5 M EDTA, incubated for 15 min. on ice. Then 8 ml of detergent lytic mix (0.1% Triton X-100, 50 mM Tris-HC1 (pH 8.0)) was added and incubation on ice was continued for 15 rain. To reduce the viscosity of the mixture 50/zl DNase I (20 mg/ml) and 1.2 ml 1 M MgC1z were added and incubated on ice for 10 min. Insoluble material was recovered by centrifugation (30 min, 20,000 × g, 4°C) and sequentially extracted with prewarmed (50°C) solutions: first with 8 ml of 1 M urea and then with 8 ml of 7 M urea, each for 3 min at 50°C. Each extraction step was followed by centrifugation (30 min, 20,000 x g, 10°C). The 7 M urea fraction containing the fusion protein was further purified by chromatography on a 5 ml Fractogel TSK DEAE 650 M (Merck, Darmstadt) column. After washing (1 × 10 ml 2.5 M NaC1, 1 × 10 ml 1 M NaC1, 5 M urea, 10 mM Tris-HCl, pH 7.4) the column (equilibrated with 50 mM NaCI, 5 M urea, 10 mM Tris-HC1, pH 7.4) was loaded with the 7 M urea fraction (8 ml) which has been adjusted to 50 mM NaCI, 5 M urea, 10 mM Tris-HCl (pH 7.4). The column was washed with 25 ml 50 mM NaCI, 5 M urea, 10 mM Tris-HC1 (pH 7.4) and eluted with a linear gradient of NaCI (20 ml of 50 mM NaC1, 5 M urea, 10 mM Tris-HC1 (pH 7.4) and 20 ml 1 M NaC1, 5 M urea, 10 mM Tris-HC1 (pH 7.4)). Fractions eluting be-

14 tween 300 and 530 mM NaC1 contained highly purified 70K fusion protein. The yield was about 1 mg. Recovery and purity of the fusion protein during the fractionation procedure was monitored by SDS-PAGE and immunoblotting. Specificity of antibodies was additionally confirmed by acid elution from the membrane and immunoblotting with natural nuclear antigens (method: Harlow et al., 1988).

Nuclear extracts Nuclear extracts from calf thymus cells were prepared at 0-4°C, all buffers contained 1 mM EDTA, 0.5 mM PMSF. Thymus glands obtained immediately after slaughtering were minced, 200 g material in 400 ml 0.01 M Tris-HC1, pH 7.3, 0.25 M saccharose was homogenized in a Waring blender at low speed for 3 s. The material was centrifuged (2500 × g, 20 min) and the sediment suspended in PBS at double volume. Nuclei were destroyed (Waring blender, low speed, 4 min) followed by extraction of soluble proteins (16 h). After centrifugation (48,000 × g, 4 h) the supernatant was fractionated by precipitation with ammoniumsulfate. The 30-60% ammoniumsulfate fraction was centrifuged (10,000 × g, 30 min), the pelleted proteins resuspended in a small volume of 0.01 M potassium phosphate (pH 7.2) and dialysed. Following centrifugation (10,000 × g, 20 min) the supernatant was fractionated on DEAE-cellulose (Whatman) equilibrated with 0.01 M potassium phosphate (pH 7.2) by stepwise elution with increasing NaC1 concentrations. The 0.3 M NaC1 fraction containing UsnRNP (CTE) was concentrated in an Amicon concentrator and stored at -70°C. To destroy RNP antigens maintaining Sm activity CTE was digested with RNase A (Worthington) (1 : 10 w/w, PBS, 6 mM MgCl 2, 37°C, 30 min). After centrifugation (10,000 ×'g, 5 min) the supernatant (CTERNase) was stored at 70oc. Anti-m3G-affinity purified U1-U4/U6 snRNP particles from HeLa cell nuclear extract were kindly provided by R. Liihrmann, Marburg (Bringmann et al., 1986). Proteins were extracted with phenol followed by precipitation with 4 vols. of aceton and reconstituted in Laemmli's buffer (Laemmli, 1970). Proteins were separated on 9% or 12.5% polyacrylamide (5 /zg protein/cm gel) -

and transferred to Immobilon-P membranes (Millipore) overnight (100 mA, 21 mM Tris, 160 mM glycine buffer, pH 8.3). After drying and cutting strips were stored at - 70°C. Following saturation with blocking buffer (10% non-fat dry milk, 1% BSA, 150 mM NaC1, 0.3% Tween 20, 10 mM Tris-HCl, pH 7.4), strips were incubated with sera diluted with blocking buffer (5 × 10 -3 to 5 × 10 5) for 2 h followed by washing (0.3% BSA, 150 mM NaC1, 10 mM Tris-HC1, pH 7.4, 0.5% Triton X, 3 × 5 min) and incubation with conjugate (anti-IgG/M F(ab')2-POD 1/1000 in blocking buffer, Dako) for 1 h. After washing, bound antibodies were visualized by o-dianisidine (Fluka) (0.5 mg/ml in 10 mM Tris-HCl, pH 7.4, 83/zM imidazole, 0.06% H 2 0 2, 30 min).

Enzyme immunoassays For determination of antibodies against 70K protein of UlsnRNP a quantitative enzyme immunoassay was established by means of the MS-2 polymerase 70K fusion protein. Optimal concentrations for coating, dilution of conjugates and patients sera were established after appropriate chess board titrations. The 70K fusion protein was applied to microtiter plates (Immunolon, Dynatech) at concentrations of 1/xg/ml, the natural antigens CTE and CTERNase were used at concentrations of 10 /xg/ml. The antigens were diluted in 25 mM MES (morpholinoethanolsulfonic acid) pH 6.0, 1% cyclohexyl-3-(2-morpholinoethyl)-carbodiimid-methyl-4-toluolsulfate. Coating was performed with 100/×l/well for 16 h at 4°C. All antigens were arranged on each plate in the same order (one row serum blank, two rows 70K protein, one row CTE, one row CTERNase). The plates were blocked with 300 /xl PBS, 0.5% BSA (16 h, 4°C), centrifuged to complete dryness and stored in plastic bags for up to 6 weeks at 4°C without significant loss of antibody binding activity. The assay was performed with 200 /xl serum/well (diluted 1/300 in PBS, 0.05% Tween, 0.05% BGG, 0.01% BSA). After incubation (90 min, room temperature) and washing (5 × PBS, 0.05% Tween), plates were incubated with rabbit anti-human I g G / A / M - P O D (Dako, 1/3000 dilution in PBS, 0.05% Tween, 0.01% BGG, 0.05% BSA, 200 /×l/welt, 90 min, room temperature) and washed (3 × PBS, 0.05% Tween, 3 × PBS).

15

B

A kDa

a

b

c

a

d

b

c

d

200

92.5 69

46

30

Fig. 1. Expression and purification of 70K fusion protein. (A) Coomassie blue staining, (B) immunoblot. (a) protein of non-induced bacteria, (b) protein of induced bacteria, (c) 7 M urea extract, (d) 70K fusion protein after DEAE chromatography.

Bound antibodies were visualized with OPD (1.5 m g / m l in 0.1 M phosphate buffer pH 6.0, 4%o H 2 0 2 , 10 min, 25°C), enzyme reaction was stopped by adding 50/xl 4 M H2SO 4. ODs were measured bichromatically at 492 n m / 6 2 0 nm (Multiscan, Flow). Calculation of antibody concentrations (anti-70K protein) was performed with a standard curve prepared from a serum containing antibodies to 70K, A and C protein of U l s n R N P exclusively as determined by immunoblotting and R N A precipitation. This serum showed > 2.5-fold OD compared to negative controls at dilutions of 1/9600 to 1/19,200. 400 U / m l were assigned to a 1/300 dilution. Tests for antibodies against CTE or CTERNas e were regarded positive at > 2.5 fold OD compared to that of three negative control sera. The ELISAs were checked with sera of known antibody speci-

ficity which were analyzed at dilutions from 10-1 to 10 -7 . For control of reproducibility inter- and intra-assay variance were analyzed using ODs and U / m l obtained from standard curves and reference sera. To avoid errors resulting from plate to plate variability each plate was run with its own standard curve.

Results

MS-2 polymerase fusion protein Synthesis of the MS-2 polymerase 70K fusion protein occurred in E. coli 537 transformed with the expression vector pEx34b, containing cDNA sequences for the 380 N terminal amino acids of the 70K protein under control of the inducible A-PL promotor (Fig. 1). The fusion protein re-

16 TABLE I ANTIBODY SPECIFITY OF CONTROL SERA AND THEIR REACTION WITH THE VARIOUS ANTIGEN USED IN THIS STUDY Antibody specifity

Reference system

N tested

Recombinant 70K protein Immunoblot ELISA

Monospecific sera Anti-70K Anti-A Anti-C Anti-B'B Anti-D

RP, RP, RP, RP, RP,

10 5 5 10 5

+ ~ 0 0 0

+ ¢ ¢ ¢ 0

+ + + + +

¢ + +

Anti-U1RNP Anti-Sm Anti-SS-A Anti-SS-B Anti-Scl-70 Anti-Jo-1

CDC, CDC, CDC, CDC, CDC, CDC,

Atlanta Atlanta Atlanta Atlanta Atlanta Atlanta

1 1 1 1 1 1

+ ¢ ¢ 0 ¢ ¢

+ ¢ 0 0 0 ¢

+ + (+ ) (+ ) ¢ (+ )

¢ + (+ ) (+ ) ¢ (+ )

Anti-UlsnRNP/Sm Anti-UlsnRNP Anti-U2snRNP Anti-Sm Anti-SS-A Anti-SS-B Anti-SS-A/-SS-B Anti-Scl-70 Anti-Jol anti-Ku Anti-PMScl Anti-ds-DNA Anti-ss-DNA Anti-histone Anti-centromeres

RP, IB, DD RP, IB, DD RP, IB RP, IB, DD, SP RP, IB*, DD, rA RP, IB*, DD, rA DD, IB* IB *, rA IB, DD DD DD, IB Farr assay, Crith. test Farr assay, Crith. test IB IF, IB

20 20 1 5 5 5 20 5 5 2 5 10 10 10 10

+ + 0 O O O O O O 0 O O 0 O O

+ + O O O 0 O O O 0 0 O O • O

+ + + + (+) (+) (+ ) 0 3/5 + 0 2/5 ( + ) 0 O (+ ) O

+ 0 0 + (+) (+) (+ ) 0 3/5 + 0 O O O (+ ) ¢

IB, rA, DD IB, rA, DD IB, rA, DD IB, rA, DD IB, rA, DD

CTE ELISA

CTERNase ELISA 0

IB, immunoblot (HeLa cell nuclear extract); RP, RNA precipitation (Wil-2 cell nuclear extract); DD agar gel, agar gel double diffusion (calf thymus nuclear extract); (+), borderline positive, OD between 2.5-3.0× of negative control; 0, negative; IB*, immunoblot with native and recombinant antigen respectively; rA, recombinant antigen ELISA; SP, synthetic peptide epitope (B'B, D) ELISA; IF, indirect immunofluorescence, metaphase chromosomes.

a c t e d with a n t i - 7 0 K p r o t e i n a n t i b o d i e s b u t n o t with s e r a o f o t h e r a n t i b o d y specificities o r with s e r a f r o m h e a l t h y d o n o r s ( T a b l e I). P u r i f i c a t i o n of t h e fusion p r o t e i n s u c c e e d e d a f t e r e x t r a c t i o n with u r e a a n d ion e x c h a n g e c h r o m a t o g r a p h y . Imm u n o r e a c t i v e p r o t e i n s w e r e e l u t e d in t h e 3 5 0 - 5 5 5 m M NaC1 fractions. T h e y w e r e f r e e of c o n t a m i n a t i n g E. coli p r o t e i n s as c o u l d b e s e e n a f t e r C o o m a s s i e b l u e staining of S D S - P A G E s . I n imm u n o b l o t s t h e p u r i f i e d p r o t e i n s r e a c t e d with a n t i - 7 0 K a n t i b o d y positive s e r a only. A n t i b o d i e s e l u t e d f r o m b l o t t i n g strips b o u n d to 70K p r o t e i n

b a n d s in i m m u n o b l o t s o f n a t u r a l H e L a cell nuc l e a r extracts a n d vice versa. T h e p u r i f i e d r e c o m b i n a n t p r o t e i n f r a c t i o n consists of t h r e e m o l e c u lar species with a p p a r e n t m o l e c u l a r weights o f 75, 53 a n d 47 k D a , respectively, as d e t e r m i n e d by S D S - P A G E a n d i m m u n o b l o t analyses. T h e size o f t h e 75 k D a b a n d c o r r e s p o n d s m o s t likely to t h e size of t h e e x p e c t e d e n t i r e r e c o m b i n a n t p r o tein a s s u m i n g t h e 380 N t e r m i n a l a m i n o acids of t h e 70K p r o t e i n b r i n g a b o u t an a n o m a l o u s m i g r a tion b e h a v i o r of t h e fusion b e t w e e n M S - 2 polym e r a s e a n d 70K p r o t e i n in S D S - P A G E similar to

17

that of the natural 70K protein (Query et al., 1989). Smaller immunoreactive proteins represent probably degradation products.

ELISA for detection of antibodies to 70K protein of UlsnRNP For detection of antibodies microtiter plates were coated with 70K fusion protein (1 /zg/ml). The E L I S A was p e r f o r m e d including appropriate controls as outlined in material and methods. Specificity was checked with a panel of reference sera of various antibodies specificities (Table I). Positive reactions were only seen with sera containing anti-70K antibodies but not with sera containing antibodies of other specificities and with A N A negative sera (n = 50) of healthy persons. Sera containing high titre antibody against 70K protein revealed O D s five times higher than negative sera even at dilutions of 10 -5 to 10 -6. Best discrimination between negative and positive sera was obtained at dilutions of 1/300. As can be seen from Fig. 2 in a panel of nonselected sera the slopes of titration curves are congruent except that of the a n t i - U 1 R N P reference serum of CDC. The antibodies of the latter serum show an epitope specificity different from most of the anti-70K sera (Guldner et al., 1988) which may cause the variant slope. Antibody concentration in patients sera was calculated with a standard curve p r e p a r e d from a serum positive at dilutions of 1 : 9600 to 1 : 19,200 ( O D > 2.5 times that of negative controls). This serum revealed O D s of about 1.7 at a 1 / 3 0 0 dilution to which 400 U / m l were assigned. Based on this standard an antibody binding of < 6 U / m l was found in A N A negative sera from healthy persons (n = 50). For the a n t i - U i R N P reference serum of C D C (Atlanta, lot no. 82-0010) we measured a concentration of 50 U / m l . The interassay variance was calculated from O D s of standard curves of consecutive days. An example of 20 consecutive days is given in Fig. 3. There was no significant change of the values for a 1 year estimate. A second interassay variance was calculated from the U / m l obtained with a control serum which was included in each run. The coefficient of variation was 9.6% for a m e a n antibody concentration of 101 _+ 9.7 U / m l . Despite the good correlation of standard curves from

00 25-

20-

1s-

~o-

'\

1/~

11~

1]~0

1600

IT20~ 12L00 1/~1)

1] f'm IIg200 I~LO0 176200

DILUTION

Fig. 2. Dilution curves of A N A and anti-UlsnRNP negative and anti-UlsnRNP positive sera. Best discrimination was obtained at dilutions of 1/300. The anti-RNP reference serum of CDC (R) differs in slope as compared to other sera. ANA p o s i t i v e s e r a : • • • • v ; A N A n e g a t i v e s e r a : © [ ] z~ v + ×.

OB ~2/520

nm

1.8:

15-

!0

05

01

Fig. 3. Statistical evaluation of 20 standard curves obtained in 20 consecutive days. Three different lots of plates were used; all reagents were prepared at the day of use. 2: mean; o-: standard deviation; VK: coefficient of variation of standard ODs (1/300 = 400 U / m l , 1/19,200 = 6.25 U / m l ) . Intra-assay variance (n = 40) was 9.9.

18 T A B L E 11 R E S U L T S O F S I M U L T A N E O U S L Y P E R F O R M E D ELISAs W I T H 10,883 S E R A F O R A N T I B O D I E S R E A C T I N G W I T H R E C O M B I N A N T 70K P R O T E I N A N D C A L F T H Y M U S E X T R A C T B E F O R E (CTE) A N D A F T E R R N a s e D I G E S T I O N (CTE RNase). Five additional sera with different results are not outlined here (see text). Group 1 2 3 4

n 319 474 967 9,123

%

70 K Protein

CTE

CTERNase

2.9 4.3 8.8 83.9

Positive Positive Negative Negative

Positive Positive Positive Negative

Negative Positive Positive Negative

several days obtained with different lots of microtiter plates each E L I S A plate was run with its own standard curve. Intra-assay variance was calculated for three different charges of coated plates. In 32 tests with a control serum of 99.8 _+ 4.7 U / m l the coefficient of intra-assay variance was 4.7%. In screening for antibodies reacting with C T E a n d / o r CTERNas e O D s > 2.5 times the m e a n O D of three negative controls were regarded as positive. Sera containing antibodies to Sm reacted with both C T E and CTEm~as e, those containing antibodies to R N P with C T E only. Reactivity of other reference sera with CTE/CTERNas e is shown in Table I.

Comparative ELISAs and immunoblots of patients set'a

Within 2 years 10,888 sera of patients with suspected or manifest rheumatic diseases were analyzed (Table II). Antibodies to recombinant 70K protein were detected in 793 sera of which 319 reacted with C T E only (group 1), 474 with both C T E and CTERNase (group 2). No reaction with 70K protein but with C T E and CTERNa~ e was found in 967 sera (group 3). No antibodies could be detected in 9123 sera (group 4). Five sera showed a different behavior: in one the reaction was restricted to CTE, in two to CTERNase, these p h e n o m e n a were not further investigated. Two sera reacted with recombinant 70K protein, but not with C T E / C T E a N a s e. In immunoblot analyses and agar gel double diffusion these sera showed no antibodies against U l s n R N P proteins. According to current definition group 1 sera (Table II) contain antibodies to U l s n R N P (anti-

R N P and anti-ENA) but not to Sin. The sera were analyzed by immunoblot with proteins of affinity purified U s n R N P particles (Table III). In the immunoblot assay, only 275 sera (86%) reacted with the 70K protein. Of the remaining 44 negative sera, 13 contained antibodies to A, and two sera antibodies against A and B'B proteins, 29 showed no binding to U s n R N P proteins. Almost 90% of group 1 sera reacted with the Sm epitope B'B in blots, however, they did not react with the natural Sm epitopes in E L I S A (CTE~Na~e). In 60% of the sera the concentration of anti-70K protein antibodies exceeded 100 U / m l , the remaining 40% showed a uniform distribution over a range from 12.5 to 60 U / m l . G r o u p 2 sera (n = 474) contain antibodies with a n t i - U l s n R N P and anti-Sm specificity. In immunoblots 349 sera of this group (74%) bound to 70K protein, no reactivity was found with 125 sera (26%), 100 of which showed antibodies against other U s n R N P proteins (Table IV). Antibody concentration was less than 40 U / m l in 50% of the sera, only 27% showed more than 100

T A B L E III I M M U N O B L O T A N A L Y S E S O F 319 S E R A (TABLE II, G R O U P 1) R E V E A L I N G A N T I B O D I E S T O T H E REC O M B I N A N T 70K P R O T E I N A N D C T E BY ELISA

Anti-70K

Immunoblot positive

Immunoblot negative

n = 275

n = 44

(86%)

(14%)

% of sera positive for other U s n R N P proteins Anti-A Anti-C Anti-B'B Anti-D

216 185 216 8

(89%) (76%) (89%) (3%)

15 0 2 0

(34%) (5%)

19 TABLE IV IMMUNOBLOT ANALYSES OF 474 SERA (TABLE II, GROUP 2) REVEALING ANTIBODIES TO THE RECOMBINANT 70K PROTEIN, CTE AND CTERNase BY ELISA Immunoblot positive

Immunoblot negative

Anti-70K

n = 349

n = 125

Anti-A Anti-C Anti-B'B Anti-D

268 201 320 284

(74%)

(26%)

% of sera positive for other UsnRNP proteins (84%) (63%) (100%) (89%)

52 21 100 68

(42%) (17%) (80%) (54%)

U / m l . T h e m e a n c o n c e n t r a t i o n of a n t i - 7 0 K protein in this g r o u p was lower t h a n in g r o u p 1. T h e results of i m m u n o b l o t s p e r f o r m e d with sera n e g a t i v e for a n t i b o d i e s against the r e c o m b i n a n t 70K p r o t e i n b u t r e a c t i n g with C T E a n d CTERNas e (group 3) are shown in T a b l e V. O f these 967 sera 329 (34%) h a r b o r e d a n t i b o d i e s to

U s n R N P proteins. A n t i b o d i e s against 70K protein only were p r e s e n t in t e n sera (1%), 13 sera (1.3%) b o u n d to 70K p r o t e i n as well as to o t h e r p r o t e i n s (A, B'B, C, D protein), 306 sera (31.6%) r e a c t e d with U s n R N P p r o t e i n s different from 70K protein. Overall, 2.4% of sera negative in a n t i - 7 0 K p r o t e i n E L I S A (n = 23) were now reactive by i m m u n o b l o t t i n g . T h e y showed a weak staining implicating a low a n t i b o d y c o n c e n t r a t i o n b u t i m m u n o b l o t s p e r f o r m e d with U s n R N P proteins s e p a r a t e d o n 9% polyacrylamide revealed the characteristic triplet of 70 k D a bands. A n t i bodies to A a n d / o r C p r o t e i n were f o u n d in 267 (28%), n o a n t i b o d i e s to U s n R N P p r o t e i n s could be d e t e c t e d in the r e m a i n i n g 638 sera (66%). B e c a u s e of the possibility of d i s c r e p a n t results o b t a i n e d with i m m u n o b l o t a n d E L I S A we tested w h e t h e r a n d to which extent E L I S A negative sera (70K p r o t e i n , C T E , CTERN . . . . g r o u p 4) c o n t a i n a n t i b o d i e s against U s n R N P proteins. A r a n d o m sample of 200 E L I S A negative sera with negative A N A test (indirect i m m u n o f l u o r e s c e n c e o n rat

TABLE V IMMUNOBLOT OF 967 SERA (TABLE II, GROUP 3) REVEALING ANTIBODIES TO CTE AND CTERNase BUT NOT AGAINST THE RECOMBINANT 70K PROTEIN BY ELISA Definition

n

Antigen specificity

Anti-UlsnRNP

132 (14%)

70K 70K + A A C A+C

10 4 99 9 10

10.6%

70K + B'B 70K + A + B' B A + B' B A+D 70K + A + B' B + C A+B'B+C 70K+A+ B'B+D A+B'B+D A+B'B+C+D

1 3 52 9 4 42 1 9 37

5.7%

28 6 5

0%

638

0%

Anti-UlsnRNP and anti-Sm

Anti-Sm

Other specificities

158 (16%)

39 (4%)

638 (66%)

B'B B'B+D D No antibodies reacting with UsnRNP proteins

Anti-70 K protein positive immunoblot only

2.4%

20 T A B L E VI IMMUNOBLOT ANALYSES OF ELISA NEGATIVE S E R A (70K P R O T E I N , CTE, CTERNase) W I T H P O S I T I V E (n = 100) A N D N E G A T I V E (n = 200) A N A T E S T In the E L I S A and A N A negative group all the blot signals showed a very weak staining, in the E L I S A negative A N A positive group half of the sera showed weak staining

Anti-70K Anti-A Anti-C Anti-B'B Anti-D Blot pos.

E L I S A negative A N A positive n = 100

E L I S A negative A N A negative n = 200

0 28 0 15 13

2 10 0 19 5

0% 28% 0% 15% 13% 38%

1% 5% 0% 9.5% 2.5% 17%

liver, kidney and stomach, Hep-2 cells) and of 100 ELISA negative sera with A N A titer 1 in > 320 and speckled pattern were analyzed with immunoblots. The results are outlined in Table VI. Two sera (ANA and ELISA negative) reacted with proteins in the 70 kDa region but not with other U l s n R N P proteins. Both sera did not precipitate natural CTE in agar gel double diffusion. A remarkable proportion of the ELISA negative sera, however, contained antibodies against A, B'B and D protein. With exception of very few cases of anti-A protein containing sera, all exhibited weak staining, implicating low antibody concentration. The discrimination between positive and negative was sometimes rather difficult.

Discussion According to criteria proposed by some investigators (Sharp et al., 1972, 1976; Alarcdn-Segovia et al., 1987; Kasukawa et al., 1987) the presence of antibodies against U l s n R N P is a prerequisite for the diagnosis of MCTD. Accordingly antiU l s n R N P would have a 100% sensitivity and a 100% negative predictive value for MCTD. It should be mentioned, however, that neither the distinctiveness of the syndrome MCTD as originally defined by Sharp et a|. (1972) nor the predictive value of U l s n R N P antibodies are univer-

sally accepted (Reichlin, 1976; Lemmer et al., 1982; McHugh et al., 1990). Nevertheless, as shown by immunoblot analyses of sera from patients with MCTD, high titers of antibodies against U l s n R N P are almost constantly associated with antibodies against the 70K protein (Habets et al., 1983, 1985, 1989a; Pettersson et al., 1984, 1986; Van Venroij 1987; Williams et al., 1988; Combe et al., 1989). Thus screening for antibodies against a recombinant 70K protein may satisfy most clinical questions as efficient as testing for anti-UlsnRNP. A negative anti-70K protein assay excludes the diagnosis of MCTD in all probability, a positive test, however, does not suggest MCTD definitively because these antibodies may also occur in patients with SLE and in other rare cases of rheumatic diseases. An ELISA for antibody screening was developed with a MS-2 polymerase 70K fusion protein. Natural antibodies against MS-2 polymerase are not found in humans, therefore nonspecific reactions caused by the fusion protein, as described for/3-galactosidase fusion proteins (Habets et al., 1989a; Clair et al., 1990) are not to be expected and parallel tests with MS-2 polymerase can be omitted. The test showed a good discrimination between negative controls and sera containing anti-70K protein in dilutions up to 10 -5 to 10 -6 (Fig. 2). Interassay and intra-assay variance are in acceptable limits (Fig. 3). The test allows to quantify antibody concentration by means of a standard curve. The clinical relevance of quantitative analyses cannot be foreseen. However, the concentration of anti-70K protein seems to fluctuate remarkably during the course of disease (Clair et al., 1990; De Rooij et al., 1990). Such phenomena as well as a shift in antibody specificity (anti-Sm towards anti-U1RNP) have already been observed with the other detection systems (Barada et al. 1980; Walsh et al., 1981; Nishikai et al., 1984; Fisher et al., 1985; Houtman et al., 1986). Quantitative analyses may further give some support in differential diagnosis because in MCTD antibody concentrations higher than in SLE were supposed (Williams et al., 1988; Yakeda et al., 1989; Clair et al., 1990). Our results may follow this line because lower concentrations of antibodies were measured in the group of anti-Sm positive sera (Table II, group 2) in which SLE pa-

21 tients have to be expected. However, in individual cases this discrimination may be difficult because some SLE patients exhibit very high antibody concentrations (Clair et al., 1990; Ehrfeld et al., 1991). The recombinant 70K protein ELISA was compared with other ELISAs using partially purified natural UsnRNP particles (CTE, CTERNase) and immunoblots. CTE/CTERNas e antigens reacted not only with anti-UlsnRNP/anti-Sm sera but also with sera containing antibodies against SS-A (Ro), SS-B (La), Jo-1 (histidyl-tRNA synthetase), PM-Scl or histones, respectively. Compared to the antibodies against UsnRNP proteins (OD up to 20 × OD of negative controls) these antibodies showed rather weak reactions which did not exceed 3.5 × OD of negative controls. As expected most of the positive results were obtained with these antigens (n = 1760), but there was a considerable number of sera which contained no antibodies against UsnRNP proteins (n = 638, Table V). Because of the other reactivities of CTE antigens mentioned above some positive results may be caused by other antibodies. Thus ELISA studies for detection of anti-RNP using similarly prepared antigens have to be regarded with precaution because the number of true anti-RNP antibodies may have been overestimated. The anti-70K protein ELISA (n = 793 positive sera) was found to be more sensitive than immunoblotting (n = 647 positive sera). Only 79% of positive results obtained with ELISA could be confirmed by immunoblotting. These figures agree with those reported by Habets et al. (1989a) from a small collective of 35 sera using a /3-galactosidase 70K fusion protein. On the other hand, the proportion of false negative results in ELISA with recombinant antigen amounts to 2.8% compared to immunoblot analyses. With exception of two, each of the 818 sera positive for anti-70K protein antibodies (ELISA a n d / o r immunoblot) reacted also with the natural CTE antigens. To find out whether there exist sera with anti70K protein antibodies not reacting in either of these ELISAs (recombinant 70K protein, CTE/CTERNase), 300 ELISA negative sera (Table II, group 4) were analyzed by immunoblot. In a significant proportion of these sera antibodies

against UlsnRNP proteins could be detected (38% in ANA positive, 17% in ANA negative sera). Antibodies reacting with the 70 kDa protein region were seen in two ANA negative sera (0.7%), which extrapolated to the total number of group 4 sera (n = 9123) would result in 64 false negatives. The reactions, however, were rather weak, antibodies against other UlsnRNP proteins were not present. The two sera did not react in agar gel double diffusion with natural antigens. Thus the presence of anti-70K protein antibodies remains questionable. None of the assays used in this study were able to detect all of the possible anti-70K protein antibodies occurring in nonselected patients' sera. In a smaller group (n = 23) a positive reaction was only seen in immunoblots (group 3 sera, random sample). Most of these sera showed a weak staining of the 70 kDa region. In order to increase the specifcity for the detection of the 70K protein in the immunoblot assay affinity purified UsnRNPs were used and SDS-PAGEs of lower acrylamide concentration were run to produce the characteristic triplet of 70K protein bands, which may be caused by different degrees of phosphorylation or by as yet unknown posttranslational processes of the 70K protein (Woppmann et al., 1990). Despite the triplet in the 70 kDa region generated by most of these sera a nonspecific binding to other proteins comigrating in that region cannot be excluded. Even affinity purified UsnRNPs may contain contaminating proteins (Lfihrmann, personal communication). In sera, however, which contained antibodies against additional UlsnRNP proteins a specific binding may be considered. The fusion protein used here lacks 13% of the C-terminus (amino acid: 381-437). As could be demonstrated by epitope mapping all anti-70K protein antibodies reacted within the N terminal half of the molecule (Guldner et al., 1988; Nyman et al., 1990). Due to the much higher number of sera studied here the presence of antibodies in some sera reacting only with the C terminal of the molecule cannot be excluded. Preliminary investigations with a full-length 70K fusion protein suggest, however, that this may not be the main cause. It is also conceivable that the recombinant 70K protein lacks conformation epitopes present

22

in the natural protein and which are not irreversibly destroyed by SDS-PAGE. A larger number of sera revealed a negative immunoblot but a positive reaction with the recombinant 70K protein (n = 162). All but two sera revealed also antibodies against the CTE antigens. 41% thereof did not react with CTERNase which can be regarded as additional indication that antibodies against an UlsnRNP protein were present. The mean antibody concentration of the immunoblot positive sera exceeded that of immunoblot negative sera (150 U / m l for immunoblot positive, 60 U / m l for immunoblot negative). This lower antibody concentration of the latter group may explain the differing results. Finally, epitopes of the 70K protein could be masked by the binding to nylon membranes. With the use of this recombinant 70K protein assay a high proportion of anti-UlsnRNP antibodies according to the current definition may be missed. Out of 967 sera positive in the antiCTE/anti-CTERNas e assay but negative for anti70K protein 267 (27.6%) showed antibodies reacting with A a n d / o r C protein. Using the 70K protein ELISA only, 26.5% of all sera exhibiting anti-UlsnRNP activity (anti-70K a n d / o r anti-A a n d / o r anti-C; see Tables II, V) would not have been detected. If we include the anti-A a n d / o r anti-C sera positive in immunoblot but negative in CTE ELISA this number may be considerably higher. Preliminary results indicate that this disadvantage can be overcome using a mixture of recombinant antigens (70K, A, C proteins). The ELISA with recombinant 70K protein may be regarded as an advance for screening antibodies against UlsnRNP. Our results, however, demonstrate that it should not be used exclusively until the clinical significance of antibodies against A and C protein as well as the cross-reactivity of antibodies against A and B'B proteins caused by the prolin rich motifs (Habets et al., 1989b; Renz et al., 1989b) are elaborated. The future use of specific assays with recombinant antigens (Renz et al., 1989a; de Rooij et al., 1990) will necessarily cause the revision of the old terms anti-Sm and anti-RNP (ENA) and lead to a redefinition of the clinical and diagnostic significance of the antibodies reacting with individual proteins of UlsnRNP.

Acknowledgements We thank Professor R. Liihrmann, University of Marburg, for kindly supplying affinity purified UsnRNP particles. This work has been partially supported by a grant of the Bundesministerium fiir Forschung und Technologie (BMFT) of the Federal Republic of Germany.

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