An enzyme-linked immunosorbent assay for antistriational antibodies associated with myasthenia gravis and thymoma: Comparison with indirect immunofluorescence

Journal o[ Imm,mologwal Methods. 59 (1983) 301 - 314

30 I

Elsevier Biomedical Press

An Enzyme-Linked Immunosorbent Assay for Antistriational Antibodies Associated with Myasthenia Gravis and Thymoma: Comparison with Indirect Immunofluorescence 1 Jon A. Carrano, Nigel R. Swanson and Roger L. Dawkins Department of Chnical lmmunoloD'. Royal Perth and Str Charles Gatrdner Hospitals and The [. nit'ersttr of 1t "estern ,4 ustraha. Perth. 1~~,4. 6000,4 ustraha

(Reco,~ed 26 June 1982. accepted 10 December 1982)

Autoantibod~es to the striations of skeletal muscle (AStrA) detected b5 immunofluorescence are useful m the diagnosis of a thymoma associated gith myasthenia gravis (MG). W~th the retention of de,.eloping a better method, an enzyme-linked immunosorbent assay (ELISA) has been e~,aluated in 147 MG patients and 200 healthy controls. An additional 107 patients vqth various autoimmune diseases and autoant~bodies were also tested. With a crude actom3osm preparation, the ELISA ga~e similar results to Jmmunofluorescence. ',lz. positP, es in 42% of MG patients, but in all with a histologicall~ proven th~moma. Less than It~. of the heahh~ controls were positive but false posmxes were found in patients gith hxer disease and anti-smooth muscle antibodies. After treatment of rheumatoid arthritis with D-pemcillamine the tltre of AStrA ma.~ rise. The ELISA gas shown to be sensitive and reproducible, but immunofluorescence is a more practical method of distinguishing between the different categories of anti-muscle antibodies. ELISA should pro~e particularl) useful for quantitation and sequentml monitoring. Ke}~'ords: myasthenta gracts -- autoannbodtes -- E L I S A -- #nmunt~lTuorescence- o - p e m c d l a m t n e skeletal muscle

Introduction A n t i s t r i a t i o n a l a n t i b o d i e s ( A S t r A ) a r e t h o u g h t to be o f v a l u e in the d i a g n o s i s o f a t h y m o m a associated with m y a s t h e n i a gravis ( M G ) but existing m e t h o d s suffer from serious limitations. A l t h o u g h i m m u n o f l u o r e s c e n c e on skeletal muscle has been used f o r 20 y e a r s ( S t r a u s s et ai., 1960), i n t e r p r e t a t i o n is c o m p l i c a t e d b y n o n s p e c i f i c b i n d i n g o f i m m u n o g l o b u l i n to c o n t r a c t i l e p r o t e i n s , o c c a s i o n a l c r o s s r e a c t i v i t y w i t h s m o o t h m u s c l e ( F a i r f a x a n d G r o s c h e I - S t e w a r t , 1977; M c D o n a l d et al., 1977: T o h et Publication Number 8117 of the Department of Clinical Immunology. 0022-1759/83/0000-0000/$03.00 '~. [983 Elsevier Science Publishers

302 al., 1978), and by the complexity of the patterns produced by different sera (Peers et al., 1977). Some of the difficulties related to substrate artifacts can be overcome by the use of glycerinated myofibrils but a recent serum exchange programme has revealed that many laboratories still experience difficulties (unpublished observations). Haemagglutination methods have been used (Djanian et al., 1964: Dawkins et al., 1975b) but these also suffer problems with standardisation and nonspecific reactions, lmmunodiffusion has been reported to allow detection of only a proportion of MG-associated AStrA (Rule et ai., 1973a; McDonald et al., 1979). The need for an improved assay has been emphasised by the repeated observation that D-penicillamine therapy induces AStrA in patients with rheumatoid arthritis (RA) (Dawkins et al., 1975a: Camus et al., 1976; Zilko et al., 1977, 1980). and that HLA-associated genetic factors influence apparent immune responsiveness to contractile proteins (Zilko et al., 1980: Carrano et al., 1982a). In mice, genetic factors are also important in determining the titre of antibody following immunisation ~ith crude myosin (Carrano et al.. 1982b). Recently, it has been shown that enzyme-linked immunosorbent assays (ELISA) can be used to detect antibody to myofibrillar proteins (Kurki. 1978: Biral et al., 1979; Charlemagne et al., 1979; Billeter et al., 1980). We have developed an ELISA assay and evaluated its suitability for routine diagnostic procedures. Experience with sera of patients treated with o-peniciilamine and the relationship between HLA and titre will be reported separately.

Materials and Methods C o n t r o l sera

Two hundred control sera were obtained from the core group of the population of Busselton. This group was age and sex matched to be representative of the total survey group of more than 3000 adult subjects and has been used for numerous other studies including HLA. A, B, C. D R typing, and the measurement of anti-DNA and anti-acetylcholine receptor antibodies. When the total group ~as surveyed for AStrA by immunofluorescence the prevalence of a positive result ~as found to be only 0.6% (Hawkins et al., 1979). Test sera

Sera from 147 patients with adult onset MG were selected for investigation. These included 128 sera from patients, of various racial groups, submitted to the Eighth Histocompatibility Workshop (Dawkins, 1980). Selection was based on a clinical history of M G and an anti-acetyicholine receptor antibody level of at least 2 U. Less than I of 100,000 individuals in a normal population would be expected to have an antibody level of greater than 2 U (Dawkins et al., 1981). The Caucasoid group included 63 Australian, 51 Italian, 26 American and 6 English patients. Sera from 13 Japanese, 8 Chinese and 17 American Negroid patients made up the other racial groups.

303 Patients were classified according to associated thymic pathology. Only cases v- ith histopathological evidence of thymoma (31%), thymic hyperplasia (55%,) or normal histology (14~) were so classified. The remainder (48 cases) were recorded as unknown. Additional sera from patients v-'ith autoantibody associated diseases v-'ere assayed to investigate their influence on anti-4M activit3 (see belong). These, submitted for routine autoantibody screening, included sera from 6 cases of thymoma (without MG), 31 of systemic lupus erythematosus (SLEI, 14 of polymyositis (PM), 13 of active chronic hepatitis (ACH), 5 of acute viral hepatitis (AVHI, 5 of alcoholic liver disease (ALD) and 33 of RA, some of which had received D-penicillamine therapy.

Indirect immunofluorescence Sera were classified as AStrA positive or negative by screening at a dilution of I in 10 on a composite substrate including guinea pig skeletal muscle and rat heart. B3 these means other autoantibodies such as anti-smooth muscle ~'ASM) and the zebra type of skeletal muscle antibody (McDonald et al.. 1977, 1979) v-'ere distinguished from AStrA. Positive sera were then tested on glycerinated fibres to alloy-' assessment of strength (I + , 2 + , 3 + corresponding to titres of 1/5, 1/25, and 1/125 or greater~ and pattern (Peers et al., 1977). The pattern previousl3 described as unclassifiable is nov-' termed complex.

Extraction of contractile proteins A crude actomyosin extract was prepared from human skeletal muscle according to the method of Rule et al. (1973a). Gastrocnemius muscle dissected at post mortem was either used immediately or stored at - 2 0 ° C . Approximately 30 g of muscle was finely diced on ice, washed 3 times by centrifugation in cold saline (4°C), then homogenised sparingly in 3 vols. of cold 0.5 M KCI, 0.1 M K_, HPO4 adjusted to pH 6.9. After adding a further 2 vols. of this extraction buffer and stirring for 15 min at 4°C, the suspension was centrifuged at 16,000 × g for 30 rain. The supernatant was filtered through gauze and added v-'ith stirring to 10 vols, of cold distilled water. A floccular precipitate formed which settled on standing to allow aspiration of most of the supernatant. The remaining solution was then spun at 4000 × g for 15 rain, v-'ashed tv-'ice by centrifugation in 0.05 M KCI and redissolved in 20 ml of 0.5 M KCI, pH 6.7. Undissolved protein was removed by centrifugation at 700 × g for 10 min. The supernatant, designated 4M, was stored with 0.05% N a N 3 at 4°C. The protein 3held from 30 g of muscle varied from approximately 20 to 40 mg.

Gel electrophoresis The 4M extract was analysed by SDS polyacrylamide gel electrophoresis using 10% acrylamide gels after the method of Weber and Osborne (1969). Samples of 4M (20-200 #g), with 10 #1 bromophenol blue, 50 #1 glycerol and 10 #1 2mercaptoethanol added, were incubated at 60°C for 15 min prior to loading. Gels were stained overnight in Coomassie blue R250 and destained by diffusion in acetic a c i d : e t h a n o l : w a t e r ( 1 : 3 : 8 v / v / v ) . Crude myosin (McDonald et al.. 1979) and

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commercial molecular weight markers (Pharmacia, Uppsala) were used for molecular weight calibration of the 4M bands (Fig. 1). Gels were scanned at 600 nm in a Transidyne 2955 scanning densitometer with curve integrator (Transidyne General Corporation, MI, U.S.A.). The 3 preparations of 4M were heterogeneous but all gave peaks corresponding to myosin, C-protein, actin, tropomyosin and troponin. Contamination of 4M with DNA and RNA was suggested by ethidium bromide staining of the gels and confirmed by comparison of banding patterns bv electrophoresis after DNAse and RNAse treatment of the muscle extract. Samples of 4M, before and after enzyme treatment, were electrophoresed by a modification of the horizontal agarose gel technique described by McDonell et al. (1977).

Enzyme-conjugated anti-immunoglobulins A goat was immunised with human gammaglobulin (Cohn Fraction II, Sigma Chemicals, U.S.A.) and DEAE cellulose purified human lgG, emulsified in complete Freund's .adjuvant. The resulting antiserum was purified by elution from glutaraidehyde insolubilised gammagiobulins using 0.1 M glycine-HCl buffer, pH

305 2.8, as described by Avrameas and Ternynck (1969). After dialysis against phosphate-buffered saline (PBS), pH 7.2, the specific antibodies were conjugated to alkaline phosphatase Type VII (Sigma Chemicals, U.S.A.) by the method of Engvall and Perlmann (1972). The conjugate had specificity for ~, and p. hea,,'y chains and light chains of immunoglobulins as defined by immunoelectrophoresis. A working dilution of I in 100 was determined by chequer board titration.

ELISA Enzyme-linked immunosorbent assays were performed as a modification of the method described by Engvall and Perlmann (1972). Two hundred microlitres 4M diluted in sodium carbonate buffer (0.1 M" pH 9.6) with N a N 3 (0.05%) were added to the wells of a pol~'inyl microtitre plate (Nunclon Delta 96 well microtest plate; A / S Nunc Kamstrup, Denmark). Plates were incubated for I h at 37°C and then overnight at 4°C. After flicking off the coating solution the plates were flushed with saline (0.15 M NaCI) containing 0.05% Tween 20 and allowed to stand for 5 min. After 3 washes the plates were drained and 200 p,I serum in PBS with 0.05% Tween 20 was added to the wells. The plates were incubated at 37°C for 60 rain, washed as before and 200 #1 of conjugate in PBS with 0.05% Tween 20 added to the w,ells. The plates were again incubated at 37°C for 60 min, washed as before, and 200 #1 of the enzyme substrate added, consisting of p-nitrophenyl phosphate (Sigma Chemicals. U.S.A.) diluted to 3 m g / m l in sodium carbonate buffer (0.05 M, pH 9.8) with ! mM MgCI_,. The plates were then incubated at 37°C and the optical density (OD) at 405 nm monitored with a Titertek Multiskan Plate Reader (Flow Laboratories, Helsinki). When a standard positive serum gave a reading of 1.80 (after approximately 40 min) all wells were read. Edge artifacts due to temperature gradients (Burt et al.. 1979; Oliver et al., 1981) ~xere overcome by floating the plastic coated microtitre plates in a 37°C water bath during incubations. Competitit:.e inhibition The specificity of antibody binding in the ELISA, was investigated by testing the ability of fluid phase 4M antigen to inhibit antibody binding to the plastic adsorbed 4M. Sera from 2 patients with MG, at a final dilution of I in 100, were incubated with varying concentrations of the 4M antigen solution. After 60 rain incubation at 37°C. the mixture was transferred to the 4M coated wells of a microtitre plate and assayed by ELISA to determine residual antibody activity. Sera were similarly preincubated with bovine serum albumin (Commonwealth Serum Laboratories, Australia) as a control for nonspecific absorption. Statistics The non-parametric Mann-Whitney U test for independent samples and the chi-square test (Siegel, 1956) were used to compare the skewed distributions of O D values in different groups. The correlation coefficient ( r ) and coefficient of variation (C = standard d e v i a t i o n / m e a n ) were used to measure between-run reproducibility (Snedecor and Cochran, 1967).

306 Results

Optimal antigen concentration and serum dilution The optimal concentration of coating antigen and serum dilution were determined by titration (Figs. 2 and 3). As shown in Fig. 2, sera strongly positive b~ immunofluorescence gave readings close to the upper limit of the measuring range at antigen concentrations as low as 32 Fg/."ml. In contrast, the low readings of a pool of sera from 10 heahhy volunteers increased slightl3 at concentrations below 8 p,g/'ml. .Accordingly, 20 p,g/ml was chosen as the standard concentration and used in the experiments described below. Serial dilutions of sera positixe for AStrA by immunofluorescence and the pool of negative sera were compared by ELISA (Fig. 3). The increased O D of the pooled negative sera at dilutions below 1 in 500 (Fig. 3) may be due to nonspecific binding of immunoglobulins to the plastic wells. Despite this, the largest positive:negative ratio occurred at a dilution of I in 20. Two of the stronger sera exceeded the measuring range at this dilution. Accordingly, to allow discrimination between stronger sera, and for purposes of economy, a dilution of I in 100 was chosen as the screening titre.

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Specificity of antibodies Five sera from patients with MG and positive AStrA by immunofluorescence were compared by ELISA (Figs. 2 and 3). Their patterns included complex. intermediate. A band and I band staining (Peers et al., 1977) but all gave similar results by ELISA. This suggests that each serum reacts with relatively major components of the 4M mixture. Individual variations may reflect additional reactions with minor components. The specificity of the ELISA was further investigated by competitive inhibition experiments. Fig. 4 shows the results of adding 4M or BSA to 2 M G sera. Incubation of 105 n g / m l of 4M with the test sera caused a 45-65% decrease in anti-4M O D values, while the same concentration of BSA had little effect. Addition of veD, low concentrations of 4M ( I - 1 0 n g / m l ) to serum of patient 2 caused an increase in OD; the reason for this is unclear but may be due to nonspecific binding of antigen/antibody complexes formed at equilibrium. DNAse and RNAse treatment of 4M induced electrophoretic changes in 4M but no change in titre was observed for A N A positive or negative sera from patients with

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Fig. 5. Comparison of ]mmunofluorescence and ELISA optical density for detecting AStrA m patients with MG (different racial groups), and a healthy control population (Caucasoids. Positive for AStrA b3 immunofluorescence: I case per po, nt (O); negative for AStrA by immunofluorescence: I case per po,nt ( © ). 5 cases per point ( @ ).

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Fig. 6. Correlation of AStrA immunofluorescence titre and ELISA optical density with thymic pathology in patients with MG. All sera tested at a dilution of Inn 100. (a) Total patient group (n = 147): thymoma positive (n = 31) {O). t h y m o m a negati~,e (n = 67) (O), unknown thymic pathology (n = 49) (zx). (b) Patients selected for htstopathological review, (n = 36): thymoma positive (n = 139 (O). thymoma negative (n = 22) {O). uncertain diagnosis (n = I) (zx).

310

MG, when compared on enzyme treated or untreated 4M (data not shown). Inhibition of the E L I S A reactivit3 was seen for certain sera after preincubation with crude nuclear extracts (data not shown). It is not vet clear whether these changes related to nonspecific binding, presence of relevant autoantibody, cross reactivity or other factors.

Between-run reproducibilio' of the ELISA Use of the E L I S A for long-term monitoring of anti-muscle antibodies relies on its between-run precision. A standard incubation period for the enzyme-substrate reaction proved inadequate. Therefore, the reaction of an aliquoted positive serum was used to terminate each run at a predetermined OD. In addition, control sera covering a range of O D values (0.2-1.5) were included in each run. With use of these techniques over a 4 month period, the between-run coefficient of variation ranged from 7% at O D 1.5 to 16% at O D 0.4, The correlation coefficient for between-run duplicates ~as greater than 0.9. Reproducibility was further improved b~ incubating the plates on a 37°C water bath to reduce thermal edge effects.

Frequen£v of anti-4M antibodies in the normal population and myasthenia grat:is The distribution of O D values for 200 sera from healthy controls are shown in Fig. 5. They form a positivel~ skewed distribution with a range of 0.09-0.83. All but one of these sera had O D levels below 0.50. This serum, with an O D of 0.83. and positive immunofluorescence, was from an elderl,, female with no evidence of MG.

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Fig. 7. C o m p a r i s o n of E L I S A optical densities for different d~sease groups. T h y m o m a (without MG) n = 6, PM n = 12, R A n = 33, SLE n = 18, liver disease (active chronic hepatius, acute viral hepatitis, alcoholic hver disease) n = 23. Positive for a u t o a n t i b o d i e s b~ i m m u n o f l u o r e s c e n c e : anti-muscle a n t i b o d i e s (AStrA, ASM, anti-heart) (A), a n t i n u c l e a r a n t i b o d i e s (D), both a n t i - m u s c l e and a n t i n u c l e a r a n t i b o d i e s ( a ). Negative for a u t o a n t i b o d i e s by i m m u n o f l u o r e s c e n c e (O). Each point represents one case.

311 The results for sera from patients with MG, organised according to racial groups, are also shown in Fig. 5. Fifty percent of the Caucasoid patients with MG had OD levels above 0.5: this differed significantly (P<< 0.001) from the frequency in healthy controls. Previously reported racial variations in the frequency of AStrA by fluorescence (Dawkins, 1980) were confirmed by ELISA. In particular, American Negroids had significantly lower OD levels than Caucasoid patients ( P < 0.005).

Comparison of ELISA and immunofluorescence Sera from 147 patients with MG were compared by the 2 assays (Fig. 6a. b). The 47 patients with immunofluorescence titres >/ I in 5 (Fig. 6a) had higher OD values than those with negative immunofluorescence ( P << 0.001) but even the negative patients had a higher mean OD level than the healthy controls ( P << 0.001) (see Fig. 5). Thus the ELISA can quantitate low titre anti-muscle antibodies which are undetectable by immunofluorescence. However, certain sera clearly positive by immunofluorescence had borderline OD values by ELISA. ELISA and irnmunofluorescence--correlation with thvmic pathology At the OD cut-off of 0.5 the ELISA detected antibodies in 30 (97%) of the patients said to have a thymoma (Fig. 6a). By contrast. 26 patients (84%) with thymoma had positive immunofluorescence. The ELISA also detected anti-muscle antibodies in 15 patients (25%) with thymic h~perplasia and negative results by immunofluorescence. A possible cause of error in attempts at such correlations is variation in the classification of thymic pathology b) the contributing laboratories. Consequently, the th2ymus sections on which the pathology reports had been made were obtained for 36 patients and reviewed by at least 2 readers lFig. 6b). One case was included as uncertain because of discrepancy in the diagnoses made by the readers. Clearly there is still considerable scatter of OD values in the various groups. However. all of the sera with immunofluorescence titres >/ 1 in 25 had OD values greater than 0.5. Moreover, all of the 13 patients with definite thymoma had OD values above this cut-off, while 7 (32%) of the patients with normal or hyperplastic thymus also had high OD. Frequency of anti-4M antibodies in other disease groups Other disease groups screened for anti-4M included systemic lupus erythematosus, rheumatoid arthritis, polymyositis, and autoimmune liver disease. The OD values for these groups and 6 patients with thymoma but without MG are shown in Fig. 7. The high frequency of OD values above 0.5 in the rheumatoid arthritis group (45%) and the thymoma patients (33%) contrasts with frequencies of 6% and 0% in patients with SLE and PM respectively. The high OD frequency in rheumatoid arthritis patients is consistent with the high frequency of AStrA detected by imrnunofluorescence in this group. A proportion of sera with anti-smooth muscle antibodies cross-react with the 4M antigen. This is evident in the sera of patients in the liver disease group.

312 Discussion

Recent inter-laborator} quality control programmes ha~e revealed poor reproducibility of indirect immunofluorescence assays for anti-muscle antibodies (unpublished observations). The ELISA reported here was developed to provide a more readily controlled and cost-effective alternative screen for these antibodies. A standardised assay is essential to realise the potential value of AStrA as a marker for thymoma in patients with MG. The data presented suggest that the ELISA meets these requirements. Between-run variation was minimised by using the reaction of standard sera to terminate the assay at predetermined OD values. Floating the microtitre plates on a 37°C water bath during incubations reduced thermal edge effects and further improved reproducibility. The ELISA confirmed the increased frequency of antibodies against contractile proteins in patients with MG. An increased antibody frequency was also observed for patients with R_,-X.Treatment with the drug D-penicillamine has been shown to induce immunofluorescent AStrA in patients with IL~ (Dawkins et al., 1975a: Camus et al., 1976; Zilko et al., 1977, 1980; Carrano et al.. 1982b). Use of ELISA to monitor autoantibody development in these patients is being investigated and will be reported separately. Antibodies against muscle antigens are commonl 3 detected in patients with liver disease (Johnson et al., 1965: Toh et al., 1978; McDonald et al., 1979; Toh, 1979). In active chronic hepatitis and acute viral hepatitis these antibodies are predominantly directed against smooth muscle antigens, however a proportion cross-react ~ith skeletal muscle contractile proteins (Fairfax and GroschelStewart, 1977: Toh et al., 1978). ELISA should be used in combination with immunofluorescence to distinguish these false positives. The ELISA readily detected low concentrations of anti-muscle antibodies, but nonetheless a proportion of sera with high titre immunofluorescent AStrA had weakly positive ELISA results. This apparent discrepanc} may reflect differences in the antigen substrates used. Disruption of conformational antigenic determinants during extraction and purification of the ELISA antigens may explain this difference. Such a mechanism has been proposed to explain the loss of antigenicity of contractile proteins and other globular proteins after purification (Rule et al.. 1973b: Reichlin, 1975). Alternatively, this disparity may reflect xenogeneic differences in the antigens used in the respective assays. Previous reports have shown minor species restrictions on the tissue specificity of AStrA (Beutner et al., 1965; N a m b a et al., 1967; Rule et al., 1973b). Comparison of the thymoma associated AStrA in the assays reported here may assist in defining the elusive antigenic specificity of these antibodies. Subgrouping of patients with MG into immunofluorescence positive and negative AStrA groups correlates well with the presence of thymoma (Dawkins, 1980). This segregation may, however, prove artificial since quantitation of anti-muscle antibodies by ELISA demonstrates a continuous distribution of antibody concentrations. It is uncertain whether this apparent distribution is composed of subpopulations of qualitatively different antibodies. The healthy control population also showed a continuous distribution of values but with one exception values did not exceed the

313 O D c u t - o f f o f 0.5. In c o n t r a s t , levels in all p a t i e n t s with M G a n d a h i s t o l o g i c a l l y c o n f i r m e d t h y m o m a e x c e e d e d this c u t - o f f , w h i l e a p r o p o r t i o n (27%) o f t h y m o m a n e g a t i v e p a t i e n t s also h a d e l e v a t e d c o n c e n t r a t i o n s (Fig. 6b). O w i n g to these limitat i o n s a high O D in the E L I S A s h o u l d not be used to c o n f i r m the d i a g n o s i s o f t h y m o m a , a l t h o u g h a low O D m a y be used to e x c l u d e this diagnosis. It s h o u l d be stressed, h o w e v e r , that little is k n o w n a b o u t the e f f e c t s o f t h y m e c t o m y a n d d r u g t h e r a p y o n the p e r s i s t e n c e of A S t r A in these p a t i e n t s . T h e E L I S A ~ill facilitate m o n i t o r i n g o f a n t i - m u s c l e a n t i b o d i e s in r e l a t i o n to t r e a t m e n t a n d d i s e a s e p r o g r e s sion.

Acknowledgements T h e a u t h o r s wish to t h a n k the f o l l o w i n g p e o p l e for advice, gifts o f r e a g e n t s a n d access to e q u i p m e n t : N, B a r t h o l o m a e u s , G. H a r n e t t , P. Kay. D. T o w n s e n d , H. W a t a n a b e a n d J, W e t h e r a l l . T h e y also t h a n k the c o n t r i b u t o r s of sera a n d h i s t o l o g i c a l slides. T h i s w o r k was s u p p o r t e d by g r a n t s f r o m the N a t i o n a l H e a l t h a n d M e d i c a l R e s e a r c h C o u n c i l of A u s t r a l i a a n d the M u s c u l a r D y s t o p h y A s s o c i a t i o n of A m e r i c a .

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