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An assay for antibodies to human acetylcholine receptor in serum from patients with myasthenia gravis
CLINICAL
IMMUNOLOGY
AND
IMMUNOPATHOLOGY
7, 36-43 (1977)
An Assay for Antibodies to Human Acetylcholine Receptor in Serum from Patients with Myasthenia Gravis
JON LINDSTROM The Salk Institute for Biological
Studies, San Diego. California 92112
Received May 17. 1976
Concentration of antibodies to acetylchohne receptor in serum from patients with myasthenia gravis was measured using receptor from human muscle labeled with [1Z51]a-bun.garotoxin as antigen. The antibodies detected bound to determinants on receptor other than the binding site for toxin (or. presumably, acetylcholine). Antibodies to receptor were found in 90% of patients with myasthenia gravis, but not in patients with other neuromuscular or autoimmune diseases. This result is consistent with the concept that myasthenia gravis is an autoimmune disease in which neuromuscular transmission is impaired by an immune response to acetylcholine receptor. Yield of receptor from human leg muscles averaged 1.1 x IO-l2 mol/g muscle. Receptor bound [1251]abungarotoxin with an apparent KD approximating 2 x lO-‘O M. Concentration of antireceptor antibodies averaged 4.4 x IO-* mol of receptor bound per liter of serum, but varied widely. Sensitivity, reproducibility, and ease of the assay suggest that it may be useful in diagnosis of myasthenia gravis and monitoring immunosuppressive therapy.
INTRODUCTION
Myasthenia gravis (MG) is an autoimmune disease characterized by muscle weakness due to impaired neuromuscular transmission. Motor nerve endings in the muscles of these patients are structurally unaltered (I), contain normal amounts of acetylcholine (2), and release acetylcholine normally in response to nerve impulses (3). However, the postsynaptic membrane of the muscle is less sensitive to acetylcholine (4), simplified in structure (I), reduced in area (l), and contains reduced numbers of acetylcholine receptor (AChR) molecules (5-7). many of which are bound with antibody (7). Blood from patients with MG contains both IgG (8 15) and lymphocytes (16- 17) which recognize AChR. Passive transfer of MG from patients to mice using antibody (18) demonstrates the importance of the humoral autoimmune response in MG. A method for measuring antibody to human AChR which is both sensitive and quantitative is described in this paper. Because of its ease and sensitivity, this method should prove valuable as a diagnostic test for MG and as a means for monitoring immunosuppressive therapy. The immunoprecipitation method described can be used to measure antibody to human AChR in the same units used to measure AChR and anti-AChR antibody in experimental autoimmune myasthenia gravis (EAMG) induced in mammals by immunization with purified fish AChR 36 Copyright All rights
@ 1977 by Academic Press. Inc of reproductmn in any form reserved
ANTIRECEPTOR
ANTIBODIES
(8, 10, 19-23), thereby permitting animals with the model disease.
IN
quantitative
MATERIALS
MYASTHENIA
comparisons
GRAVIS
31
between patients and
AND METHODS
Preparation ofAChR. Extracts containing human AChR were prepared from leg amputations. Fresh muscle was homogenized 1 min in a Waring Blendor at 4°C in 4 vol of O.lM NaCl, 0.01 M Na phosphate buffer pH 7.0, 0.01 M NaN,, then centrifuged 30 min at 105g. The resulting pellet was extracted by mild homogenization to resuspend, followed by stirring for 1 hr with 2 vol of the same buffer containing 2% Triton X-100. The supemate after centrifugation for 60 min at 105g contained solubilized AChR. AChR was diluted in “0.5% Triton buffer” composed of 0.5% Triton X-100,0.1 M NaCl, 0.01 M Na phosphate buffer pH 7.0,O.Ol M NaNa. Extracts fully retained the ability to specifically bind aBGT and antibody to AChR after being frozen for several months at -70°C. Preparation of [z251joBGT. a-Bungarotoxin (aBGT) was iodinated using chloramine T as previously described (8) to specific activities of 2-3.5 x 10” cpm/mol. Measurement of AChR concentration. Concentration of AChR was estimated by measuring [lz51](rBGT binding in two different ways. One method used chromatography on Sephadex G2OO to separate bound toxin from free. Aliquots of receptor (0.2 ml) were incubated 4 hr with 5 x 1O-g M [1251]aBGT or with [lz51]aBGT and 1O-3 M benzoquinonium (a gift from Sterling-Winthrop Pharmaceuticals) to competitively inhibit binding of [lz51]aBGT to the acetylcholine binding site of AChR. Aliquots were applied to a Sephadex G200 column (1.5 x 20 cm) equilibrated in 0.5% Triton buffer. [lz51]aBGT bound to macromolecules eluted in the void volume. Nonspecific binding occurring in the presence of 1O-3 M benzoquinonium was subtracted from the total. A faster and easier method for measuring AChR concentration was immunoprecipitation using serum from an MG patient. AChR was diluted to less than 1 x lo-lo M in l-ml aliquots of 0.5% Triton buffer plus or minus benzoquinonium 1O-3 M. Then, [1251](rBGT (2 x 1O-g M) was added at 4”C, followed in 4 hr by 5 ~1 of the patient’s serum (titer 5.4 x lo-* M estimated as described below). This gave greater than twofold excess of antibody over AChR. After incubation overnight at 4”C, goat anti-human gamma globulin serum (15 ~1 of a 15% N+SO, cut) was added to precipitate complexes of antibody-AChR-[1251]aBGT. After 4 hr at 4°C the precipitate was collected by centrifugation for 2 min in a Brinkman microfuge, washed with 1 ml of 0.5% Triton buffer, and counted in a gamma counter. [1251]aBGT bound in the presence of benzoquinonium was subtracted from the total before computing the concentration of AChR. Measurement of antibody concentration. Anti-AChR antibody concentration was measured by immunoprecipitation using [1251]cuBGT-labeled AChR as antigen. Antibody titer was expressed as moles of [1251]aBGT binding sites precipitated per liter of serum. A volume of 5 ~1 of serum in 1 ml of 2 X lo-lo M AChR, 3 x lo-lo M [lz51]aBGT gave sufficient excess of [U51](rBGT-AChR for many sera to ensure that the assay remained in the linear portion of the precipitin curve. Sera with titers 2 1O-8 M were assayed again using 0.5 ~1 of serum and 4.5 ~1 of normal
38
JON
L.INDS’I
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serum as carrier. Sera with titers a IO-’ M were assayed using 0.05 ~1 of MG serum and 5 ~1 of normal serum. Sera were assayed in triplicate in microfuge tubes. To correct for the small amount of lz51 nonspecifically trapped in the pellet (< 1% of the total), triplicate assays for each serum were also done in the presence of IO-‘I M benzoquinonium. Sera were incubated with [Iz51](yBGT-AChR overnight at 4°C. Sufficient goat serum was added to give complete precipitation in 4 hr of [lz51]aBGT-AChR-antibody complexes formed by 5 ~1 of a standard MG patient’s serum. Precipitates were pelleted by centrifugation (2 min in a microfuge), washed in I ml of 0.5% Triton buffer, and counted. Standard deviation of triphcate assays was less than IO% of the mean value. The value for 125I pelleted in the presence of benzoquinonium was subtracted from the assay value. Then the value for moles of [ 12sI]aBGT binding sites precipitated per liter of serum was multiplied by a factor to correct the results to conditions of complete labeling of AChR by [1251]crBGT. This factor was determined using standard sera assayed simultaneously as described in results. RESULTS
AChR was solubihzed from human skeletal muscle using the nonionic detergent Triton X-100. Concentration of AChR in the extracts averaged 1.0 ? 0.57 X lO-s M (12 preparations) [1251]aBGT binding sites protectable by benzoquinonium. Yield of AChR averaged 1 .I ? 0.46 x lo-‘* moI/g muscle. AChR labeled with [1251]~BGT sedimented on sucrose gradients as a single component of approximately 9.5s (8), approximating the size of AChR purified from Electrophorus (24). Binding of anti-AChR antibody to AChR did not significantly inhibit binding of [1251]~BGT. This was shown by determining the concentration of [1251]cuBGT binding sites in five preparations of AChR by each of two methods. Using chromatography on Sephadex G200 to measure [lz51@BGT binding gave an average value I .90 x IO-g M. A similar value (I .83 x 1O-9 M) was obtained by immunoprecipitation of [1251]aBGT-AChR-anti-AChR antibody complexes formed in the presence of excess anti-AChR antibodies. Antibody bound to complexes of AChR [‘251]cuBGT was detected by precipitating the complexes with goat antisera to human gamma globulin and measuring lz51 in the precipitate. Figure I shows that, when [ ‘251]toxin-AChR was in sufficient excess over antibody added, precipitation was linear with increasing amounts of serum. Titers of sera were expressed as moles of [1251]~BGT binding sites precipitated per liter of serum. In order to conserve human AChR and minimize nonspecific binding so that very low concentrations of antibody could be detected. AChR was used at 2 x IO-lo M in the assay and [lz51]aBGT was used at 3 x IO-lo M. Because this low concentration of toxin is near the KL, for toxin binding to AChR labeled with antibody (Fig. 2), not all AChR sites were labeled with toxin. In order to determine the absolute amount of AChR precipitated, it was necessary to extrapolate to conditions of complete labeling. Thus, all values were multiplied by a correction factor equal to the maximum concentration of toxin binding sites in the preparation divided by the concentration observed using 3 x IO-lo M toxin. Another way to obtain this correction factor was to assay each preparation of AChR with standard sera from MG patients and determine the ratio between the apparent titer
ANTIRECEPTOR
ANTIBODIES
pl
IN
MYASTHENIA
GRAVIS
39
MG SERUM
I. Precipitation of [1251]aBGT-AChR by serum from an MG patient. AChR (3 x 10-r” M) labeled with [rZSI]aBGT (1 x 1tP M) was used an antigen in duplicate l-ml aliquots in microfuge tubes. At each point sufftcient normal serum was added to bring the total serum in each assay tube to 5 ~1. After overnight incubation at 4°C goat anti-human gamma globulin was added to precipitate gamma globulin including that bound to [1251]aBGT-AChR. A background value of 496 cpm, determined using 5 ~1 of normal serum, was subtracted from all values. FIG.
of these sera and the titer obtained with a preparation already corrected to complete AChR labeling. Both methods yielded approximately the same correction factors which averaged 2.6 + 0.71. Thus, the small differences between preparations of human AChR appeared to result mostly from variation in affinity for cvBGT rather than variations in antigenicity. Titers of antibodies to human AChR in sera from patients with MG and other diseases are shown in Table 1 and in Fig. 3. Anti-AChR antibody was detected only in patients with MG or neonatal MG and not in normals or patients with other neurological or autoimmune diseases. Persons without MG exhibited a narrow range of titers closely approximating zero at the limits of resolution of the assay. Only one serum in this group (0.4% of the sample) had a titer greater than 0.5 x 1O-9 M. Patients with MG exhibited a wide range of titers of which 91% were greater than 0.5 x 1O-s M. Senstivity of the assay was indicated by the observation that the average value for patients with MG (4.4 x 1O-8 M) exceeded that for the non-MG group (1.6 x lo-lo IV) by 280-fold. DISCUSSION
The use of solubilized AChR labeled with [1251]~BGT as antigen permits quantitation of antibody in the same units by which AChR is measured. The average serum concentration of anti-AChR antibodies in patients with MG (4.4 x lo-* M) is such that the number of antibodies in the serum would be larger than the number of AChR present in normal muscle,’ and in many patients with high antibody titers
’ If one assumes a blood volume of 5 liters containing 50% serum, 1.1 x lo-’ mol of anti-AChR would be present in serum. If a 1.50pound individual were 50% muscle, then at 1.1 x lo-‘” mol of AChRIg, 3.7 X
40
JON
13
-:i;
LINDSTROM
h
0
_I
2
.3 BOUND T FREE
.4
.5
.6
.7
FIG. 2 Binding of aBGT to AChR from human muscle. Duplicate microfuge tubes containing 0. l-ml aliquots of AChR extract were made up to 1 ml with 0.5% Triton buffer and [‘Y]aBGT at concentrations between 1 and 20 x l&r0 M. Replicate tubes were prepared containing, in addition, 1CP M benzoquinonium. After 4 hr at 4°C 10 ~1 of a standard MG serum (anti-AChR antibody titer of 5.1 x W8M) was added to each tube. After overnight at 4°C goat anti-human gamma globulin (40~1 of 15% NaxSO, cut) was added. After 4 hr at 4°C the precipitates were pelleted. washed once and radioactivity determined in a gamma counter. Values in the presence of benzoquinonium were subtracted from those in its absence. The slope of this Scatchard plot indicates a KDfor toxin binding of 2.05 x lo-lo M while the intercept indicates a concentration of AChR in the assay of 1.39 x IO-“’ M.
and. reduced amounts of AChR the excess must be very large. These anti-AChR antibodies appear to be immunoglobulin G on the basis of their sedimentation on sucrose gradients (8) and their precipitation by anti-immunoglobulin G antibodies. Only antibodies directed at determinants other than the acetylcholine binding site are detected by the assay method described, since this site is protected by the [lWI]> bound to the AChR used as antigen. Binding of antibodies to [lz51]crBGT-AChR complexes did not displace [1251](rBGT bound to the AChRs. This indicates that these antibodies are not competitive antagonists of the acetycholine binding site. However, when AChR is in the membrane antibody binding might exert an allosteric effect on the acetylcholine binding site. Bender et al. (15) have reported that some sera from patients with MG prevented binding of aBGT to human muscle endplates as measured by the subsequent binding of antibody to CXBGT and peroxidase labeled anti-antibody. Because this method is quite indirect, the possibility remains that ACh, and perhaps even (uBGT, could continue to bind to AChR after antibody was bound, but that anti-aBGT, and
10% mot of AChR would be present in muscle. A nerve-muscle junction in a normal human contains about 6.3 x 10-l’ mol of AChR, whereas nerve-muscle junctions in MG patients average only about 1.2 x 10-l’ mol(5). Thus, a patient with an average antibody titer and an average reduction in amount of AChR would contain sufficient antibody in his seurm to bind 16 times the amount of AChR present in his muscles.
ANTIRECEF’TOR
ANTIBODIES
IN
MYASTHENIA
41
GRAVIS
FIG. 3. Distribution of anti-AChR antibody titers. Data from Table 1 is plotted to show the incidence of various concentrations of anti-AChR antibody in the sera tested. (A) Sera from patients diagnosed as having MC. There is a broad range of titers, 91% greater than 0.5 x lO-9 M with the median in the lo-20 x 10-gM group. (B) Sera from normal individuals and patients with neuromuscular and autoimmune diseases other than MG. There is a narrow range of titers approximating 0 within the limits of the assay. Only one serum (0.4%) is greater than 0.5 x 1O-8 M with the median in the O-O.2 x lO-9 M titer group.
anti-anti-cYBGT could not. In support of this possibility, Mittag (13) found that serum from patients with MG did not inhibit binding of [1251]aBGT to AChR in plasma membranes from denervated muscle. Similarly, we (25) found that AChR ANTIBODIES
TO HUMAN
Number of patients
Condition Myasthenia gravis Neonatal myasthenia gravis Other neurological diseases (Eaton-Lambert syndrome (Amyotrophic lateral sclerosis (Various muscular dystrophies (Multiple sclerosis (Others Autoimmune and endocrine diseases (Polymyositis (Sjogren’s Syndrome (Systemic lupus erythematosus (Others Thymoma, no myasthenia Normal
TABLE 1 AChR IN SERA FROM PATIENTS Antibody titer (mol [rz51]~BGT binding sites bound per liter of serum x 10-9) Average
Range
142 4 106
44 106 0.10
(o-840) (6.9-376) (o-0.42)
105
0.18
(o-0.56)
2 42
0.083 0.26
(O-O. 17) (O-0.41)
19)
20) 27) 6) 34)
11) 21)
11)
62)
42
JON
LINDSI‘KOM
in electric organ cells labeled with antibodies from a goat with EAMG could still bind [1251]aBGT. The incidence of antibody detected in patients with MG usmg human AChR as antigen according to the method described in this paper is higher than others have reported using heterologous AChR (from rat and Torprdo) an antigen ( 12.14). This is due, in part, to the species specificity of antibodies to receptor. Sera from MG patients cross-react less than 15% with AChR from rat and undetectably (under these conditions) with AChR from Electwphorrrs (8). There is some antigenic similarity between AChR from human muscl’e and Elcctrophorrrs demonstrable by a 1.0% cross-reaction between goat antisera to ElwtrophorrtLs AChR and AChR from human muscle (Lindstrom and Bridgman. unpublished). Goat antisera to AChR from Elwtrophorus cross-react only 3% with AChR from Torptdo c.dijiwnica. All of this cross-reacting antibody is directed at determinants other than the ACh binding site (10). Rat antisera to AChR from E1rctrophorrr.t cross-react oni!, l-3% with AChR from rat (8,21). The identity of the determinants in common between AChR of various species is unknown. Detection of antibodies to AChR in patients with MC; IS significant in several respects. The presence of antibodies to AChR unique to patients with MG indicates the autoimmune nature of this disease. The presence of antibodies to AChR in the serum of neonatal myasthenics suggests that this transient form of myasthenia occurring in the newborn of some myasthenic mothers results from maternal antibodies and resolves as the maternal antibodies are removed from the infant. The concentration of antibody varies over a wide range in patients with MG. Analysis of the clinical features of patients included in this study will be published elsewhere (9). One conclusion from this analysis is that antibody concentration does not correlate closely with disease intensity in the population studied. Studies of changes in serum antibody titer in individual patients during changes in intensity of MG have not been reported in detail, although we have encountered a number of cases where exacerbations are accompanied by increased titers and relative remissions by decreased titers (Lindstrom, Seybold. Lambert, Dawkins. Keesey. and others. unpublished). The concentration of anti-AChR antibodies accumulated at the endplate and the alterations in AChR function and metabolism that result may not be directly proportional to the con-centration of antibodies to AChR in the serum. Failure to detect anti-AChR antibodies in some of the 10% of the patients diagnosed to have MG probably results from the inability to distinguish very low concentrations of antibodies, since many patients have titers in their serum not greatly above the background level. The assay method for antibodies to AChR described provides an objective diagnostic test for MG which can be used along with electromyography and subjective evaluation of muscle weakness and fatigability. Because the method is both sensitive and quantitative it should also provide a means to monitor immunosuppressive therapy. ACKNOWLEDGMENTS I thank Brett Einarson and Mac Campbell for technical assistance and DI-\ Vanda Lxnnon Marjorie Seybold for valuable discussions. Sera were kindly provided hy Dr<. S. Whittingham. Seybold. D. Daune, E. Lambert. P. Dyck. J. Peter. E. Tan. M. Auspaugh. D. Dalessio. 1.. Blakey.
and M. M,
ANTIRECEPTOR
ANTIBODIES
IN
MYASTHENIA
GRAVIS
43
Cherrington, W. DeBolt, P. Ebeling, L. Myers, and J. Rosenberg. Human muscle was obtained through the kind cooperation of the Departments of Pathology at these San Diego hospitals: Alvarado Community, Mercy, Scripps Memorial, Sharp Memorial, University, and Veterans Administration, This work was supported by grants from N.I.H. (NS 11323) and the Muscular Dystrophy Association.
REFERENCES I. Engel. A. G., and Santa, T., Ann. N. Y. Acad. Sci. 183, 46, 1971. 2. Ito, Y., Miledi. R., Molenaar, P. C., Vincent, A., Polak. R. L.. van Gilder. M.. and Davis. Proc.
3. 4. 5. 6. 7. 8. 9.
Roy.
Sot.
Ser. B 192, 475, 1976. Elmquist, D., Ann. N. Y. Acad.
J. N..
London
Lambert. E. H., and Sci. 183, 183, 1971. Albuquerque, E. X., Rash, J. E., Mayer, R. F., and Satterfield, J. R., Exp. Neurol. 51,536, 1976. Fambrough. D.. Drachman, D.. and Satyamurti, S., Science 182, 293, 1973. Engel. A. G.. Lindstrom, J. M., Lambert, E. H., and Lennon. V. A., Neurology 26, 371. 1976. Lindstrom, J.. and Lambert, E., in preparation. Lindstrom, J., Lennon, V., Seybold, M., and Whittingham, S., N. Y. Acad. Sci. 274, 254, 1976. Lindstrom, J. M.. Seybold, M. E.. Lennon, V.. Whittingham. S.. and Duane. D., Nrurology, in press. 10. Lindstrom, J., J. Supramolecular Structure 4, 389, 1976. 1 I. Almon, R., Andrew, C., and Appel, S., Science 186, 55. 1974. 12. Appel. S. H.. Almon. R. R.. and Levy. N.. N. Enyl. J. Med. 293, 760, 1975. 13. Mittag, T., Kornfeld, P., Tormay, A., and Woo, C.. Nar. J. Med. 294, 691. 1976. 14. Aharonov, A., Abramsky. O., Tarrab-Hazdai, R.. and Fuchs, S.. Lancef 1, 340, 1975. 1.5. Bender, A., Ringle. S.. Engel. W., Daniels. M., and Vogel, Z., Lancet 1, 607, 1975. 16. Abramsky. 0.. Aharonov. A., Webb, C., and Fuchs. S., Immune/. 19, 11. 1975. 17. Richman. D. P., Patrick, J., and Amason, B. G. W.. N. Engl. J. &fed. 294, 694, 1976. 18. Toyka. K.. Drachman. D. B., Pestronk. A.. and Kao, I., Science 190, 397, 1975. 19. Patrick. J., and Lindstrom, J.. Science 180, 871, 1973. 20. Patrick, J.. Lindstrom. J.. Gulp, B., and McMillan, J.. Proc. Nat. Acad. Sri. USA 70, 3334, 1973. 21. Lennon. V. A., Lindstrom, J. M., and Seybold, M. E.. J. Exp. Med. 141, 1365, 1975. 22. Lindstrom. J., Einarson, B., Lennon, V., and Seybold, M., J. Exp. Med. 144, 1976, in press. 23. Lindstrom, J., Engel, A., Seybold, M., Lennon, V., and Lambert, E., J. Exp. Med. 144, 1976, in press. 24. Lindstrom. J., and Patrick, J., In “Synaptic Transmission and Neuronal Interaction” (M. V. L. Bennet. Ed.), pp. 191-216, Raven Press, New York, 1974. 25. Lindstrom. J., Einarson, B., and Francy, M., J. Supramolecular Structure, in press.
IMMUNOLOGY
AND
IMMUNOPATHOLOGY
7, 36-43 (1977)
An Assay for Antibodies to Human Acetylcholine Receptor in Serum from Patients with Myasthenia Gravis
JON LINDSTROM The Salk Institute for Biological
Studies, San Diego. California 92112
Received May 17. 1976
Concentration of antibodies to acetylchohne receptor in serum from patients with myasthenia gravis was measured using receptor from human muscle labeled with [1Z51]a-bun.garotoxin as antigen. The antibodies detected bound to determinants on receptor other than the binding site for toxin (or. presumably, acetylcholine). Antibodies to receptor were found in 90% of patients with myasthenia gravis, but not in patients with other neuromuscular or autoimmune diseases. This result is consistent with the concept that myasthenia gravis is an autoimmune disease in which neuromuscular transmission is impaired by an immune response to acetylcholine receptor. Yield of receptor from human leg muscles averaged 1.1 x IO-l2 mol/g muscle. Receptor bound [1251]abungarotoxin with an apparent KD approximating 2 x lO-‘O M. Concentration of antireceptor antibodies averaged 4.4 x IO-* mol of receptor bound per liter of serum, but varied widely. Sensitivity, reproducibility, and ease of the assay suggest that it may be useful in diagnosis of myasthenia gravis and monitoring immunosuppressive therapy.
INTRODUCTION
Myasthenia gravis (MG) is an autoimmune disease characterized by muscle weakness due to impaired neuromuscular transmission. Motor nerve endings in the muscles of these patients are structurally unaltered (I), contain normal amounts of acetylcholine (2), and release acetylcholine normally in response to nerve impulses (3). However, the postsynaptic membrane of the muscle is less sensitive to acetylcholine (4), simplified in structure (I), reduced in area (l), and contains reduced numbers of acetylcholine receptor (AChR) molecules (5-7). many of which are bound with antibody (7). Blood from patients with MG contains both IgG (8 15) and lymphocytes (16- 17) which recognize AChR. Passive transfer of MG from patients to mice using antibody (18) demonstrates the importance of the humoral autoimmune response in MG. A method for measuring antibody to human AChR which is both sensitive and quantitative is described in this paper. Because of its ease and sensitivity, this method should prove valuable as a diagnostic test for MG and as a means for monitoring immunosuppressive therapy. The immunoprecipitation method described can be used to measure antibody to human AChR in the same units used to measure AChR and anti-AChR antibody in experimental autoimmune myasthenia gravis (EAMG) induced in mammals by immunization with purified fish AChR 36 Copyright All rights
@ 1977 by Academic Press. Inc of reproductmn in any form reserved
ANTIRECEPTOR
ANTIBODIES
(8, 10, 19-23), thereby permitting animals with the model disease.
IN
quantitative
MATERIALS
MYASTHENIA
comparisons
GRAVIS
31
between patients and
AND METHODS
Preparation ofAChR. Extracts containing human AChR were prepared from leg amputations. Fresh muscle was homogenized 1 min in a Waring Blendor at 4°C in 4 vol of O.lM NaCl, 0.01 M Na phosphate buffer pH 7.0, 0.01 M NaN,, then centrifuged 30 min at 105g. The resulting pellet was extracted by mild homogenization to resuspend, followed by stirring for 1 hr with 2 vol of the same buffer containing 2% Triton X-100. The supemate after centrifugation for 60 min at 105g contained solubilized AChR. AChR was diluted in “0.5% Triton buffer” composed of 0.5% Triton X-100,0.1 M NaCl, 0.01 M Na phosphate buffer pH 7.0,O.Ol M NaNa. Extracts fully retained the ability to specifically bind aBGT and antibody to AChR after being frozen for several months at -70°C. Preparation of [z251joBGT. a-Bungarotoxin (aBGT) was iodinated using chloramine T as previously described (8) to specific activities of 2-3.5 x 10” cpm/mol. Measurement of AChR concentration. Concentration of AChR was estimated by measuring [lz51](rBGT binding in two different ways. One method used chromatography on Sephadex G2OO to separate bound toxin from free. Aliquots of receptor (0.2 ml) were incubated 4 hr with 5 x 1O-g M [1251]aBGT or with [lz51]aBGT and 1O-3 M benzoquinonium (a gift from Sterling-Winthrop Pharmaceuticals) to competitively inhibit binding of [lz51]aBGT to the acetylcholine binding site of AChR. Aliquots were applied to a Sephadex G200 column (1.5 x 20 cm) equilibrated in 0.5% Triton buffer. [lz51]aBGT bound to macromolecules eluted in the void volume. Nonspecific binding occurring in the presence of 1O-3 M benzoquinonium was subtracted from the total. A faster and easier method for measuring AChR concentration was immunoprecipitation using serum from an MG patient. AChR was diluted to less than 1 x lo-lo M in l-ml aliquots of 0.5% Triton buffer plus or minus benzoquinonium 1O-3 M. Then, [1251](rBGT (2 x 1O-g M) was added at 4”C, followed in 4 hr by 5 ~1 of the patient’s serum (titer 5.4 x lo-* M estimated as described below). This gave greater than twofold excess of antibody over AChR. After incubation overnight at 4”C, goat anti-human gamma globulin serum (15 ~1 of a 15% N+SO, cut) was added to precipitate complexes of antibody-AChR-[1251]aBGT. After 4 hr at 4°C the precipitate was collected by centrifugation for 2 min in a Brinkman microfuge, washed with 1 ml of 0.5% Triton buffer, and counted in a gamma counter. [1251]aBGT bound in the presence of benzoquinonium was subtracted from the total before computing the concentration of AChR. Measurement of antibody concentration. Anti-AChR antibody concentration was measured by immunoprecipitation using [1251]cuBGT-labeled AChR as antigen. Antibody titer was expressed as moles of [1251]aBGT binding sites precipitated per liter of serum. A volume of 5 ~1 of serum in 1 ml of 2 X lo-lo M AChR, 3 x lo-lo M [lz51]aBGT gave sufficient excess of [U51](rBGT-AChR for many sera to ensure that the assay remained in the linear portion of the precipitin curve. Sera with titers 2 1O-8 M were assayed again using 0.5 ~1 of serum and 4.5 ~1 of normal
38
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lwhl
serum as carrier. Sera with titers a IO-’ M were assayed using 0.05 ~1 of MG serum and 5 ~1 of normal serum. Sera were assayed in triplicate in microfuge tubes. To correct for the small amount of lz51 nonspecifically trapped in the pellet (< 1% of the total), triplicate assays for each serum were also done in the presence of IO-‘I M benzoquinonium. Sera were incubated with [Iz51](yBGT-AChR overnight at 4°C. Sufficient goat serum was added to give complete precipitation in 4 hr of [lz51]aBGT-AChR-antibody complexes formed by 5 ~1 of a standard MG patient’s serum. Precipitates were pelleted by centrifugation (2 min in a microfuge), washed in I ml of 0.5% Triton buffer, and counted. Standard deviation of triphcate assays was less than IO% of the mean value. The value for 125I pelleted in the presence of benzoquinonium was subtracted from the assay value. Then the value for moles of [ 12sI]aBGT binding sites precipitated per liter of serum was multiplied by a factor to correct the results to conditions of complete labeling of AChR by [1251]crBGT. This factor was determined using standard sera assayed simultaneously as described in results. RESULTS
AChR was solubihzed from human skeletal muscle using the nonionic detergent Triton X-100. Concentration of AChR in the extracts averaged 1.0 ? 0.57 X lO-s M (12 preparations) [1251]aBGT binding sites protectable by benzoquinonium. Yield of AChR averaged 1 .I ? 0.46 x lo-‘* moI/g muscle. AChR labeled with [1251]~BGT sedimented on sucrose gradients as a single component of approximately 9.5s (8), approximating the size of AChR purified from Electrophorus (24). Binding of anti-AChR antibody to AChR did not significantly inhibit binding of [1251]~BGT. This was shown by determining the concentration of [1251]cuBGT binding sites in five preparations of AChR by each of two methods. Using chromatography on Sephadex G200 to measure [lz51@BGT binding gave an average value I .90 x IO-g M. A similar value (I .83 x 1O-9 M) was obtained by immunoprecipitation of [1251]aBGT-AChR-anti-AChR antibody complexes formed in the presence of excess anti-AChR antibodies. Antibody bound to complexes of AChR [‘251]cuBGT was detected by precipitating the complexes with goat antisera to human gamma globulin and measuring lz51 in the precipitate. Figure I shows that, when [ ‘251]toxin-AChR was in sufficient excess over antibody added, precipitation was linear with increasing amounts of serum. Titers of sera were expressed as moles of [1251]~BGT binding sites precipitated per liter of serum. In order to conserve human AChR and minimize nonspecific binding so that very low concentrations of antibody could be detected. AChR was used at 2 x IO-lo M in the assay and [lz51]aBGT was used at 3 x IO-lo M. Because this low concentration of toxin is near the KL, for toxin binding to AChR labeled with antibody (Fig. 2), not all AChR sites were labeled with toxin. In order to determine the absolute amount of AChR precipitated, it was necessary to extrapolate to conditions of complete labeling. Thus, all values were multiplied by a correction factor equal to the maximum concentration of toxin binding sites in the preparation divided by the concentration observed using 3 x IO-lo M toxin. Another way to obtain this correction factor was to assay each preparation of AChR with standard sera from MG patients and determine the ratio between the apparent titer
ANTIRECEPTOR
ANTIBODIES
pl
IN
MYASTHENIA
GRAVIS
39
MG SERUM
I. Precipitation of [1251]aBGT-AChR by serum from an MG patient. AChR (3 x 10-r” M) labeled with [rZSI]aBGT (1 x 1tP M) was used an antigen in duplicate l-ml aliquots in microfuge tubes. At each point sufftcient normal serum was added to bring the total serum in each assay tube to 5 ~1. After overnight incubation at 4°C goat anti-human gamma globulin was added to precipitate gamma globulin including that bound to [1251]aBGT-AChR. A background value of 496 cpm, determined using 5 ~1 of normal serum, was subtracted from all values. FIG.
of these sera and the titer obtained with a preparation already corrected to complete AChR labeling. Both methods yielded approximately the same correction factors which averaged 2.6 + 0.71. Thus, the small differences between preparations of human AChR appeared to result mostly from variation in affinity for cvBGT rather than variations in antigenicity. Titers of antibodies to human AChR in sera from patients with MG and other diseases are shown in Table 1 and in Fig. 3. Anti-AChR antibody was detected only in patients with MG or neonatal MG and not in normals or patients with other neurological or autoimmune diseases. Persons without MG exhibited a narrow range of titers closely approximating zero at the limits of resolution of the assay. Only one serum in this group (0.4% of the sample) had a titer greater than 0.5 x 1O-9 M. Patients with MG exhibited a wide range of titers of which 91% were greater than 0.5 x 1O-s M. Senstivity of the assay was indicated by the observation that the average value for patients with MG (4.4 x 1O-8 M) exceeded that for the non-MG group (1.6 x lo-lo IV) by 280-fold. DISCUSSION
The use of solubilized AChR labeled with [1251]~BGT as antigen permits quantitation of antibody in the same units by which AChR is measured. The average serum concentration of anti-AChR antibodies in patients with MG (4.4 x lo-* M) is such that the number of antibodies in the serum would be larger than the number of AChR present in normal muscle,’ and in many patients with high antibody titers
’ If one assumes a blood volume of 5 liters containing 50% serum, 1.1 x lo-’ mol of anti-AChR would be present in serum. If a 1.50pound individual were 50% muscle, then at 1.1 x lo-‘” mol of AChRIg, 3.7 X
40
JON
13
-:i;
LINDSTROM
h
0
_I
2
.3 BOUND T FREE
.4
.5
.6
.7
FIG. 2 Binding of aBGT to AChR from human muscle. Duplicate microfuge tubes containing 0. l-ml aliquots of AChR extract were made up to 1 ml with 0.5% Triton buffer and [‘Y]aBGT at concentrations between 1 and 20 x l&r0 M. Replicate tubes were prepared containing, in addition, 1CP M benzoquinonium. After 4 hr at 4°C 10 ~1 of a standard MG serum (anti-AChR antibody titer of 5.1 x W8M) was added to each tube. After overnight at 4°C goat anti-human gamma globulin (40~1 of 15% NaxSO, cut) was added. After 4 hr at 4°C the precipitates were pelleted. washed once and radioactivity determined in a gamma counter. Values in the presence of benzoquinonium were subtracted from those in its absence. The slope of this Scatchard plot indicates a KDfor toxin binding of 2.05 x lo-lo M while the intercept indicates a concentration of AChR in the assay of 1.39 x IO-“’ M.
and. reduced amounts of AChR the excess must be very large. These anti-AChR antibodies appear to be immunoglobulin G on the basis of their sedimentation on sucrose gradients (8) and their precipitation by anti-immunoglobulin G antibodies. Only antibodies directed at determinants other than the acetylcholine binding site are detected by the assay method described, since this site is protected by the [lWI]> bound to the AChR used as antigen. Binding of antibodies to [lz51]crBGT-AChR complexes did not displace [1251](rBGT bound to the AChRs. This indicates that these antibodies are not competitive antagonists of the acetycholine binding site. However, when AChR is in the membrane antibody binding might exert an allosteric effect on the acetylcholine binding site. Bender et al. (15) have reported that some sera from patients with MG prevented binding of aBGT to human muscle endplates as measured by the subsequent binding of antibody to CXBGT and peroxidase labeled anti-antibody. Because this method is quite indirect, the possibility remains that ACh, and perhaps even (uBGT, could continue to bind to AChR after antibody was bound, but that anti-aBGT, and
10% mot of AChR would be present in muscle. A nerve-muscle junction in a normal human contains about 6.3 x 10-l’ mol of AChR, whereas nerve-muscle junctions in MG patients average only about 1.2 x 10-l’ mol(5). Thus, a patient with an average antibody titer and an average reduction in amount of AChR would contain sufficient antibody in his seurm to bind 16 times the amount of AChR present in his muscles.
ANTIRECEF’TOR
ANTIBODIES
IN
MYASTHENIA
41
GRAVIS
FIG. 3. Distribution of anti-AChR antibody titers. Data from Table 1 is plotted to show the incidence of various concentrations of anti-AChR antibody in the sera tested. (A) Sera from patients diagnosed as having MC. There is a broad range of titers, 91% greater than 0.5 x lO-9 M with the median in the lo-20 x 10-gM group. (B) Sera from normal individuals and patients with neuromuscular and autoimmune diseases other than MG. There is a narrow range of titers approximating 0 within the limits of the assay. Only one serum (0.4%) is greater than 0.5 x 1O-8 M with the median in the O-O.2 x lO-9 M titer group.
anti-anti-cYBGT could not. In support of this possibility, Mittag (13) found that serum from patients with MG did not inhibit binding of [1251]aBGT to AChR in plasma membranes from denervated muscle. Similarly, we (25) found that AChR ANTIBODIES
TO HUMAN
Number of patients
Condition Myasthenia gravis Neonatal myasthenia gravis Other neurological diseases (Eaton-Lambert syndrome (Amyotrophic lateral sclerosis (Various muscular dystrophies (Multiple sclerosis (Others Autoimmune and endocrine diseases (Polymyositis (Sjogren’s Syndrome (Systemic lupus erythematosus (Others Thymoma, no myasthenia Normal
TABLE 1 AChR IN SERA FROM PATIENTS Antibody titer (mol [rz51]~BGT binding sites bound per liter of serum x 10-9) Average
Range
142 4 106
44 106 0.10
(o-840) (6.9-376) (o-0.42)
105
0.18
(o-0.56)
2 42
0.083 0.26
(O-O. 17) (O-0.41)
19)
20) 27) 6) 34)
11) 21)
11)
62)
42
JON
LINDSI‘KOM
in electric organ cells labeled with antibodies from a goat with EAMG could still bind [1251]aBGT. The incidence of antibody detected in patients with MG usmg human AChR as antigen according to the method described in this paper is higher than others have reported using heterologous AChR (from rat and Torprdo) an antigen ( 12.14). This is due, in part, to the species specificity of antibodies to receptor. Sera from MG patients cross-react less than 15% with AChR from rat and undetectably (under these conditions) with AChR from Electwphorrrs (8). There is some antigenic similarity between AChR from human muscl’e and Elcctrophorrrs demonstrable by a 1.0% cross-reaction between goat antisera to ElwtrophorrtLs AChR and AChR from human muscle (Lindstrom and Bridgman. unpublished). Goat antisera to AChR from Elwtrophorus cross-react only 3% with AChR from Torptdo c.dijiwnica. All of this cross-reacting antibody is directed at determinants other than the ACh binding site (10). Rat antisera to AChR from E1rctrophorrr.t cross-react oni!, l-3% with AChR from rat (8,21). The identity of the determinants in common between AChR of various species is unknown. Detection of antibodies to AChR in patients with MC; IS significant in several respects. The presence of antibodies to AChR unique to patients with MG indicates the autoimmune nature of this disease. The presence of antibodies to AChR in the serum of neonatal myasthenics suggests that this transient form of myasthenia occurring in the newborn of some myasthenic mothers results from maternal antibodies and resolves as the maternal antibodies are removed from the infant. The concentration of antibody varies over a wide range in patients with MG. Analysis of the clinical features of patients included in this study will be published elsewhere (9). One conclusion from this analysis is that antibody concentration does not correlate closely with disease intensity in the population studied. Studies of changes in serum antibody titer in individual patients during changes in intensity of MG have not been reported in detail, although we have encountered a number of cases where exacerbations are accompanied by increased titers and relative remissions by decreased titers (Lindstrom, Seybold. Lambert, Dawkins. Keesey. and others. unpublished). The concentration of anti-AChR antibodies accumulated at the endplate and the alterations in AChR function and metabolism that result may not be directly proportional to the con-centration of antibodies to AChR in the serum. Failure to detect anti-AChR antibodies in some of the 10% of the patients diagnosed to have MG probably results from the inability to distinguish very low concentrations of antibodies, since many patients have titers in their serum not greatly above the background level. The assay method for antibodies to AChR described provides an objective diagnostic test for MG which can be used along with electromyography and subjective evaluation of muscle weakness and fatigability. Because the method is both sensitive and quantitative it should also provide a means to monitor immunosuppressive therapy. ACKNOWLEDGMENTS I thank Brett Einarson and Mac Campbell for technical assistance and DI-\ Vanda Lxnnon Marjorie Seybold for valuable discussions. Sera were kindly provided hy Dr<. S. Whittingham. Seybold. D. Daune, E. Lambert. P. Dyck. J. Peter. E. Tan. M. Auspaugh. D. Dalessio. 1.. Blakey.
and M. M,
ANTIRECEPTOR
ANTIBODIES
IN
MYASTHENIA
GRAVIS
43
Cherrington, W. DeBolt, P. Ebeling, L. Myers, and J. Rosenberg. Human muscle was obtained through the kind cooperation of the Departments of Pathology at these San Diego hospitals: Alvarado Community, Mercy, Scripps Memorial, Sharp Memorial, University, and Veterans Administration, This work was supported by grants from N.I.H. (NS 11323) and the Muscular Dystrophy Association.
REFERENCES I. Engel. A. G., and Santa, T., Ann. N. Y. Acad. Sci. 183, 46, 1971. 2. Ito, Y., Miledi. R., Molenaar, P. C., Vincent, A., Polak. R. L.. van Gilder. M.. and Davis. Proc.
3. 4. 5. 6. 7. 8. 9.
Roy.
Sot.
Ser. B 192, 475, 1976. Elmquist, D., Ann. N. Y. Acad.
J. N..
London
Lambert. E. H., and Sci. 183, 183, 1971. Albuquerque, E. X., Rash, J. E., Mayer, R. F., and Satterfield, J. R., Exp. Neurol. 51,536, 1976. Fambrough. D.. Drachman, D.. and Satyamurti, S., Science 182, 293, 1973. Engel. A. G.. Lindstrom, J. M., Lambert, E. H., and Lennon. V. A., Neurology 26, 371. 1976. Lindstrom, J.. and Lambert, E., in preparation. Lindstrom, J., Lennon, V., Seybold, M., and Whittingham, S., N. Y. Acad. Sci. 274, 254, 1976. Lindstrom, J. M.. Seybold, M. E.. Lennon, V.. Whittingham. S.. and Duane. D., Nrurology, in press. 10. Lindstrom, J., J. Supramolecular Structure 4, 389, 1976. 1 I. Almon, R., Andrew, C., and Appel, S., Science 186, 55. 1974. 12. Appel. S. H.. Almon. R. R.. and Levy. N.. N. Enyl. J. Med. 293, 760, 1975. 13. Mittag, T., Kornfeld, P., Tormay, A., and Woo, C.. Nar. J. Med. 294, 691. 1976. 14. Aharonov, A., Abramsky. O., Tarrab-Hazdai, R.. and Fuchs, S.. Lancef 1, 340, 1975. 1.5. Bender, A., Ringle. S.. Engel. W., Daniels. M., and Vogel, Z., Lancet 1, 607, 1975. 16. Abramsky. 0.. Aharonov. A., Webb, C., and Fuchs. S., Immune/. 19, 11. 1975. 17. Richman. D. P., Patrick, J., and Amason, B. G. W.. N. Engl. J. &fed. 294, 694, 1976. 18. Toyka. K.. Drachman. D. B., Pestronk. A.. and Kao, I., Science 190, 397, 1975. 19. Patrick. J., and Lindstrom, J.. Science 180, 871, 1973. 20. Patrick, J.. Lindstrom. J.. Gulp, B., and McMillan, J.. Proc. Nat. Acad. Sri. USA 70, 3334, 1973. 21. Lennon. V. A., Lindstrom, J. M., and Seybold, M. E.. J. Exp. Med. 141, 1365, 1975. 22. Lindstrom. J., Einarson, B., Lennon, V., and Seybold, M., J. Exp. Med. 144, 1976, in press. 23. Lindstrom, J., Engel, A., Seybold, M., Lennon, V., and Lambert, E., J. Exp. Med. 144, 1976, in press. 24. Lindstrom. J., and Patrick, J., In “Synaptic Transmission and Neuronal Interaction” (M. V. L. Bennet. Ed.), pp. 191-216, Raven Press, New York, 1974. 25. Lindstrom. J., Einarson, B., and Francy, M., J. Supramolecular Structure, in press.