A solid-phase enzyme immunoassay for anti-acetylcholine receptor antibody in myasthenia gravis patients

A solid-phase enzyme immunoassay for the measurement of anti-acetylcholine receptor antibodies in the sera of patients with myasthenia gravis is reported. Sufficient amounts of acetylcholine receptor for the sensitive detection of anti-acetylcholine receptor antibody were directly fixed to Costar serocluster 96-well EIA plates coated with poly-L-lysine hydrobromide. The solid-phase enzyme immunoassay detected anti-acetylcholine receptor antibodies in 91% of the myasthenia gravis patients including 4 out of 4 ocular type myasthenia patients, anti-acetylcholine receptor antibodies of which were not detectable by the immunoprecipitation assay. Correlation between antibody titers measured by enzyme immunoassay and the immunoprecipitation assay was significant.     

Modulation of anti-acetylcholine receptor antibody specificities and of experimental autoimmune myasthenia gravis by synthetic peptides.

Synthetic peptides corresponding to selected sequences from the nicotinic acetylcholine receptor (AChR) were employed to identify possible antigenic determinants within the receptor which can modulate the anti-AChR response and experimental autoimmune myasthenia gravis (EAMG). Immunization of rabbits with peptides T alpha 73-89, T alpha 351-368, T delta 354-367 and H alpha 351-368, prior to AChR inoculation, affected the course of EAMG in six out of eight rabbits. These six protected rabbits survived three inoculations of AChR and survived for at least five months after the third injection with AChR, whereas control rabbits died following one or two injections of AChR. The survival of peptide-preimmunized rabbits injected with AChR seemed to correlate with the antibody specificities in immunoblots. Following AChR inoculation there was a shift in reactivity, from a subunit-restricted response, to reactivity with all subunits of the receptor. This shift was delayed in protected rabbits. This may indicate that the reactivity with the entire Torpedo receptor molecule represents a loss of tolerance to AChR which culminates in the autoimmune disease, EAMG.     

Cell types required for anti-acetylcholine receptor antibody synthesis by cultured thymocytes and blood lymphocytes in myasthenia gravis.

In most young myasthenia gravis patients, the thymic medulla contains germinal centres. Thymocytes from these cases spontaneously synthesize anti-acetylcholine receptor autoantibody (anti-AChR) in culture; after irradiation they may also selectively stimulate anti-AChR antibody production by autologous blood lymphocytes. By depleting cortical or mature thymic T cells by complement killing, we now show that neither of these responses depends on thymic T cells, unlike the total IgG response to pokeweed mitogen which is T cell-dependent and shows T/B cell synergy. The results suggest that much of the spontaneous anti-AChR production is by autonomous thymic plasma cells, which may be HLA-DR-. The ability to stimulate autologous blood lymphocytes does not require viable HLA-DR+ thymic cells but appears to depend on rare antigen presenting cells from the germinal centres. In preliminary experiments, blood T cells were apparently also necessary.     

T and B lymphocytes in myasthenia gravis.

Peripheral blood lymphocytes from seventeen non-thymectomized and nine thymectomized patients with myasthenia gravis (MG) and thirteen healthy controls were examined for the presence of surface markers characteristic of T and B lymphocytes by rosette formation with sheep red blood cells (SRBC). T cells were identified by their capacity to spontaneously form rosettes with SRBCs. The percentage of B lymphocytes was determined by the erythrocyte antibody complement (EAC) rosette-forming test. The EAC complex was prepared with either whole rabbit anti-SRBC serum or with the IgM fraction of rabbit anti-SRBC serum. The two kind of erythrocyte complement rosette-forming cells (EAC-RFC) are designated erythrocyte-haemolysin-complement RFC (EA(H)C-RFC), and erythrocyte-IgM-complement RFC (EA(M)C-RFC). The percentage of total lymphocytes and T cells was not altered in MG patients. The percentage of 'active' T cells, which have been considered to be more actively involved in cellular immunity, was also similar in MG patients and controls. A significant increase in EA(H)C-RFC occurred in both thymectomized and non-thymectomized MG patients, while in B cells detected by EA(M)C-RFC no alterations were found. The increase in EA(H)C-RFC in lymphocytes from MG patients may be due to an increase in the 19S antibody-forming B lymphocytes or to an increase in T cells which have Fc receptors on their surface.     

Detection of anti-acetylcholine receptor factors in serum and thymus from patients with myasthenia gravis.

Since the blood and thymus of patients with myasthenia gravis may contain inhibitors of neuromuscular transmission that affect acetylcholine receptors of striated muscle, we used denervated rat muscle to test for inhibitors in 43 serums and 18 thymus glands from such patients. Seven per cent of serums inhibited the binding of 125l alpha-bungarotoxin to triton-solubilized receptors; 65 per cent interfered with binding of toxin-labeled receptors to concanavalin-A coupled to Sepharose gel, and 85 per cent formed IgG-receptor complexes detectable by immunoprecipitation. Serum inhibitory activity varied widely among patients with similar clinical manifestations and was not correlated with duration of myasthenia gravis or thymectomy. Among thymus extracts, 44 per cent were inhibitory in the concanavalin-A binding assay, whereas 72 per cent contained anti-receptor IgG. Thus, serums from patients with myasthenia gravis contain more than one anti-receptor factor.     

Are spontaneous anti-idiotypic antibodies against anti-acetylcholine receptor antibodies present in myasthenia gravis?

The presence of anti-acetylcholine receptor anti-idiotypic antibodies in sera from 102 myasthenia gravis patients and from 33 first-degree relatives was investigated by: (a) Enzyme linked immunosorbent assay (ELISA) using monoclonal antibodies raised against human acetylcholine receptor, (b) immunoprecipitation of 125I-monoclonal anti-acetylcholine receptor antibodies; (c) inhibition of anti-acetylcholine receptor monoclonal antibody binding to the receptor and/or (d) inhibition of autologous and heterologous anti-acetylcholine receptor antibody binding to the receptor. No clear evidence for the presence of abnormal levels of spontaneous anti-idiotypic antibodies to anti-acetylcholine receptor antibodies was found.     

Anti-acetylcholine receptor antibody related idiotypes in myasthenia gravis

Anti-acetylcholine receptor antibody associated idiotypes were defined by six murine monoclonal antibodies raised against purified receptor antibodies. Four of the monoclonal antibodies bound to idiotopes located within or close to the antigen binding site of the anti-receptor antibodies; the other two monoclonal antibodies were directed against framework determinants. These monoclonal antibodies recognized idiotopes present on immunoglobulins in 14-60% of patients presenting myasthenia gravis, indicating substantial idiotype sharing. These idiotopes were also found in patients with no detectable anti-receptor antibody activity in their serum. In all patients studied, the pattern of idiotypes fluctuated considerably during the course of the disease regardless of clinical symptoms. This suggests continuous modulation of the autoimmune process in myasthenia gravis.     

Acetylcholine receptor antibody in patients with myasthenia gravis

The determination of acetylcholine receptor antibody (AChR Ab) titer by an enzyme-linked immunosorbent assay (ELISA) in patients with myasthenia gravis was introduced. The optimal conditions were determined by chequerboard determination. The specificity was confirmed by inhibition tests. The sensitivity is 9 p mole. The comparison of AChR Ab titers among 49 myasthenic patients, 19 non-myasthenic neurological patients and 20 healthy blood donors has shown that it is a highly sensitive, specific, reproducible, rapid, simple and inexpensive method for determining AChR Ab and that it is highly valuable for the diagnosis of myasthenia gravis.     

In vitro anti-acetylcholine receptor antibody synthesis by myasthenia gravis patient lymphocytes: Correlations with thymic histology and thymic epithelial-cell interactions

In vitro anti-acetylcholine receptor antibody (anti-AChR Ab) production by peripheral blood lymphocytes (PBL) and thymic lymphocytes was investigated in 52 patients with myasthenia gravis (MG). There was a positive correlation betweenin vitro anti-AChR Ab synthesis andin vivo titers. A relationship between the rates of synthesis by PBL and histological abnormalities of the thymus was also observed. Patients with hyperplastic thymus tended to produce the largest amountsin vitro, while those with an involuted thymus produced little or none. Production in thymoma patients is likely to correlate with the histological nature of the thymus associated with the tumor.In vitro Ab synthesis was modulated by the depletion of a cell subset for half of the patients tested. Finally, anti-AChR Ab production by thymocytes but not by PBL is enhanced by the addition of autologous or allogeneic thymic epithelial cells, suggesting a possible role of thymic epithelial cells in the autosensitization against AChR occurring in the thymus.     

Analysis of false negative results in the immunoassay for anti-acetylcholine receptor antibodies in myasthenia gravis

Possible causes for the failure of immunoassays to detect anti-acetylcholine receptor activity in serum from confirmed myasthenia gravis (MG) patients were investigated. A more sensitive assay, using Protein A to trap immune complexes (ARIA), was applied to 65 MG sera which were negative in the usual assay and to 42 normal human sera. Normal and negative MG sera had antibody (Ab) activity in the same range (50-70 pM). Titers present in 70% of normal sera appeared to be specific antireceptor antibodies as defined by tests for antigen specificity. Thus, higher sensitivity assays did not improve discrimination of MG from normals. In a second group of 108 MG sera studied, 48 were negative by the usual assay criteria in a rat acetylcholine receptor immunoassay. Further detailed analysis of this negative group showed that 3/48 had IgG3 antibody not detectable in the test, 14/48 had Ab's recognizing human receptor determinants exclusively, 29/48 had toxin blocking Ab's not determined by immunoassays, and 6/48 were negative in all tests. The results indicate that the exclusive occurrence of toxin-blocking antibodies in MG subjects is a major factor contributing to false negatives in the ARIA test. Estimates of the amount of Ab's with this functionality indicated that they are present in very much smaller amounts than other classes of anti-receptor Ab's. Degree of blocking activity in patient serum showed a fair correlation with severity of disease. Thus, blocking antibodies appear capable of causing all degrees of disease severity in the absence of other types of antireceptor Ab's. The development of a sensitive and quantitative in vitro assay for blocking antibodies combined with the usual immunoassay would be a major improvement for a MG diagnostic test, with greater than 94% positivity predicted.     

Inhibition of T cell proliferation specific for acetylcholine receptor epitopes related to myasthenia gravis with antibody to T cell receptor or with competitive synthetic polymers.

Long-term T cell lines and clones of C3H.SW origin specific to synthetic peptides representing immunogenic epitopes of the human aetylcholine receptor alpha-subunit were established. Using these lines and clones, it was possible to characterize the T cell recognition process of myasthenic epitopes. Testing a panel of N-and/or C-terminal truncated peptides it could be demonstrated that the deletion of the two C-terminal amino acids of peptides p195-212 and p259-271 resulted in a loss or reduction of the stimulatory capacity of the peptides towards the specific T cell lines. In contrast, no substantial effect on the stimulation of the line could be observed by shortening peptide p195-212 by up to five amino acids at the N-terminal end. The proliferation of T cell lines and clones specific to peptide p195-212 was inhibited by a mAb directed against the V beta 8 region of the T cell receptor. Furthermore, it was possible to block the peptide-specific proliferative responses of the lines and clones by the I-Ab restricted synthetic polypeptide (T, G)-A--L but not by the I-Ak restricted polypeptide antigen (H,G)-A--L. Similarly, p195-212 inhibited the proliferative response of the TCSW259-271 T cell line and p259-271 inhibited the specific proliferative response of the TCSW195-212 line. Moreover, the C-terminal shortened peptides inhibited significantly the in vitro stimulation of the T cell lines by the immunogenic peptides p195-212 and p259-271. The inhibition by the synthetic peptides or by (T,G)-A--L may be due to competitive blockade of the MHC binding site for the T cell line stimulating AChR peptides.     

Modulation of the anti-acetylcholine receptor response and experimental autoimmune myasthenia gravis by recombinant fragments of the acetylcholine receptor

Myasthenia gravis (MG) is a neuromuscular disorder of man caused by a humoral response to the acetylcholine receptor (AChR). Most of the antibodies in MG and in experimental autoimmune myasthenia gravis (EAMG) are directed to the extracellular portion of the AChR α subunit, and within it, primarily to the main immunogenic region (MIR). We have cloned and expressed recombinant fragments, corresponding to the entire extracellular domain of the AChR α subunit (Hα1 – 210), and to portions of it that encompass either the MIR (Hα1 – 121) or the ligand binding site of AChR (Hα122 – 210), and studied their ability to interfere with the immunopathological anti-AChR response in vitro and in vivo. All fragments were expressed as fusion proteins with glutathione S-transferase. Fragments Hα1 – 121 and Hα1 – 210 protected AChR in TE671 cells against accelerated degradation induced by the anti-MIR monoclonal antibody (mAb)198 in a dose-dependent manner. Moreover, these fragments had a similar effect on the antigenic modulation of AChR by other anti-MIR mAb and by polyclonal rat anti-AChR antibodies. Fragments Hα1 – 121 and Hα1 – 210 were also able to modulate in vivo muscle AChR loss and development of clinical symptoms of EAMG, passively transferred to rats by mAb 198. Fragment Hα122 – 210 did not have such a protective activity. Our results suggest that the appropriate recombinant fragments of the human AChR may be employed in the future for antigen-specific therapy of myasthenia.     

Acetylcholine receptor antibody characteristics in myasthenia gravis. III. Patients with low anti-AChR antibody levels.

Twenty-five myasthenia gravis (MG) patients whose anti-acetylcholine receptor (anti-AChR) titres against denervated human leg AChR fell in the range of less than 0.1-2.0 nM were tested against AChR preparations from normal human leg muscle, and from human extra-ocular muscle, and the results compared with those of sera with titres greater than 2.0 nM. Seven sera failed to react appreciably with any of the AChR. Many of the remaining 18 low titre sera reacted better with normal leg and ocular AChR than with denervated AChR. In contrast, sera from patients with high anti-AChR titres generally reacted better with denervated, AChR, and as a group this was significantly different from the low titre patients. Sera reacting better with ocular AChR are not restricted to patients with purely ocular symptoms. The results indicate a subgroup of myasthenia gravis patients who have low titres, tend to be male and have relatively mild disease. The aetiology of the autoimmune disorder in this subgroup may differ from that in other MG patients.     

Acetylcholine receptor antibody characteristics in myasthenia gravis. II. Patients with penicillamine-induced myasthenia or idiopathic myasthenia of recent onset.

Anti-acetylcholine receptor (anti-AChR) antibody characteristics including light chain, IgG subclass, avidity for denervated human acetylcholine receptor and reaction with various human and mammalian AChR preparations were examined in 11 patients who developed myasthenia during penicillamine treatment of rheumatoid arthritis. Results were compared with those already reported in 35 patients with generalized idiopathic myasthenia gravis (MG). We found significant differences in the avidity and the light chain of the anti-AChR. However, anti-AChR characteristics in 12 patients with recent onset (less than 4 months'' duration) idiopathic MG did not differ significantly from those in patients with penicillamine-induced MG. In the patients with generalized MG a trend was found towards higher percentage of kappa light chain and higher anti-AChR avidity with duration of disease. Anti-acetylcholine receptor antibodies in penicillamine-induced myasthenia gravis therefore appear to be similar to those of idiopathic myasthenia gravis of recent onset.     

Specificities of antibody to acetylcholine receptor in rabbits with experimental myasthenia gravis.

The injection of acetylcholine receptor (AChR) purified from Narke electroplax japonica induced 'experimental autoimmune myasthenia gravis' (EAMG) in rabbits. Serial measurements of anti-AChR antibody titre using Narke AChR and rabbit AChR as antigen revealed that the intensity of myasthenia had a rather closer correlation with titre to Narke AChR than that to rabbit AChR. Antibody reacting to rabbit AChR was almost completely adsorbed out with torpedo receptor conjugated to agarose. Serum concentrations of antibody protein measured by affinity chromatography ranged from 54 to 896 microgram/ml serum and were well correlated with the intensity of myasthenia. The results suggested that in rabbits immunized with heterologous AChR the cross-reaction of anti-heterologous AChR antibody with rabbit AChR caused myasthenia rather than an immune response specific to rabbit AChR dose.     

Abnormal T cell receptor V gene usage in myasthenia gravis: prevalence and characterization of expanded T cell populations

The usage of T cell receptor (TCR) Vα/Vβ chains on cells from 38 patients with myasthenia gravis (MG) was determined by flow cytometry. There was a decreased number of cells expressing Vβ2 in CD8⁺ and Vβ3 in CD4⁺ cells in patients compared with healthy individuals. Abnormal expansions of T cells using particular TCR Vα/Vβ gene products were found in 18/38 patients. A significantly higher usage of Vβ13 was observed but there was no restriction with regard to other TCR Vα/Vβ. Expanded cells belonging to both CD4⁺ and CD8⁺ were present in MG patients while restricted to the CD8⁺ population in healthy individuals. To elucidate the role of the expanded populations, we studied characteristics of the expanded and non-expanded T cells from MG patients who had persistent T cell expansions over more than 2 years. The cells were analysed with regard to phenotype, cytokine secretion, cytokine mRNA expression and reactivity with the autoantigen, the acetylcholine receptor. The characteristics of the expanded populations in MG clearly differed from those found in healthy individuals. More cells in the CD4⁺ expanded populations expressed HLA-DR and there was also a tendency for higher expression of CD25, CD28 and CD57. The number of cells spontaneously secreting cytokines was higher in the expanded populations. A dominant Th1-type cytokine secretion and mRNA expression was noted. Autoantigen-reactive CD4⁺ T cells were largely restricted to the expanded populations.     

Study of two different subpopulations of anti-acetylcholine receptor antibodies in a rabbit with experimental autoimmune myasthenia gravis

Two different subpopulations of anti-acetylcholine receptor antibodies were studied during the evolution of experimental autoimmune myasthenia in one rabbit immunized with Torpedo acetylcholine receptor. The results show that the subpopulation of antibodies directed against the toxin-binding site of the receptor might play a role in the appearance of the paralysis observed in this particular case.