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.     

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.     

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.     

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.     

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.     

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.     

Characterization of anti-acetylcholine receptor (AChR) antibodies from mice differing in susceptibility for experimental autoimmune myasthenia gravis (EAMG).

In the murine model for EAMG we investigated the relation between disease susceptibility and fine specificity of anti-AChR antibodies obtained from high susceptible C57Bl/6 and low susceptible BALB/c mice after immunization with Torpedo acetylcholine receptor (tAChR). Anti-AChR MoAbs with fine specificity for the main immunogenic region (MIR), the alpha-bungarotoxin (alpha-BT)/acetylcholine binding sites and other extra- and intracellular epitopes were isolated from both mouse strains. In total, nine out of 38 MoAbs obtained from C57Bl/6 mice were directed against extracellular epitopes on mouse AChR in contrast to only one out of 27 MoAbs from BALB/c mice. A difference in antibody repertoire may underlie the difference in pathogenic response observed between these mouse strains. These results indicate that strain-specific differences in disease susceptibility in murine EAMG may be related to differences in the available repertoire of potential pathogenic antibodies.     

Search for cross-reactive idiotypes on monoclonal and myasthenia gravis acetylcholine receptor antibodies.

Myasthenia gravis patients have serum anti-acetylcholine receptor antibodies that compete with monoclonal antibodies for binding to epitopes on the human acetylcholine receptor. To investigate the presence of shared idiotypes we immunised syngeneic mice with each of ten well-characterised monoclonal antibodies, previously raised against purified human acetylcholine receptor, and tested the polyclonal antisera and seven monoclonal anti-idiotype antibodies, for binding to the antigen-combining site, to framework idiotopes, and by ELISA. The polyclonal sera were mostly directed against antigen-combining site idiotopes and cross-reacted only with monoclonal anti-acetylcholine receptor antibodies that bound to the same region on the acetylcholine receptor. In contrast, five of the seven IgM monoclonal anti-idiotypic antibodies raised, none of which demonstrated antigen-combining site specificity in solution, cross-reacted with mAbs binding to more than one region. None of the antisera showing reactivity with the antigen-combining site inhibited the binding of MG anti-acetylcholine receptor antibody.     

A radioimmunoassay for the quantitative evaluation of anti-human acetylcholine receptor antibodies in myasthenia gravis.

A radioimmunoassay was developed for the quantitative evaluation of antibodies to the acetylcholine receptor in the serum of myasthenic patients. AcChR was extracted from human muscle. A detailed preparation of the 125I-labelled alpha-Bgt-AcChR complex used as antigen is reported. Usually, an average of 20 pmol were obtained form 100 g muscle. This preparation is stable for 1 month in presence of an inhibitor of proteolysis and sufficient for performing about fifteen assays. The labelled complex was incubated with increasing amounts of sera and precipitated with anti-human IgG serum. Titres were expressed in pmol 125I-labelled alpha-Bgt-AcChR complex precipitated per ml serum. Out of thirty-nine sera tested thirty-six had positive titres ranging from 0-1-46 pmol/ml. No anti-AcChR were detected in the sera from twenty-seven patients used as controls.     

Characterization of human anti-acetylcholine receptor monoclonal autoantibodies from the peripheral blood of a myasthenia gravis patient using combinatorial libraries

Myasthenia gravis (MG) is an autoimmune disease caused by autoantibodies against the nicotinic acetylcholine receptor (AChR). Using phage-display technology we have characterized the largest panel of anti-AChR monoclonal antibodies thus far isolated from a single patient. Despite having been isolated with either Torpedo AChR or a human peptide, the recombinant antibodies shared with the donor's serum the ability to recognize human AChR expressed in its native configuration on the surface of TE671 cells. Their specificity for the main immunogenic region (MIR) of the AChR was demonstrated using a synthetic peptide corresponding to the region 67-76 of the human AChR alpha subunit and by inhibition of a highly pathogenic rat anti-MIR monoclonal antibody (mAb35). This work demonstrates the value of combinatorial libraries in isolating pathogenic autoantibodies from peripheral blood lymphocytes. Future genetic, structural, and functional analyses of the monoclonal antibodies reported herein should enhance our understanding of the pathogenesis of MG.     

Evans blue as counterstain in the demonstration of muscle antibodies by immunofluorescence in myasthenia gravis

The binding of muscle antibodies to skeletal muscle was studied by immunofluorescence techniques. The non-specific fluorescence was not significantly reduced by absorption of the conjugate by tissue powder but was abolished by counterstaining with a 0.1% Evans blue. Possible explanations for this effect are discussed. By this method, immunofluorescence may become more useful in the demonstration of muscle antibodies.     

Unexpected cross-reactivity between myosin and a main immunogenic region (MIR) of the acetylcholine receptor by antisera obtained from myasthenia gravis patients.

Antibodies obtained from the plasma of patients with myasthenia gravis (MG) were found to contain reactivity against both the classic target antigen, the acetylcholine receptor, as well as muscle myosin. This observation was consistent with several previously published reports. However, it was also observed in the present study that much of the dual reactivity contained in MG plasma was due to the ability of individual clonotypic species of anti-receptor antibodies to also bind myosin. Furthermore, the cross-reactivity demonstrated by these antibodies appeared to involve a main immunogenic region of the acetylcholine receptor and an enzymatically important region in the head of the myosin heavy chain. This observation appears to provide new explanations for the epitope-restricted antibody response seen in MG patients.     

Use of Torpedo-mouse hybrid acetylcholine receptors reveals immunodominance of the alpha subunit in myasthenia gravis antisera.

The nicotinic acetylcholine receptor (AChR), a pentameric complex of alpha 2 beta gamma delta subunits, is the autoantigen in the human autoimmune disease myasthenia gravis (MG). Anti-AChR antibodies are found in approximately 90% of MG patients and using indirect methods (competitive binding to solubilized AChR), peptides, or synthetic peptides, the majority of these antibodies have been shown to bind to the AChR alpha subunit. In order to determine directly the AChR subunit specificities of MG antibodies, we employed as antigens a novel set of hybrid AChR composed of species cross-reacting and non-cross-reacting subunits stably expressed in fibroblasts. Sequence similarities of homologous subunits among species can vary widely, with mammalian subunits having 87%-96% identity and Torpedo-mammalian subunits having 54%-80% identity. These findings are reflected in antigenic specificities, with human anti-AChR antisera frequently recognizing mouse AChR but rarely recognizing Torpedo. By establishing separate cell lines stably expressing all-Torpedo, all-mouse, and different combinations of Torpedo and mouse subunits, we were able to provide the first direct evidence of a predominant anti-alpha subunit specificity in MG antisera. Functional hybrid AChR stably expressed in an intact cell membrane provide us with a system that best mimics the in vivo environment of the MG antibody in a binding assay. Such a system allows us to investigate a perplexing observation in the field: a poor correlation between the patient's clinical status and antibody titer. Those antibodies which can interfere with AChR function, such as ones with the ability to cross-link AChR and induce their accelerated internalization and degradation (antigenic modulation) might represent a subpopulation of MG antibodies important in disease induction or maintenance. In this report, we demonstrate that wild-type and hybrid AChR expressed in fibroblasts can be antigenically modulated by intermolecular cross-linking antibodies as AChR are in native muscle cells. Because we can monitor dynamic interactions between AChR and MG antibodies, this system may allow us to define crucial pathogenic epitopes in MG by expressing hybrid, chimeric, and mutant AChR.     

The reactivity of the antistriational antibodies associated with thymoma and myasthenia gravis.

Using immunofluorescence forty-nine sera giving striational staining of skeletal and cardiac muscle have been tested on glycerinated myofibrils. By comparing immunofluorescence and phase contrast it has been found that these antibodies are rather heterogeneous but at least three different staining patterns can be identified. Some patterns may be associated with the presence of a thymoma or may be induced by penicillamine treatment of patients with rheumatoid arthritis. Routine screening for antistriational antibodies may lead to the recognition of undiagnosed myasthenia gravis and thymoma. Antistriational autoantibodies may be useful in the further study of the structure and function of the sarcomere.     

A modified assay for antibody against the nicotinic acetylcholine receptor in myasthenia gravis.

Myasthenia gravis is an autoimmune disease which may be detected by the presence of serum antibodies against the nicotinic acetylcholine receptor at the neuromuscular junction. Immunoprecipitation assays have been developed to measure these immunoglobulins and calculate titres. These assays require the labelling of the receptor with 125I-alpha-bungarotoxin which binds irreversibly. However, the standard immunoprecipitation assay may significantly underestimate the titres of some myasthenic patients. We have discovered patients with antibodies specific for the alpha-bungarotoxin binding site of purified rat muscle receptor. If labelled toxin is already present on the receptor, these antibodies are unable to bind to the protein. This phenomenon may lead to underestimates of the actual antibody titre. To circumvent this problem, we have designed a modified immunoprecipitation assay to evaluate titres.     

'Split tolerance' induction by intrathymic injection of acetylcholine receptor in a rat model of autoimmune myasthenia gravis; implications for the design of specific immunotherapies.

Experimental autoimmune myasthenia gravis (EAMG) in the Lewis rat, induced by a single injection of acetylcholine receptor (AChR) protein, is a model used to study human myasthenia gravis (MG). The production of anti-AChR antibodies in the animal model and human MG is T cell-dependent, and AChR-specific T cells have been considered as a potential target for specific immunotherapy. Intrathymic injection of antigens induces antigen-specific tolerance in several T cell-mediated autoimmune models. We examined the effect of intrathymic injection of AChR on T cell responses and the production of antibodies to AChR in EAMG rats. Primed lymph node cells from rats receiving intrathymic injection of AChR exhibited reduced proliferation to AChR with marked suppression of interferon-gamma (IFN-gamma) secretion in the antigen-stimulated culture, compared with those of rats injected with PBS. However, neither anti-Narke AChR nor anti-rat AChR antibody production was suppressed or enhanced in intrathymically AChR-injected animals compared with that of animals injected intrathymically with PBS or perithymically with AChR. This 'split tolerance' may be attributable to the suppression of type-1 T helper cells (Th1). Our results suggest that the suppression of Th1 function alone may not be sufficient for the prevention of antibody-mediated autoimmune diseases.     

Acetylcholine receptor-reactive antibodies in experimental autoimmune myasthenia gravis differing in disease-causing potential: subsetting of serum antibodies by preparative isoelectric focusing.

Antibodies obtained from the sera of Lewis rats demonstrating impaired neuromuscular function following immunization with purified acetylcholine receptor (AChR) were fractionated by preparative isoelectric focusing. Upon passive transfer of fractionated anti-receptor antibodies into immunologically naive, healthy recipient rats it was observed that two main subsets of AChR-specific antibody could be identified. One subset, representing about one-third of the expressed clonotypic antibody repertoire, was capable of directly perturbing AChR-dependent neuromuscular function following transfer. A second subset, demonstrated no detectable ability to induce disease symptoms following transfer. Although the anti-AChR antibodies were produced by immunization with Torpedo AChR, the inability of some antibody fractions to perturb AChR function was not explained by their inability to react with AChR of mammalian origin. Furthermore, the ability to transfer symptoms did not correspond with a particular antibody isotype (although the response was dominated by IgG2a) and did not depend solely on high relative binding avidity (benign reactivities of high relative binding avidity were also observed). Nonetheless, an anti-AChR antibody subset can be directly identified and purified from immune serum that is likely to contain reactivities that are most directly responsible for neuromuscular disease symptoms demonstrated by rats with experimental autoimmune myasthenia gravis.     

Neonatal myasthenia gravis: antigenic specificities of antibodies in sera from mothers and their infants.

Transient neonatal myasthenia gravis (MG) is a human model of passively transferred MG. In an effort to understand the characteristics of the most pathogenic antibodies in MG, we studied the fine antigenic specificities of anti-AChR antibodies in sera from 21 MG mothers (nine of which had transiently transferred the disease) and 17 of their infants. Although in a few cases significant differences in antibody specificities were observed between mothers and infants, whether myasthenic or not, generally the antigenic specificities of the antibodies in sera from infants were very similar to those of their mothers. Furthermore, no characteristic differences were detected between the antibody repertoires of mothers who transferred the disease and those who did not.