IgG1 antibodies to acetylcholine receptors in ‘seronegative’ myasthenia gravis†

Abstract

Only around 80% of patients with generalized myasthenia gravis (MG) have serum antibodies to acetylcholine receptor [AChR; acetylcholine receptor antibody positive myasthenia gravis (AChR-MG)] by the radioimmunoprecipitation assay used worldwide. Antibodies to muscle specific kinase [MuSK; MuSK antibody positive myasthenia gravis (MuSK-MG)] make up a variable proportion of the remaining 20%. The patients with neither AChR nor MuSK antibodies are often called seronegative (seronegative MG, SNMG). There is accumulating evidence that SNMG patients are similar to AChR-MG in clinical features and thymic pathology. We hypothesized that SNMG patients have low-affinity antibodies to AChR that cannot be detected in solution phase assays, but would be detected by binding to the AChRs on the cell membrane, particularly if they were clustered at the high density that is found at the neuromuscular junction. We expressed recombinant AChR subunits with the clustering protein, rapsyn, in human embryonic kidney cells and tested for binding of antibodies by immunofluorescence. To identify AChRs, we tagged either AChR or rapsyn with enhanced green fluorescence protein, and visualized human antibodies with Alexa Fluor-labelled secondary or tertiary antibodies, or by fluorescence-activated cell sorter (FACS). We correlated the results with the thymic pathology where available. We detected AChR antibodies to rapsyn-clustered AChR in 66% (25/38) of sera previously negative for binding to AChR in solution and confirmed the results with FACS. The antibodies were mainly IgG1 subclass and showed ability to activate complement. In addition, there was a correlation between serum binding to clustered AChR and complement deposition on myoid cells in patients’ thymus tissue. A similar approach was used to demonstrate that MuSK antibodies, although mainly IgG4, were partially IgG1 subclass and capable of activating complement when bound to MuSK on the cell surface. These observations throw new light on different forms of MG paving the way for improved diagnosis and management, and the approaches used have applicability to other antibody-mediated conditions.

Autoimmune disorders of the neuromuscular junction

“…We were then able to detect the presence of antibodies in about 60% of the sera that were negative for binding to AChR in solution. [27] It appears that this "cell-based" method [ Figure 3] can also be applied to measuring antibodies to MuSK, and should provide a more sensitive assay for all MG patients. [27] The Lambert Eaton Myasthenic Syndrome…”

The role of humoral autoimmunity in gastrointestinal neuromuscular diseases

“…Thirdly, the antigen is specifically expressed at a high concentration, increasing assay sensitivity. Cell binding assays have been employed to detect autoantibodies in a variety of diseases and have been demonstrated to be more sensitive than traditional immunoprecipitation assays in screening for antibodies in well characterised disorders such as neuromyotonia Leite et al 2008).…”

“…embryonic kidney fibroblasts) transfected to express channels or receptors of interest represents a means by which the binding of antibodies to the external surface of functionally active channels / receptors may be investigated (Blaes et al 2000). This may be done by incubating cells expressing the target of interest with patient serum or by extracting the cells for fluorescent-labelled immunoprecipitation, and is perhaps now the assay of choice in detecting anti-channel antibodies, not only because functionality is strongly implied, but also because such assays may be more sensitive (Leite et al 2008). …”

“…This confirmed our method of transfection was robust. Secondly, successful expression of M 2 mAChR was confirmed by immunofluorescently staining transfected cells with a commercial anti-M 2 mAChR monoclonal antibody (Leite et al 2008). It should be noted that only commercial antibodies raised against intracellular epitopes of the receptor were available at this time and as such it was necessary to permeabilise the transfected cells prior to antibody incubation.…”

“…With these conditions satisfied one may feel confident in using the assay to screen sera for antibodies against the target in question. Indeed, this assay is increasingly becoming the assay of choice in detecting autoantibodies as it has been shown to be more sensitive than traditional radioimmunoprecipitation assays and is closer to the in vivo conditions where autoantibodies meet membrane bound, fully processed channels and receptors (Leite et al 2008).…”