Myasthenia gravis (MG) is an autoimmune disorder of the neuromuscular junction (NMJ) in which autoantibodies disrupt signaling between nerve and muscle, producing the fatigable, fluctuating weakness that characterizes the disease.¹

Diagnosis of MG is based on a combination of findings, including clinical presentation, electrophysiological studies, imaging, and autoantibody testing.² A positive antibody result does two things at once: it confirms clinical suspicion, and it increasingly determines which therapies a patient can access. Complement inhibitors and neonatal Fc receptor antagonists have changed the treatment landscape, and eligibility for many of these agents, and for the trials evaluating them, is tied to a defined antibody serostatus.³ A patient who has MG but whose antibodies go undetected may be excluded from a therapy that would help.

Antibody targets in MG

Roughly 80% of patients with generalized MG carry antibodies against nicotinic acetylcholine receptors (AChR).^1,2 A smaller group, generally reported around 5–8%, carries antibodies against muscle-specific kinase (MuSK), a receptor tyrosine kinase that organizes AChR clustering at the NMJ.⁴  MuSK-positive patients are clinically distinct, often with prominent bulbar and respiratory involvement, and they respond differently to treatment, so accurate subtyping matters beyond simple confirmation.⁴ In addition to AChR and MuSK, antibodies to low-density lipoprotein receptor-related protein 4 (LRP4) and to agrin account for a smaller share of MG patients.^1,2

Conventional testing for AChR and MuSK antibodies leaves approximately 10% of patients with MG without a detectable antibody.⁵ These patients are described as double-seronegative, though the label may reflect the limits of the assay as much as the absence of antibody. In some cases, antibodies are present but bind in a way conventional assay formats do not capture.³

Antibody testing methodologies

Radioimmunoprecipitation assay

The radioimmunoprecipitation assay (RIPA) has been the long-standing reference method for the detection of antibodies against AChR and MuSK.⁶ It is quantitative and extensively validated, but it carries operational constraints. The requirement of working with radioactive reagents limits where RIPAs can be performed.

For AChR antibodies in particular, a second limitation concerns antigen presentation. RIPA measures antibody binding to AChRs that have been solubilized from tissue or cell extract and complexed with radiolabeled α-bungarotoxin.⁷ However, at the NMJ in vivo, AChRs are instead packed at high density and clustered by rapsyn.⁸ Antibodies that depend on that arrangement, or that bind with low affinity, may go undetected.⁶

ELISA

ELISA is a solid-phase, non-radioactive assay that offers higher throughput for anti-AChR and anti-MuSK detection. In the ELISA format, AChR antigen is immobilized on the plate rather than presented in the densely clustered arrangement found at the NMJ.⁷ Antibodies that bind only to a clustered receptor have been shown to escape detection by RIPA,⁸ and by design neither RIPA nor ELISA reconstitutes that arrangement. At the same time, comparisons of ELISA against clustered cell-based assays in seronegative cohorts remain limited.

For AChR antibody detection, reported sensitivity varies considerably by kit and by cutoff selection, and comparative studies have found meaningful performance differences between assays, so results from one platform should not be assumed to generalize to another. Specificity is generally high, typically in the 90% to 100% range, though this often comes at some cost to sensitivity depending on where the cutoff is set.⁷ In addition, ELISA also provides quantitative antibody levels, which may be useful for monitoring.^9,10

Cell-based assays

Cell-based assays (CBA) present AChR and MuSK on the surface of living or fixed cells rather than as purified or immobilized protein. Human cells are transfected to express the target antigen on their surface. Patient serum is applied, and bound IgG is detected by immunofluorescence, either by microscopy or by flow cytometry.^11,12

For AChR, the CBAs co-express the receptor subunits together with rapsyn, the intracellular protein that clusters AChRs at the NMJ, producing a dense, clustered array of receptors. Presenting AChRs in clustered form allows detection of some low-affinity and clustering-dependent antibodies that RIPA may not capture. ^8,12

There are two formats of CBA; live CBAs use unfixed cells and may offer the highest analytical sensitivity, but they are technically demanding and have largely remained in specialized and research laboratories. Fixed CBAs, in which transfected cells are fixed onto slides, are more reproducible and are commercially available, which has brought the method within reach of routine diagnostic laboratories.¹²

Method comparison data: CBA vs. RIPA

A study from Mirian et al. found that a fixed CBA for AChR antibodies showed specificity comparable to RIPA alongside significantly higher sensitivity for MG, identifying anti-AChR antibodies in 21% of patients previously classified as seronegative. A further analysis of fixed CBAs for AChR and MuSK antibodies found high positive agreement and 100% negative agreement with RIPA among patients with clinically confirmed MG. Taken together, these findings indicate that CBAs deliver performance equivalent or superior to RIPA without requiring radioactive materials.¹³

Additional evidence supporting the use of CBAs comes from a prospective, multicenter, double-blind study that compared fixed CBA, RIPA, and ELISA head-to-head in patients with suspected MG.⁶ Among 2,043 patients with confirmed MG and 229 non-MG controls, AChR antibodies were detected by CBA in 1,478 patients; by RIPA in 1,310; and by ELISA in 1,280 — corresponding to sensitivities of 72.3%, 64.1%, and 62.7%. Specificity was 97.8% for both CBA and RIPA and 94.8% for ELISA.¹⁴

Spagni et al. compared fixed and live CBAs directly. In RIPA-positive samples, sensitivity for AChR antibodies was 98.5% for fixed CBA and 100% for live CBA, a difference that did not reach significance. For MuSK, both formats reached 100% sensitivity and 100% specificity. The live format retained an advantage in samples negative by both RIA and fixed CBA, particularly those with low antibody levels.¹²

A 2025 clinical-practice evaluation examined the positive predictive value (PPV) of fixed CBA testing for AChR and MuSK antibodies after the assay was adopted as first-line testing at a single center. Of 770 patients tested, 109 (14%) were positive, and 108 were confirmed as true positives on clinical review, giving a PPV of 99% in a population already selected for suspected MG. In a referral population where MG is actively suspected, which is the setting where most of this testing is ordered, a positive fixed CBA result was reliable enough to act on.¹⁵

Positioning CBAs in the testing algorithm

As a first-line test, a fixed CBA offers sensitivity for AChR antibodies at least equal to RIPA, comparable specificity, and the operational advantage of eliminating radioisotope handling. For laboratories evaluating a move away from radioactive methods, this is a meaningful consideration.^12,13,15

As a reflex test, CBA has a clear role in patients who are negative by RIA or ELISA but remain clinically suspicious. These are the patients in whom a CBA is most likely to change the diagnostic outcome, though reported yields vary considerably across published cohorts, reflecting differences in how the seronegative population was defined and which assay format was used.^3,8,13

Conclusion

Autoantibody testing is central to the diagnosis of MG, and because treatment access frequently depends on serostatus, the accuracy of that testing carries direct clinical consequences. Cell-based assays for anti-AChR address a specific and long-standing weakness of conventional serology by presenting the target antigen in native, clustered, membrane-bound form. The result is improved sensitivity for AChR antibodies, a meaningful diagnostic yield in patients previously labeled seronegative, and comparable specificity with established methods. With reproducible fixed CBAs now commercially available and live CBAs anchoring reference testing for the most difficult cases, laboratories are positioned to identify more true cases of MG and to deliver the answers that increasingly guide therapy.

References

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2. Tannemaat MR, Huijbers MG, Verschuuren JJGM. Myasthenia gravis-Pathophysiology, diagnosis, and treatment. Handb Clin Neurol. 2024;200:283-305. doi:10.1016/B978-0-12-823912-4.00026-8. 

3. Masi G, Li Y, Karatz T, et al. The clinical need for clustered AChR cell-based assay testing of seronegative MG. J Neuroimmunol. 2022;367:577850. doi:10.1016/j.jneuroim.2022.577850.

4. Rodolico C, Bonanno C, Toscano A, Vita G. MuSK-associated myasthenia gravis: Clinical features and management. Front Neurol. 2020;11:660. doi:10.3389/fneur.2020.00660.

5. Abuhammad A, Ayyad M, Albandak M, et al. Double-seronegative myasthenia gravis: clinical characteristics, treatment, and outcomes - a systematic review of case reports and case series. BMC Neurol. 2026;26(1):107. doi:10.1186/s12883-026-04644-8.

6. Mousavi A, Kumar P, Frykman H. The changing landscape of autoantibody testing in myasthenia gravis in the setting of novel drug treatments. Clin Biochem. 2024;133-134:110826. doi:10.1016/j.clinbiochem.2024.110826. 

7. Diogenes L, Dellavance A, Baldo DC, et al. Detection of autoantibodies against the acetylcholine receptor, evaluation of commercially available methodologies: Fixed cell-based assay, radioimmunoprecipitation assay and enzyme-linked immunosorbent assay1. J Neuromuscul Dis. 2024;11(3):613-623. doi:10.3233/JND-230210.

8. Leite MI, Jacob S, Viegas S, et al. IgG1 antibodies to acetylcholine receptors in 'seronegative' myasthenia gravis. Brain. 2008;131(Pt 7):1940-52. doi:10.1093/brain/awn092. 

9. Marcuse F, Brandts L, Moens D, et al. The association between anti-acetylcholine receptor antibody level and clinical improvement in myasthenia gravis. Eur J Neurol. 2022;29(4):1187-1197. doi:10.1111/ene.15238.

10. Kojima Y, Uzawa A, Ozawa Y, et al. Rate of change in acetylcholine receptor antibody levels predicts myasthenia gravis outcome. J Neurol Neurosurg Psychiatry. 2021;92(9):963-968. doi:10.1136/jnnp-2020-325511.

11. Lazaridis K, Tzartos SJ. Autoantibody specificities in myasthenia gravis; Implications for improved diagnostics and therapeutics. Front Immunol. 2020;11:212. doi:10.3389/fimmu.2020.00212. 

12. Spagni G, Gastaldi M, Businaro P, et al. Comparison of fixed and live cell-based assay for the detection of AChR and MuSK antibodies in myasthenia gravis. Neurol Neuroimmunol Neuroinflamm. 2022;10(1):e200038. doi:10.1212/NXI.0000000000200038. 

13. Mirian A, Nicolle MW, Edmond P, Budhram A. Comparison of fixed cell-based assay to radioimmunoprecipitation assay for acetylcholine receptor antibody detection in myasthenia gravis. J Neurol Sci. 2022;432:120084. doi:10.1016/j.jns.2021.120084. 

14. Li Z, Zhang C, Chang T, et al. A multicentre, prospective, double-blind study comparing the accuracy of autoantibody diagnostic assays in myasthenia gravis: the SCREAM study. Lancet Reg Health West Pac. 2023;38:100846. doi:10.1016/j.lanwpc.2023.100846.

15. Li CMF, Nicolle MW, Sangam K, Yang L, Budhram A. Testing for acetylcholine receptor and muscle-specific tyrosine kinase antibodies by fixed cell-based assay in clinical practice: Positive predictive value for myasthenia gravis. Eur J Neurol. 2025;32(3):e70123. doi:10.1111/ene.70123.