A Stable Cell Line Expressing Clustered AChR: A Novel Cell-Based Assay for Anti-AChR Antibody Detection in Myasthenia Gravis

Abstract

Cell-based assays (CBAs) and radioimmunoprecipitation assay (RIPA) are the most sensitive methods for identifying anti-acetylcholine receptor (AChR) antibody in myasthenia gravis (MG). But CBAs are limited in clinical practice by transient transfection. We established a stable cell line (KL525) expressing clustered AChR by infecting HEK 293T cells with dual lentiviral vectors expressing the genes encoding the human AChR α1, β1, δ, ϵ and the clustering protein rapsyn. We verified the stable expression of human clustered AChR by immunofluorescence, immunoblotting, and real-time PCR. Fluorescence-activated cell sorting (FACS) was used to detect anti-AChR antibodies in 103 MG patients and 58 healthy individuals. The positive results of MG patients reported by the KL525 was 80.6% (83/103), 29.1% higher than the 51.4% (53/103) of RIPA. 58 healthy individuals tested by both the KL525 CBA and RIPA were all negative. In summary, the stable expression of clustered AChR in our cell line makes it highly sensitive and advantageous for broad clinical application in CBAs.

Detection of Antibodies against the Acetylcholine Receptor in Patients with Myasthenia Gravis: A Comparison of Two Enzyme Immunoassays and a Fixed Cell-Based Assay

“…We previously demonstrated that the F-CBA for MG diagnosis did not accurately identify low anti-AChR and anti-MuSK levels, which were detected by an ELISA [26]. Mirian et al showed that F-CBAs have similar specificity and higher sensitivity compared to RIPAs but lower performance than L-CBAs [22]. Also, Spagni et al found that L-CBAs are more sensitive than F-CBAs [29].…”

“…Various analytical methods are available for serological analysis, including the radioimmunoprecipitation assay (RIPA), enzyme-linked immunosorbent assay (ELISA), dot-blot testing and a commercial biochip based on a fixed cell-based assay (F-CBA), which measures antibodies against AChR and MuSK simultaneously [16][17][18][19][20][21][22][23]. F-CBAs and live cell-based assays (L-CBA) are reported to have higher sensitivity compared to RIPAs or ELISAs.…”

Detection of Autoantibodies Against the Acetylcholine Receptor, Evaluation of Commercially Available Methodologies: Fixed Cell-Based Assay, Radioimmunoprecipitation Assay and Enzyme-Linked Immunosorbent Assay1

“…In the anti-AChR CBA used in this study, cell fixing could affect, up to some degree, the receptor integrity and clustering, which would in turn affect autoantibody binding and impair detection, although here only one patient with MG and RIPA-Pos showed no reactivity in the CBA. It has been suggested that live cells expressing clustered nAChR to detect the AAbs can show a sensitivity even higher than RIPA itself, because the unfixed transfected cells better resembles the physiological expression of the AChR by myocytes [13,[27][28][29]. However, the maintenance of live-cell cultures expressing AChR requires special facilities and expertise, which also restricts the adoption of this methodology in most clinical laboratories.…”

Complement activation profiles in anti‐acetylcholine receptor positive myasthenia gravis

“…Studies in such larger series could additionally focus on the various AChR epitopes recognized by the patients' Abs, which can vary between MG subgroups [23] and might affect complement activation. Additionally, our SNMG patients were not tested in the cell‐based assays that are currently confined to specialized research centres [24–26]; as they reportedly have higher sensitivities [27, 28], especially for Abs specific for clustered AChRs [22], they should be used in future studies on SNMG patients.…”

“…Additionally, they also found no correlation of plasma levels of the altered complement compo- Studies in such larger series could additionally focus on the various AChR epitopes recognized by the patients' Abs, which can vary between MG subgroups [23] and might affect complement activation. Additionally, our SNMG patients were not tested in the cell-based assays that are currently confined to specialized research centres [24][25][26]; as they reportedly have higher sensitivities [27,28],…”