Establishment And Application Of A Cell-based Assay Expressing Clustered Acetylcholine Receptor

Background and aimsMyasthenia gravis(MG)is an autoimmune disorder affecting the signaling of neurotransmitters at neuromuscular junction.But circulating acetylcholine receptor antibody(anti-AChR)with low affinity,accounting for roughly 10% of MG,remains undetected by radioimmunology assay(RIPA),the most sensitive serologic test clinically available.The current cell-based assay(CBA)is sensitive in detecting such antibodies but is also limited in clinical application attributable to its complete reliance on transient transfection.To technically bridge this gap,a CBA expressing clustered AChR(KL525 stable cell line)was established in this study by lentiviral infection with validated sensitivity and specificity in the detection of anti-AChR.MethodsThe CBA expressing clustered AChR in a stable manner was constructed by infecting human embryonic kidney 293T(HEK 293T)cells with lentiviral vectors expressing the genes of human AChR ?1,?1,?,?subunits and rapsyn protein.The expression of clustered AChR was examined by immunofluorescence,immunoblots and PCR.59 patients with confirmed MG and 47 individuals without MG were recruited in this study.Fluorescence-Activated Cell Sorting(FACS)were utilized to determine the sensitivity and specificity of KL525 stable cell line in detecting anti-AChR.ResultsThe stable expression of human AChR ?1,?1,?,? subunits and rapsyn in KL525 stable cell line generated by this study was verified by immunofluorescence,immunoblots and PCR.The sensitivity of KL525 stable cell line in detecting anti-AChR in MG patients was 86.4%(51/59),20.3% higher than RIPA.The sensitivity of KL525 stable cell line in SNMG patients was 60%(12/20).The specificity of both KL525 stable cell line and RIPA was 100%(47/47).The results of KL525 stable cell line were potentially associated with symptoms,clinical subtype,MG Foundation of America(MGFA)classification and therapeutic strategies.ConclusionsCompared with RIPA,KL525 stable cell line significantly improved the sensitivity in the detection of anti-AChR compared.KL525 stable cell line also remedied the technical defects of previous CBA completely depending on the transient transfection.Therefore,the CBA stably expressing clustered AChR established by lentiviral infection is more advantageous than current serologic tests.