Stable Expression of Human Muscle-Specific Kinase in HEp-2 M4 Cells for Automatic Immunofluorescence Diagnostics of Myasthenia Gravis

Abstract

Muscle-specific kinase (MuSK) belongs to the nicotinic acetylcholine receptor complex which is targeted by pathogenic autoantibodies causing Myasthenia gravis. While up to 95% of patients with generalized Myasthenia gravis were shown to be positive for acetylcholine receptor-specific autoantibodies, up to 70% of the remaining patients develop autoantibodies against MuSK. Discrimination of the autoantibody specificity is important for therapy of Myasthenia gravis. Recently, the new automatic fluorescence assessment platform AKLIDES has been developed for immunofluorescence-based diagnostics of autoimmune diseases. In order to establish an AKLIDES procedure for the detection of MuSK-specific autoantibodies (anti-MuSK), we developed a recombinant HEp-2 cell clone expressing the human MuSK cDNA. Here we show at the mRNA and protein level that the cell clone HEp-2 M4 stably expresses human MuSK. We provide evidence for a localization of MuSK at the cell membrane. Using cell clone HEp-2 M4 on the AKLIDES system, we investigated 34 patient sera that were previously tested anti-MuSK positive by radioimmunoassay as positive controls. As negative controls, we tested 29 acetylcholine receptor-positive but MuSK-negative patient sera, 30 amytrophic lateral sclerosis (ALS) patient sera and 45 blood donors. HEp-2 M4 cells revealed a high specificity for the detection of MuSK autoantibodies from 25 patient sera assessed by a specific pattern on HEp-2 M4 cells. By using appropriate cell culture additives, the fraction of cells stained positive with anti-MuSK containing sera can be increased from 2–16% to 10–48%, depending on the serum. In conclusion, we provide data showing that the novel recombinant cell line HEp-2 M4 can be used to screen for anti-MuSK with the automatic AKLIDES system.

Bacterial production and purification of immunoreactive paraneoplastic neurological syndrome autoantigen Ma2

“…Albeit our data with a restricted number of patient sera indicate that purified recombinant Ma2 could be used for sensitive and specific autoantibody detection in PNS patients, further studies with increased sample numbers are required. In addition, we will compare the performance of anti-Ma2 dot blot immunoassays with cell-based assays such as previously developed for diagnostics of Myasthenia gravis [21][22][23].…”

The CytoBead assay – a novel approach of multiparametric autoantibody analysis in the diagnostics of systemic autoimmune diseases

“…In addition, granulocytes can be used as a substrate for ANCA detection, and Crithidia luciliae as a specific target for anti-dsDNA-AAb [11,20,39,40] . For the detection of organ-specific AAb, primate or rat tissue sections from the esophagus, liver, stomach or kidney as well as recombinant autoantigens expressed in specific cell lines are used [41,42] .…”

Der CytoBead-Assay – Eine neue Möglichkeit der multiparametrischen Autoantikörperanalytik bei systemischen Autoimmunerkrankungen

“…Darüber hinaus können Granulozyten als Substrat für den ANCA-Nachweis und Crithidia luciliae als spezifischer anti-dsDNA-AAK Test genutzt werden [11,20,39,40]. Für den Nachweis von organspezifischen AAK werden Gewebeschnitte von Ratten oder Affen aus Speiseröhre, Leber, Magen oder Niere verwendet als auch rekombinant in Zellen exprimierte Autoantigene [41,42].…”