title: 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.
authors.affiliation: Faculty of Science, Brandenburg University of Technology Cottbus-Senftenberg, Senftenberg, Germany ; Institute of Immunology, Technical University Dresden, Dresden, Germany.
authors.forename: Sandra
authors.initials: S
authors.lastname: George
authors.affiliation: Faculty of Science, Brandenburg University of Technology Cottbus-Senftenberg, Senftenberg, Germany.
authors.forename: Silvia
authors.initials: S
authors.lastname: Paulick
authors.affiliation: GA Generic Assays GmbH, Dahlewitz/Berlin, Germany.
authors.forename: Ilka
authors.initials: I
authors.lastname: Knütter
authors.affiliation: Institute of Immunology, Technical University Dresden, Dresden, Germany.
authors.forename: Nadja
authors.initials: N
authors.lastname: Röber
authors.affiliation: GA Generic Assays GmbH, Dahlewitz/Berlin, Germany.
authors.forename: Rico
authors.initials: R
authors.lastname: Hiemann
authors.affiliation: Faculty of Science, Brandenburg University of Technology Cottbus-Senftenberg, Senftenberg, Germany ; GA Generic Assays GmbH, Dahlewitz/Berlin, Germany.
authors.forename: Dirk
authors.initials: D
authors.lastname: Roggenbuck
authors.affiliation: Institute of Immunology, Technical University Dresden, Dresden, Germany.
authors.forename: Karsten
authors.initials: K
authors.lastname: Conrad
authors.affiliation: Faculty of Science, Brandenburg University of Technology Cottbus-Senftenberg, Senftenberg, Germany.
authors.forename: Jan-Heiner
authors.initials: JH
authors.lastname: Küpper
mesh_terms: D001323:Autoantibodies / Q000097:blood / Q000276:immunology; D001331:Automation; D002460:Cell Line; D049109:Cell Proliferation; D048430:Cell Shape; D005455:Fluorescent Antibody Technique; D006801:Humans; D009157:Myasthenia Gravis / Q000097:blood / Q000175:diagnosis* / Q000201:enzymology* / Q000276:immunology; D009928:Organ Specificity; D020794:Receptor Protein-Tyrosine Kinases / Q000378:metabolism*; D011950:Receptors, Cholinergic / Q000378:metabolism*
chemical_list: D001323:Autoantibodies; D011950:Receptors, Cholinergic; C095714:MUSK protein, human; D020794:Receptor Protein-Tyrosine Kinases