Development of a new immunoanalytical fluorescence-based technology for autoantibody detection in Myasthenia Gravis patients. Comparison of different antigen sources and characterization of reference sera

MANUELLI PN; GONZÁLEZ MAGLIO DH; AGUIRRE F; VILLA A; LEONI J; BARRANTES FJ; PAZ ML

Congreso; LXIV Reunión Anual Sociedad Argentina de Inmunología; 2016

Myasthenia Gravis (MG) is an autoimmune disease mediated by pathogenic autoantibodies (ACRA) directed against the nicotinic acetylcholine receptor (nAChR). The radioimmunoassay (RIA), an expensive and environmentally harmful method, is the current reference assay to detect ACRA, the serological markers of the disease. We aim to develop and validate a new methodology for ACRA measurement by flow cytometry, simple and eco-friendly, using polystyrene microbeads, nAChR, and fluorescent probes.Various sources of nAChR were compared: RD human muscle cells, bovine muscle (bm) and Torpedo marmorata electric organ (Tm). Immunofluorescence (IF) was not sufficiently sensitive to detect antigens in crude extracts. Antigen purified from bm extract by anion exchange chromatography and affinity chromatography-purified Tm nAChR were subsequently used for the IF detection. We also characterized different ACRA + sera from MG patients (n=5, A to E), confirming their reactivity by RIA.The new analytical technique to assay the antigen-antibody interaction relied on coating the surface of 4 µm polystyrene beads with purified nAChRs from Tm or bm (70 μg/100 cm2). Coating efficiency was verified by fluorescent microscopy. Flow cytometry was subsequently performed to measure the MFI (mean fluorescent intensity) of 1x105 antigen-coated-beads incubated with 1 μM α-BTX-AlexaFluor488 (BTX), human normal serum pool (HNS) or ACRA + serum (1:100) and FITC-labelled secondary antibodies (1:200).Fluorescence microscopy images showed that the beads were effectively coated with nAChR, as the receptor could be recognized by fluorescent-labelled α-BTX, a highly specific neurotoxin for the nAChR. Flow cytometry analyses showed positive results for both types of coated beads. For Tm, MFI: autofluorescence vs BTX (p