IDEHU   05542
INSTITUTO DE ESTUDIOS DE LA INMUNIDAD HUMORAL PROF. RICARDO A. MARGNI
Unidad Ejecutora - UE
congresos y reuniones científicas
Título:
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.
Autor/es:
GONZALEZ MAGLIO, D.; LEONI, J.; MANUELLI, P; VILLA, A.; PAZ, M.L.; AGUIRRE, F.; BARRANTES, F.
Lugar:
Mar del Plata
Reunión:
Congreso; LXIV REUNIÓN ANUAL DE LA SOCIEDAD ARGENTINA DE INMUNOLOGÍA (SAI); 2016
Resumen:
Myasthenia Gravis (MG) is an autoimmune disease mediatedby pathogenic autoantibodies (ACRA) directed against the nicotinicacetylcholine receptor (nAChR). The radioimmunoassay (RIA), an expensive andenvironmentally harmful method, is the current reference assay for ACRA, the serological markers of the disease.We aim to develop and validate a new methodology to detect ACRA by flow cytometry, a simple andeco-friendly technique, using polystyrene microbeads, nAChR, and fluorescentprobes.Various sources of nAChR were compared: RD human musclecells, bovine muscle (bm) and Torpedomarmorata electric organ (Tm). Immunofluorescence(IF) was not sensitive enough to detect antigens in crude extracts. Antigen purifiedfrom bm extract by anion exchange chromatography and affinitychromatography-purified Tm nAChR were subsequently used for the IF detection. Wealso characterized different ACRA + sera from MGpatients (n=5, A to E), their reactivity confirmed by RIA.Thenew analytical technique to assay the antigen-antibody interaction relied oncoating the surface of 4 µm polystyrene microbeads with purified nAChRs from Tm or bm (70 μg/100 cm2). Coating efficiency wasverified by fluorescent microscopy and flowcytometry was subsequently performed registering the MFI (meanfluorescent intensity) of 1x105 coated-beadsincubated 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 beadswere effectively coated with nAChR, as the receptor could be recognized byfluorescent-labelled α-BTX, which is a neurotoxin highly specific for the nAChR.Flow cytometry analyses showed positive results forboth types of coated beads. For Tm, MFI: autofluorescence vs BTX (p<0.05), HNSvs. serum C, D, E (p<0.001) A, B (p<0.05). For bm, MFI: autofluorescencevs BTX (p<0.01), HNS vs. serum A, C, D (p<0.001) E (p<0.01) B (p<0.05).