Cell- free fluorescent microbead-based autoantibody diagnostic test for Myasthenia Gravis

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

Otro; Colloquium of the Alexander von Humboldt Foundation; 2018

Alexander von Humboldt Foundation

The nicotinic acetylcholine receptor (nAChR) is a pentameric receptor with two isoforms: the neuronal and the muscle-type. Myasthenia gravis (MG) is an autoimmune disease with patients who display autoantibodies (ACRA) against muscle nAChR. Here we present the initial validation of a new fluorescence methodology to detect ACRA using nAChR immobilized onto polystyrene microbeads.The first step of the procedure involved coating the surface of 4 µm beads with nAChR purified from T. marmorata (70 μg/100 cm2). Coating efficiency was tested by fluorescent microscopy: 1x104 coated-beads were immobilized onto poly-L-lysine coated glass coverslips and incubated with α-BTX-AlexaFluor488or -AlexaFluor555 (1 μM), or two anti-nAChR primary antibodies: anti-α1 (1:400) mAb35 and serum from a clinically-diagnosed MG patient (1:100), followed by FITC-labeled secondary antibodies. Fluorescence microscopy images showed that beads were effectively coated with nAChR, as revealed by the two fluorescent methodologies. To verify that the antigen-coated beads were also labeled in solution, samples containing 1.5x105 beads were submitted to flow cytometry and their mean fluorescent intensity (MIF) recorded. Flow cytometry analyses confirmed the successful coating of the beads (MFI: autofluorescence=1.9 vs BTX=6.7; p