Immobilized nicotinic acetylcholine receptor as a tool for the detection of autoantibodies in the development of a new immunoanalytical fluorescence-based technology.

PAZ, M.L.; GONZALEZ MAGLIO, D.; BARRANTES, F.; AGUIRRE, F.; LEONI, J.; MANUELLI, P.; VILLA, A.

Buenos Aires

Congreso; 2nd FALAN Congress; 2016

The nicotinic acetylcholine receptor (nAChR) is a pentamericreceptor with twoisoforms: the neuronal and the muscle-type. Myasthenia gravis (MG) is an autoimmune disease with patientswho displayautoantibodies (ACRA) against muscle nAChR. Here we present the initial validation of a new fluorescencemethodology to detect ACRA using nAChR immobilized onto polystyrene microbeads.Thefirst step of the procedure involved coating the surfaceof 4 µm beads with nAChR purified from T. marmorata (70 μg/100 cm2).Coating efficiency was tested by fluorescent microscopy: 1x104 coated-beadswere immobilized onto poly-L-lysine coated glass coverslips and incubated withα-BTX-AlexaFluor488or -AlexaFluor555 (1 μM), or two anti-nAChRprimary antibodies: anti-α1 (1:400) mAb35 and serum from aclinically-diagnosed MG patient (1:100), followed by FITC-labeled secondary antibodies.Fluorescence microscopy images showed thatbeads were effectively coated with nAChR, as revealed by the two fluorescentmethodologies. To verify that the antigen-coated beads were also labeledin solution, samples containing 1.5x105 beads were submitted to flowcytometry and their mean fluorescent intensity (MIF) recorded. Flowcytometry analyses confirmed the successful coating of the beads (MFI: autofluorescence=1.9vs BTX=6.7; p<0.05 and vs. mAb35=27.6; p<0.001), and hence the possibleapplication of the technique for the detection of ACRA (human normal serum =10.0vs. MG serum=31.1; p<0.001)