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Open Access Research article

An immunoassay that distinguishes real neuromyelitis optica signals from a labeling detected in patients receiving natalizumab

Ismael Sánchez Gomar1, María Díaz Sánchez2, Antonio José Uclés Sánchez2, José Luis Casado Chocán2, Reposo Ramírez-Lorca1, Ana Serna1, Javier Villadiego1, Juan José Toledo-Aral13 and Miriam Echevarría14*

Author Affiliations

1 Instituto de Biomedicina de Sevilla (IBiS), Hospital Universitario Virgen del Rocío/CSIC/Universidad de Sevilla, Av. Manuel Siurot s/n, Seville 41013, Spain

2 Unidad de Gestión Clínica de Neurociencias, Servicio de Neurología del Hospital Universitario Virgen del Rocío, Seville, Spain

3 Centro de Investigación Biomédica en Red sobre Enfermedades Neurodegenerativas (CIBERNED), Instituto de Salud Carlos III, Madrid, Spain

4 Centro de Investigación Biomédica en Red sobre Enfermedades Respiratorias (CIBERES), Instituto de Salud Carlos III, Madrid, Spain

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BMC Neurology 2014, 14:139  doi:10.1186/1471-2377-14-139

Published: 1 July 2014

Abstract

Background

Cell-based assays for neuromyelitis optica (NMO) diagnosis are the most sensitive and specific methods to detect anti-aquaporin 4 (AQP4) antibodies in serum, but some improvements in their quantitative and specificity capacities would be desirable. Thus the aim of the present work was to develop a sensitive quantitative method for detection of anti-AQP4 antibodies that allows clear diagnosis of NMO and distinction of false labeling produced by natalizumab treatment.

Methods

Sera from 167 individuals, patients diagnosed with NMO (16), multiple sclerosis (85), optic neuritis (24), idiopathic myelitis (21), or other neurological disorders (13) and healthy controls (8), were used as the primary antibody in an immunofluorescence assay on HEK cells transfected with the M23 isoform of human AQP4 fused with enhanced green fluorescent protein. Cells used were freshly transfected or stored frozen and then thawed just before adding the serum.

Results

Microscopic observation and fluorescence quantification produced similar results in fresh and frozen samples. Serum samples from patients diagnosed with NMO were 100% positive for anti-AQP4 antibodies, while all the other sera were negative. Using serum from patients treated with natalizumab, a small and unspecific fluorescent signal was produced from all HEK cells, regardless of AQP4 expression.

Conclusions

Our cell-based double-label fluorescence immunoassay protocol significantly increases the signal specificity and reduces false diagnosis of NMO patients, especially in those receiving natalizumab treatment. Frozen pretreated cells allow faster detection of anti-AQP4 antibodies.

Keywords:
AQP4-EGFP; NMO-IgG; HEK cells; Natalizumab; Immunohistochemistry