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

BMC Neurology

Miriam Echevarría⁴  Juan José Toledo-Aral³  Javier Villadiego¹  Ana Serna¹  Reposo Ramírez-Lorca¹  José Luis Casado Chocán²  Antonio José Uclés Sánchez²  María Díaz Sánchez²  Ismael Sánchez Gomar¹ 

[1] Instituto de Biomedicina de Sevilla (IBiS), Hospital Universitario Virgen del Rocío/CSIC/Universidad de Sevilla, Av. Manuel Siurot s/n, Seville 41013, Spain;Unidad de Gestión Clínica de Neurociencias, Servicio de Neurología del Hospital Universitario Virgen del Rocío, Seville, Spain;Centro de Investigación Biomédica en Red sobre Enfermedades Neurodegenerativas (CIBERNED), Instituto de Salud Carlos III, Madrid, Spain;Centro de Investigación Biomédica en Red sobre Enfermedades Respiratorias (CIBERES), Instituto de Salud Carlos III, Madrid, Spain

关键词: Immunohistochemistry;    Natalizumab;    HEK cells;    NMO-IgG;    AQP4-EGFP;   

Others  :  848406
DOI  :  10.1186/1471-2377-14-139

 received in 2014-01-27, accepted in 2014-06-25,  发布年份 2014

【 摘 要 】

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.

【 授权许可】

   
2014 Sánchez Gomar et al.; licensee BioMed Central Ltd.

【 图 表 】

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