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

Abstract

BackgroundCell-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.MethodsSera 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.ResultsMicroscopic 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.ConclusionsOur 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.

Predictive Value of Serum Antibodies and Point Mutations of AQP4, AQP1 and MOG in A Cohort of Spanish Patients with Neuromyelitis Optica Spectrum Disorders

“…Our results demonstrate that anti-AQP4 antibodies were only detected in the serum of NMOsd patients and were never detected in the serum of patients from any other disease group analyzed or in the control subjects. Specifically, similar detection sensitivity was obtained with the commercial CBA as with the in-house developed [23] detection system. The serum of twelve out of the 18 (67%) NMOsd patients was positive for these antibodies using the commercial CBA, compared with 11 (61%) who tested positive using the protocol developed in our laboratory [23].…”

“…Specifically, similar detection sensitivity was obtained with the commercial CBA as with the in-house developed [23] detection system. The serum of twelve out of the 18 (67%) NMOsd patients was positive for these antibodies using the commercial CBA, compared with 11 (61%) who tested positive using the protocol developed in our laboratory [23]. A much lower sensitivity for detection of AQP4 antibodies was obtained with the commercial ELISA kit, with which the serum of only 7 (39%) patients tested positive.…”

“…A summary of these results, together with the demographic and clinical characteristics of the patients is presented in Table 1. First, we began by comparing the results obtained with the CBA we developed some years ago [23] for detecting anti-AQP4 antibodies in serum, with the results obtained using the commercially-available CBA from Euroimmun. Our results demonstrate that anti-AQP4 antibodies were only detected in the serum of NMOsd patients and were never detected in the serum of patients from any other disease group analyzed or in the control subjects.…”

“…A CBA method similar to the one we developed previously [23] for the detection of anti-AQP4 IgG was used here for the detection of anti-AQP1 IgG. Figure 3 shows the results from these immunofluorescent assays, presenting a side-by-side comparison of the results obtained when AQP1 transfected cells were exposed to a commercial antibody against AQP1 (positive control) or when serum from an NMOsd patient was used as the primary antibody.…”

“…In this study we examined the presence of serum IgGs against AQP4, AQP1 and MOG using two cell-based assay (CBA) methods, one previously developed by us [23,24] and the commercial Euroimmun ® Assay widely used abroad for diagnostic purposes. ELISA determinations for the three antibodies were also performed by either commercial, as for AQP4 and MOG, or by in-house developed methods in the case of AQP1-IgG detection.…”