An immunoassay that distinguishes real neuromyelitis optica signals from a labeling detected in patients receiving natalizumab
© Sánchez Gomar et al.; licensee BioMed Central Ltd. 2014
Received: 27 January 2014
Accepted: 25 June 2014
Published: 1 July 2014
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.
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.
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.
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.
KeywordsAQP4-EGFP NMO-IgG HEK cells Natalizumab Immunohistochemistry
Neuromyelitis optica (NMO) is an inflammatory demyelinating disease of the central nervous system (CNS) that primarily affects the optic nerves and spinal cord [1, 2]. Although for long time it was considered a variant of multiple sclerosis (MS), new pathological and serological tests have helped to identify the disorder as a different disease . Lennon and colleagues  provided the main evidence for this distinction when they discovered specific immunoglobulins in the serum of NMO patients (NMO-IgG) that were usually absent in classical forms of MS. The antigen recognized for NMO-IgG is aquaporin-4 (AQP4), the most abundantly expressed aquaporin in the CNS [4–8], highly localized in astrocyte membranes facing blood vessel capillaries and in ependymal cells that line the cerebrospinal fluid-filled ventricles and layer of the meninges surrounding the brain and spinal cord . Recent studies have found convincing evidence of a direct involvement of AQP4 autoantibodies in the development of NMO disease [5, 9–11]. Magnetic resonance imaging (MRI) in NMO patients indicates that most affected areas coincide with those with higher AQP4 expression . Histopathological lesions observed in the CNS on postmortem show disappearance of AQP4 and deposition of immunoglobulins and products of complement activation in a vasculocentric pattern that coincides with the normal distribution of AQP4 [5, 12, 13]. Protocols commonly used for NMO diagnosis include MRI studies that are able to identify longitudinally extensive spinal cord lesions extending over three vertebral segments [14, 15], with optic nerve involvement and brain lesions in areas of high AQP4 expression . However, the discovery that anti-AQP4 IgG antibodies were present in serum of patients with NMO  has revolutionized the diagnosis criteria for this disease and allows more specific treatments that may help reduce the frequency of new relapses.
At least five different methods have been described for detection of anti-AQP4 antibodies in serum of patients [16–25]. Some approaches involve incubation of the serum with mouse brain slices and the signal, well fluorescent or peroxidase, comes from a secondary antibody that recognizes the AQP4 IgG bound to AQP4 [3, 4, 16, 20]. Other assays allow the detection of antibodies by incubation of serum with extracts in which AQP4 is labeled with either radioactive or fluorescent tags prior to the precipitation step [20, 21]; and also enzyme-linked immunosorbent assays are being used to detect AQP4 antibodies in patient serum [23, 24]. Finally, a cell-based assay initially described as proof of the identification of AQP4 as specific antigen target in NMO positive serum, is nowadays extensively used for routine diagnosis [21, 25].
In the present work, we adapted this last method, developing a protocol that combines expression of an AQP4-enhanced green fluorescent protein (EGFP) with the use of a red fluorescent goat anti-human secondary antibody. By this double labeling, we obtained a method with extremely high sensitivity and specificity for identifying NMO positive patients and that additionally enables quantitative comparison of antibody levels in sera samples tested at the same time. Moreover, the high signal specificity of the method we describe here allows false signals to be distinguished from those produced by sera from patients treated with natalizumab.
Subjects and serum recollection
Demographic and clinical variables of patients
Number of patients
Mean age at inclusion +/- SD (range)
Group 1: NMO
55.02 +/- 9.80
Group 2: MS
- Relapsing-remitting MS
- Secondary progressive MS
- Primary progressive MS
Group 3: idiopathic ON
35.01 +/- 11.39
-Recurrent idiopathic ON
Group 4: idiopathic myelitis
46.30 +/- 14.27
> 3 vertebral segments
≤ 3 vertebral segments
-Recurrent idiopathic myelitis
Group 5: other neurological disorders
50.45 +/- 6.11
-Myelitis associated with lupus
-ON associated with Sjögren syndrome
-Multifocal motor neuropathy
-Ischemic optic neuropathy
Group 6: healthy controls
34.45 +/- 8.31
The patients (Groups 1-5) and the healthy controls (Group 6) were recruited by the Service of Neurology at the Virgen del Rocío University Hospital and the IBiS, respectively. For all participants, written consent was obtained before their inclusion in the study, and demographic and clinical variables were recorded including gender, age at inclusion in the study, clinical diagnosis and treatments received (Table 1). The study counted with the approval of The Ethics Committee of The University Hospital Virgen del Rocío (HUVR), with the registration number 14/2010.
Plasmid construction, cell culture and cell transfection
Primers for PCR amplification of full-length human aquaporin 4 (hAQP4)
Forward : 5′-ACTCCTCGAGGGCGGTGGGGTAAGTGTGGAC -3′
Reverse: 5′-ACTCCCCGGGAATGGGTGGAAGGAAATCTGA -3′
Immunofluorescence assay and signal quantification
RT-qPCR amplification of the integrin α4 chain
Primers for qRT-PCR analysis of integrin α4
Forward primer sequence
Reverse primer sequence
5′- GGCAAGGAAGTTCCAGGTTACAT -3′
5′- ATGCTTCCTGTAATCACGTCAGAA -3′
Data are presented as mean ± standard error of the mean, and all statistical analyses were conducted using the IBM SPSS Statistics (IBM Corp., Armonk, NY), version 19.0. Data with a non-normal distribution were analyzed using analysis of variance (ANOVA) for nonparametric data, using the Kruskal-Wallis H or Mann-Whitney U tests for two or more than two groups, respectively.
Optimization of an immunofluorescence protocol for detection of anti-AQP4 antibodies
Comparative analysis of immunofluorescence assay using fresh and frozen cells
Positivity of serum from natalizumab-treated patients
Serological data for 167 subjects included in the study
Number of patients
False labeling pattern
Nowadays, the detection of AQP4 autoantibodies (NMO-IgG), as a serum biomarker for NMO, is an essential test for a final diagnosis of this disease. Our initial goal was to develop a simple detection method for use in routine practice and we began by assessing results with immunoassay procedures such as Western blots and direct detection of AQP4 by immunohistochemical analysis. After many trials, only nonspecific results were obtained with both of these approaches, forcing us to explore alternatives and we started to work on a cell-based immunofluorescence assay using transfected cells with high expression of the human AQP4 M23 isoform.
The protocol we have developed and described herein consists of a cell-based double-label fluorescence immunoassay, using two fluorophores with different emission wavelengths: green, to directly visualize the expression of human AQP4 in HEK cells; and red to visualize the antihuman-IgG that binds to AQP4 antibodies in serum. The co-localization of the two fluorescence signals evidences the presence of NMO-IgG in serum and, therefore, represents a specific reaction that confirms the NMO positive diagnosis. Only 16 of all the individuals analyzed had autoantibodies for AQP4 in their serum and therefore had an NMO positive diagnosis. Notably, quantification of fluorescence signals obtained with sera from these 16 patients also revealed high levels of NMO-IgG, markedly higher than levels detected in any other serum sample analyzed. Accordingly, the diagnosis of patients based on serum immune response agreed with the diagnosis reached by neurological examination based on the current diagnostic criteria in 100% of cases .
While to our knowledge previously reported assays to detect anti-AQP4 antibodies in NMO rely either on microscopic observation of specific immune signals (visual fluorescence or colored precipitate), or on quantification of colorimetric, fluorescent or radioactive signals , the diagnostic assay we present takes into account both localization and magnitude of the fluorescent signal. Combining these features, we have obtained a diagnostic assay with high sensitivity and specificity that would likely reduce false positive results and would improve detection of NMO-IgG when present at low levels. Quantification of the fluorescence intensity indicates NMO-IgG positivity with fluorescence signals that were clearly stronger than those obtained with controls with this assay and the consistency in repeated measurements suggests that it is a reliable and reproducible method. So far, we have not investigated variations in antibodies levels over time, but this is a future application of our method and it is plausible to suppose that the results will have predictive value for disease progression and treatment response.
Results presented here also demonstrate that freezing HEK cells expressing AQP4-EGFP after permeabilization, ready to hybridize with serum as soon as sample arrive at the laboratory, can substantially shorten the standard cell-based fluorescent immunoassay protocol, to about 3 days, without losing sensitivity or specificity. This would accelerate diagnosis from serum samples and is an approach that could potentially be used to develop rapid kits for NMO diagnosis in hospital services where cell culture and cell biology facilities are not easily available.
In a clinical context, another important finding in the present study comes from experiments in which detailed microscopic observation revealed a subgroup of sera in which, after hybridization with AQP4 HEK-transfected cells, a small fluorescent signal was observed from every single cell regardless of whether there was cellular expression of the fluorescent AQP4-EGFP protein. Surprisingly, the sera producing this distinctive and confusing staining pattern, that could lead to misdiagnosis of patients as NMO positive, all came from patients that received natalizumab treatment within the 6 months prior to blood sample collection. Quantification of the fluorescence signals corroborated a positive immune reaction, but although higher than those detected with C- (NMO-IgG (-)), signals were clearly weaker than those obtained with positive control sera (NMO-IgG (+)).
To characterize the diagnostic assay, it was of interest to assess the immune fluorescence-staining pattern in sera obtained from patients who had taken natalizumab but stopped the treatment more than 6 months before the blood test. We found that the fluorescence-staining pattern in these samples was indistinguishable from that obtained with a C- serum, indicating that long periods of time (at least 6 months) allow sufficient reduction in circulating levels of natalizumab in serum for the false labeling pattern to disappear.
Hence, while HEK cells offer very convenient features, such as high transfection efficiency and levels of expression of heterologous human AQP4, a drawback of using these cells in NMO diagnostic assays is also evidenced in the present work: specifically, the expression of integrin proteins in their membrane seems to elicit the binding of natalizumab antibodies that, in turn, can be recognized by human anti-IgG antibodies and result in a false signal for NMO diagnosis. Exploring this cross reactivity of natalizumab in different cell lines would be important to further improve the cell-based double-label fluorescent immunoassay procedure presented in this work. Moreover, a similar response to the cross reactivity observed with natalizumab could also be produced by other immunotherapies, potentially leading to false positive diagnoses of NMO. In view of these results, we should also underline the secondary effects some immunotherapies might have by binding to as yet unknown targets in the human body.
In conclusion, the use of HEK cells expressing AQP4-EGFP that are stored frozen after the blocking step make it possible to obtain a final result within a few days of receiving serum samples, accelerating the diagnosis of NMO. A limitation was found in the use of HEK cells and may be explained by false signals from the binding of natalizumab antibodies to integrins present in HEK cell membrane that, in turn, can be recognized by human anti-IgG antibodies. Nevertheless, the cell-based double-label fluorescent immunoassay protocol we present here significantly increases the signal specificity and reduces the likelihood of false diagnoses of NMO, especially in patients treated with natalizumab.
Enhanced green fluorescent protein
Human embryonic kidney cells
Magnetic resonance imaging
Central nervous system
Reverse transcription quantitative polymerase chain reaction.
We are grateful to all patients included in this study. We thank Dr. A. Saiz (Hospital Clínic, Barcelona) for sending us serum samples that were crucial for setting up our NMO diagnosis assay. Grants from the Regional Ministries of Innovation, Science and Business (P08-CTS-03574) and of Health (PI0298-2010) of the Government of Andalusia, and from the Carlos III Institute of Health (Exp. PI12/01882 and PS09/0184) to ME and JJTA funded this work. We thank the Genzyme Foundation for awarding ME one of their 2012 fellowships for work on multiple sclerosis. Authors have no conflicts of interest to declare.
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