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Clinical Usefulness of Cell-based Indirect Immunofluorescence Assay for the Detection of Aquaporin-4 Antibodies in Neuromyelitis Optica Spectrum Disorder

Clinical Usefulness of Cell-based Indirect Immunofluorescence Assay for the Detection of Aquaporin-4 Antibodies in Neuromyelitis Optica Spectrum Disorder

Eun-suk Kang, M.D.,1 Ju-Hong Min, M.D.,2 Kwang Ho Lee, M.D.,2 and Byoung Joon Kim, M.D.2

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The presence of antibodies to aquaporin-4 (AQP4) has been identified as a key characteristic of neuromyelitis optica spectrum disorder (NMOSD), an autoimmune inflammatory demyelinating central nervous system (CNS) disorder. We evaluated the performance of a cell-based indirect immunofluorescence assay (CIIFA) for detecting AQP4 antibodies using antigen prepared with a recombinant AQP4 peptide transfection technique and assessed the usefulness of CIIFA for diagnosis of NMOSD in routine clinical practice.


Forty-six serum samples from 36 patients as a comparison set and another 101 patients enrolled consecutively from a neurology clinic were included. CIIFA and fluorescence immunoprecipitation assays (FIPA) were performed. CIIFA was performed at 2 different institutions for comparison purposes.


CIIFA and FIPA sensitivity in the comparison set was 86% and 79% in neuromyelitis optica (NMO) patients and 55% and 36% in high-risk NMO patients, respectively. The semiquantitative titer measured by CIIFA correlated well with the arbitrary unit (fluorescence units [FU]) derived from FIPA (r=0.66). Titers measured by CIIFA and FIPA were elevated in NMO patients compared to high-risk NMO patients (1:240 vs. 1:180 and 8,390 vs. 4,059 FU, respectively). The frequency of AQP4 antibody detection by CIIFA in 101 consecutively enrolled patients was 100% in NMO and 23% in high-risk NMO patients, while only 4.6% in control patients, including those with multiple sclerosis.


Detection of AQP4 antibodies by CIIFA provides sensitive and highly specific diagnostic information for NMO and high-risk NMO patients, which can be used to differentiate these conditions from other demyelinating CNS diseases.

Keywords: Neuromyelitis optica, Aquaporin 4, Indirect immunofluorescence assay, Immunoprecipitation assay

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Neuromyelitis optica (NMO; also known as Devic syndrome) is a chronic inflammatory demyelinating disorder of the central nervous system (CNS), which was first described in the late 19th century by E. Devic and others [1]. It preferentially affects the optic nerves and the spinal cord, thus frequently manifests as recurrent optic neuritis (RON) and longitudinally extensive transverse myelitis (LETM) [2, 3]. The condition which is considered to be a type of neuromyelitis optica spectrum disorder (NMOSD) includes NMO and an array of high-risk NMO disorders such as Asian optic-spinal multiple sclerosis (OSMS), recurrent myelitis associated with longitudinal extensive spinal cord lesions, recurrent isolated or simultaneous bilateral optic neuritis (RON/BON), and optic neuritis (ON) or myelitis/LETM in the context of certain organ-specific and non-organ-specific autoimmune diseases or with brain lesions typically observed in cases of NMO [4, 5]. Due to its relapsing course with rare spontaneous remission, the accumulation of irreversible deficits and rapid progression of NMO often renders patients severely disabled. These characteristics highlight the need to distinguish NMO from other demyelinating CNS conditions as early as possible; however, it is often difficult to differentiate inflammatory demyelinating CNS disorders that have differing etiologies but similar clinical presentations and cerebrospinal fluid (CSF) and magnetic resonance imaging (MRI) findings. In particular, both NMO and multiple sclerosis (MS) have a relapsing-remitting course in the majority of cases, thus NMO has been typically considered to be a localized form of MS due to the lack of an NMO-specific laboratory test [3, 5, 6].

However, recent reports suggest that NMO is a distinct disease entity with a fundamentally different pathogenic mechanism than that of MS or other demyelinating diseases. An NMO-specific IgG, designated NMO-IgG, and antibodies to aquaporin-4 (AQP4) that serve as relevant antigens of NMO-IgG, are detectable in 60-90% of patients with NMO but are virtually absent in patients with MS and other inflammatory and non-inflammatory neurological diseases [7-10]. This evidence of autoantibody-mediated NMO pathogenesis enabled the development of therapeutic strategies targeted to the humoral arm of the immune system. Treatments based on B cell- and antibody-depleting strategies such as rituximab administration and plasmapheresis have been effective in combination with conventional immunosuppressive treatment, in contrast to the inefficacy of these approaches for treating MS and other diseases associated with inflammatory lesions [11-13].

In 2004, Lennon et al. demonstrated that NMO-IgG was capable of specifically binding to CNS microvessels, pia mater, subpia mater, and Virchow-Robin spaces in rodent brain tissue by using an indirect immunofluorescence method [7]. The target antigen of this autoantibody was quickly identified to be AQP4, the most abundant water channel in the CNS [8, 14]; subsequently, the immunopathogenic role of AQP4 antibodies in NMO was reported [14]. The presence of AQP4 antibodies has been verified by various methods, including visualization of AQP4-antibody immunoprecipitates by western blotting, fluorescence immunoprecipitation assay (FIPA), ELISA, and cell-based indirect immunofluorescence assay (CIIFA) [14-18]. The sensitivity and specificity of these tests were variable in approximately 60-90% and 90-100% of cases, respectively, depending on the method used. With the full native conformation of AQP4 antigen, the antibody is known to enhance the sensitivity and specificity of its binding [15]. Therefore, FIPA and CIIFA studies using human embryonic kidney (HEK) cells transfected with AQP4 as the source of antigen resulted in improved performance relative to other assays for the diagnosis of NMO and related disorders, thus differentiating them from other demyelinating disorders [15].

Recently, commercially available CIIFA was introduced by EUROIMMUN AG (Lubeck, Germany), which utilizes fixed, AQP4-transfected HEK cells on slides as an antigenic substrate. The aim of this study was to evaluate the diagnostic performance of CIIFA compared to FIPA for the detection of AQP4 antibodies and to assess the usefulness of CIIFA for the diagnosis of NMO and high-risk NMO in routine clinical practice using a commercially available CIIFA kit.

Continued at resource.

Read More: Clinical Usefulness of Cell-based Indirect Immunofluorescence Assay for the Detection of Aquaporin-4 Antibodies in Neuromyelitis Optica Spectrum Disorder

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