Antibody to aquaporin 4 in the diagnosis of neuromyelitis optica

Abstract

Background: Neuromyelitis optica (NMO) is a demyelinating disease of the central nervous system (CNS) of putative autoimmune aetiology. Early discrimination between multiple sclerosis (MS) and NMO is important, as optimum treatment for both diseases may differ considerably. Recently, using indirect immunofluorescence analysis, a new serum autoantibody (NMO-IgG) has been detected in NMO patients. The binding sites of this autoantibody were reported to colocalize with aquaporin 4 (AQP4) water channels. Thus we hypothesized that AQP4 antibodies in fact characterize NMO patients.

Methods and findings: Based on these observations we cloned human water channel AQP4, expressed the protein in a eukaryotic transcription/translation system, and employed the recombinant AQP4 to establish a new radioimmunoprecipitation assay (RIPA). Indeed, application of this RIPA showed that antibodies against AQP4 exist in the majority of patients with NMO (n = 37; 21 positive) as well as in patients with isolated longitudinally extensive transverse myelitis (n = 6; six positive), corresponding to a sensitivity of 62.8% and a specificity of 98.3%. By contrast, AQP4 antibodies were virtually absent in 291 other participants, which included patients with MS (n = 144; four positive), patients with other inflammatory and noninflammatory neurological diseases (n = 73; one positive), patients with systemic autoimmune diseases (n = 45; 0 positive), and healthy participants (n = 29; 0 positive).

Conclusions: In the largest series reported so far to our knowledge, we quantified AQP4 antibodies in patients with NMO versus various other diseases, and showed that the aquaporin 4 water channel is a target antigen in a majority of patients with NMO. The newly developed assay represents a highly specific, observer-independent, and easily reproducible detection method facilitating clinically relevant discrimination between NMO, MS, and other inflammatory diseases.

Figure 1. Characteristics of the Radioimmunoprecipitation Assay

(A) Titration curve to assess the optimal serum volumes. Five samples are plotted: three positive (two NMO, one LETM) and two negative (one MS, one healthy control [HC]). While positive sera bound up to 1,800 cpm of ³⁵S-AQP4 with maximum binding at 2.5–5 μl, negative samples showed no more than 300–400 cpm of precipitation at all serum volumes. Values represent mean cpm of five independent measurements (error bars: SD). (B) Immunoprecipitation at different concentrations of ³⁵S-AQP4 (input cpm ranging from 5,000 to 40,000) of a positive serum (NMO), a negative serum (HC), and the rabbit serum rbAQP4 (5 μl per sample).

Figure 2. Antibody Ratios Found in Patients with NMO and in Patients with Relevant Differential Diagnoses or Healthy Controls

Each individual value represents the result from a single serum sample tested twice with the AQP4 RIPA. Black horizontal bars represent mean. The dashed horizontal line indicates the cutoff at an antibody ratio of 11. Diagnostic categories: NMO, neuromyelitis optica; LETM longitudinally extensive transverse myelitis; MS/ON+, multiple sclerosis with optic neuritis; MS/ON−, multiple sclerosis without optic neuritis; ON, optic neuritis; MYL, myelitis extending over fewer than three vertebral segments on MRI; OIND, other inflammatory neurological disorders and systemic disorders with neurological involvement (e.g., neuroborreliosis, chronic inflammatory demyelinating polyneuropathy; for exact classification see legend for Table 1); OND, other noninflammatory neurological diseases (e.g., carpal tunnel syndrome); RD, rheumatological diseases without neurological involvement (for exact classification see legend for Table 1); HC, healthy controls.