J Clin Neurol.  2017 Apr;13(2):175-180. 10.3988/jcn.2017.13.2.175.

Large-Scale in-House Cell-Based Assay for Evaluating the Serostatus in Patients with Neuromyelitis Optica Spectrum Disorder Based on New Diagnostic Criteria

Affiliations
  • 1Department of Neurology, Research Institute and Hospital of National Cancer Center, Goyang, Korea. hojinkim@ncc.re.kr
  • 2Division of Translational and Clinical Research II, Research institute, National Cancer Center, Goyang, Korea.
  • 3Department of Neurology, Sungkyunkwan University School of Medicine, Seoul, Korea.
  • 4Department of Biochemistry and Molecular Biology, and Department of Biomedical Sciences, Seoul National University College of Medicine, Seoul, Korea.

Abstract

BACKGROUND AND PURPOSE
The detection of aquaporin 4-IgG (AQP4-IgG) is now a critical diagnostic criterion for neuromyelitis optica spectrum disorder (NMOSD). To evaluate the serostatus of NMOSD patients based on the 2015 new diagnostic criteria using a new in-house cell-based assay (CBA).
METHODS
We generated a stable cell line using internal ribosome entry site-containing bicistronic vectors, which allow the simultaneous expression of two proteins (AQP4 and green fluorescent protein) separately from the same RNA transcript. We performed in-house CBA using serum from 386 patients: 178 NMOSD patients diagnosed according to the new diagnostic criteria without AQP4-IgG, 63 high risk NMOSD patients presenting 1 of the 6 core clinical characteristics of NMOSD but not fulfilling dissemination in space, and 145 patients with other neurological diseases, including 66 with multiple sclerosis. The serostatus of 111 definite and high risk NMOSD patients were also tested using a commercial CBA kit with identical serum to evaluate the correlation between the 2 methods. All assays were performed by two independent and blinded investigators.
RESULTS
Our in-house assay yielded a specificity of 100% and sensitivities of 80% (142 of 178) and 76% (48 of 63) when detecting definite- and high risk NMOSD patients, respectively. The comparison with the commercial CBA kit revealed a correlation for 102 of the 111 patients: no correlation was present in 7 patients who were seronegative using the commercial method but seropositive using the in-house method, and in 2 patients who were seropositive using the commercial method but seronegative using the in-house method.
CONCLUSIONS
These results demonstrate that our in-house CBA is a highly specific and sensitive method for detecting AQP4-IgG in NMOSD patients.

Keyword

neuromyelitis optica spectrum disorder; aquaporin 4; aquaporin 4-IgG; cell-based immunofluorescence assay

MeSH Terms

Aquaporin 4
Cell Line
Humans
Methods
Multiple Sclerosis
Neuromyelitis Optica*
Research Personnel
Ribosomes
RNA
Sensitivity and Specificity
Aquaporin 4
RNA

Figure

  • Fig. 1 AQP4 overexpression in HEK293 cells using the AQP4-IRES2-EGFP vector. Western blot data confirming the successful transfection of AQP4. Lanes containing either untransfected HEK293 cells or HEK293 cells transfected with the IRES2-EGFP vector only do not show any bands. The lane containing lysate of HEK293 cells transfected with the M23-AQP4 gene shows a band at ~30 kDa. AQP4: aquaporin 4, EGFP: enhanced green fluorescent protein, HEK293: human embryonic kidney 293, IRES: internal ribosome entry site, N/C: untransfected cells, Vector: transfected IRES2-eGFP vector only.

  • Fig. 2 In-house CBA to detect AQP4-IgG in patient sera. Sera from healthy subjects (A-D), MS patients (E-H), and NMOSD patients (I-L) were added to AQP4-GFP-transfected HEK293 cells. HEK293 cells transfected with AQP4-GFP show green fluorescence in the cytosol under fluorescence microscopy (B, F, and J). When the patient serum contained AQP4-IgG, red fluorescence was detected on the HEK293 cell membrane (K) due to binding of AQ4-IgG to AQP4, which is expressed on the membrane of HEK293 cells. AQP4-IgG: aquaporin 4-IgG, CBA: cell based assay, GFP: green fluorescent protein, HEK293: human embryonic kidney 293, MS: multiple sclerosis, NMOSD: neuromyelitis optica spectrum disorder.


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