Human Serum Anti-aquaporin-4 Immunoglobulin G Detection by Cell-based Assay

Summary

Cell-based assay is a widely used method to detect serum anti-aquaporin-4 immunoglobulin G. This method could be applied to clinical diagnosis and scientific researches of neuromyelitis optical spectrum disorders.

Abstract

Anti-aquaporin-4 (AQP4) immunoglobulin G (IgG) is the core diagnostic biomarker for neuromyelitis optica spectrum disorders (NMOSD). The cell-based assay (CBA) is a widely used method to detect anti-AQP4 IgG in human serum with high sensitivity and specificity. Briefly, serum anti-AQP4 IgG is captured by AQP4-transfected cell that is fixed on the biochip then detected by a fluorescein-labelled secondary antibody. Fluorescence microscopy is utilized to visualize the fluorescence, and the intensity of fluorescence is evaluated by at least two experienced clinicians. A final diagnosis of NMOSD can be made based on the combination of anti-AQP4 IgG detection results, clinical manifestations, and neuroradiological findings. According to previous studies, CBA is more sensitive and specific than other anti-AQP4 IgG detection methods, and it can be applied to both clinical diagnosis and studies of NMOSD. The method has limitations; for example, an international scale to evaluate serum anti-AQP4 IgG titers is still lacking. Here, a detailed protocol for human serum anti-AQP4 IgG detection using CBA is described.

Introduction

Serum AQP4 IgG is a core diagnostic biomarker for neuromyelitis optica spectrum disorders (NMOSD)¹. The cell-based assay (CBA) is a widely used anti-AQP4 IgG detection method with high sensitivity and specificity. Here, a detailed protocol for CBA is introduced.

AQP4, a water channel protein, has six membrane-spanning units and two helical domains surrounding one aqueous pore². Anti-AQP4 IgG is involved in the pathogenesis of NMOSD through binding to its target AQP4, which is mainly located on the endfeet of astrocytes³. It has been shown that anti-AQP4 IgG is positive ....

Protocol

This procedure was approved by the Ethics Committee of the First Hospital of Jilin University and was performed on approximately 1,500 subjects.

1. Patient Enrollment and Blood Sample Collection

1. Apply the laboratory detection of serum anti-AQP4 IgG to clinic patients with the chief complaints and symptoms listed below. Perform physical examinations as well.
   
   Optic neuritis: Patients suffer with visual deficits, such as loss of visual fields and reduction of visual acuity.
   Acute myelitis: Patients may present with paralysis, sensory deficits and autonomic dysfunction.
   Area postrema syndrome: Patients m....

Results

Using the procedure described here, specific anti-AQP4 IgG in serum is detectable. During the procedure, pre-diluted samples, a positive control, and a negative control were added to the reaction fields, which contain transfected and untransfected areas (Figure 2). Fluorescence of the negative control in a transfected area mainly indicated the unspecific binding of secondary antibody to the transfected cells on biochips (Figure 3.......

Discussion

We have described a widely accessible method to detect anti-AQP4 IgG in human serum. Anti-AQP4 IgG is closely related to NMOSD, and establishing a reliable anti-AQP4 IgG detection method is crucial for the clinical diagnosis of NMOSD. First, anti-AQP4 IgG is specific for NMOSD. Multiple sclerosis is also an immune-mediated disease of the central nervous system and shares many similarities with NMOSD¹². However, anti-AQP4 IgG is only positive in NMOSD¹³. Second, anti-AQP4 Ig.......

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
Anti-aquaporin-4 IIFT	Euroimmun	FA 1128-2005-50	Contains biochip slides coated with AQP4-M1 transfected and untransfected EU 90 cells, fluorescein-labelled anti-human IgG, anti-AQP4 antibody as positive control, antibody negative sample, salt for PBS pH 7.2, Tween 20 and embedding medium.
CellSens Dimension	OLYMPUS	N/A	photograph software
Gel & clot activator tube	Improve medical	623040202	From a local Chinese company