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 in approximately two-thirds of NMOSD patients⁴. In the most recent international diagnostic criteria for NMOSD, anti-AQP4 IgG is considered to be a core diagnostic biomarker⁵. In this regard, it is crucial to establish a reliable protocol to detect human serum anti-AQP4 IgG and facilitate clinical diagnosis of NMOSD.

Currently, various anti-AQP4 IgG detection methods are available, such as CBA, tissue-based assay, enzyme-linked immunosorbent assay, and flow cytometry^6,7. CBA employs EU90 cells, which are transfected with human AQP4, to capture anti-AQP4 IgG. The captured anti-AQP4 IgG is detected by fluorescent secondary antibodies and subsequently visualized by microscopy. Accumulating evidence has shown that CBA is more sensitive and specific than other anti-AQP4 IgG detection methods^6,7. According to a meta-analysis, the sensitivity and specificity of CBA were shown to be 76% and 99%, which were higher than tissue-based and enzyme-linked immunosorbent assays⁶. Furthermore, a multicenter comparison of diagnostic assays of anti-AQP4 IgG was conducted⁷. A total of 193 study subjects from 15 European diagnostic centers were enrolled⁷. Four different methods were utilized to detect serum anti-AQP4 IgG⁷. It was demonstrated that CBA was more sensitive and specific than the other methods⁷. As AQP4 is expressed as two major isoforms (AQP4-M1 and AQP4-M23), anti-AQP4 IgG capture cell is transfected with either AQP4-M1 or AQP4-M23. However, which type of capture cell is better remains controversial. One investigation has supported AQP4-M1 based CBA⁸, while others have indicated that AQP4-M23 based CBA is better^7,9,10. However, AQP4-M23-based CBA may yield false positive results due to unspecific IgG binding⁸. Jarius et al.¹¹ reported that there was no significant difference in anti-AQP4 IgG detection rates between AQP4-M1 and AQP4-M23-based CBAs.

In summary, serum anti-AQP4 IgG is a core biomarker for NMOSD. CBA has higher specificity and sensitivity than other anti-AQP4 IgG detection methods. It remains controversial whether AQP4-M1- or AQP4-M23-based CBA is better. In this article, a detailed protocol for AQP4-M1-based CBA is described, which can apply to clinical diagnosis and studies of NMOSD.

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 may have symptoms of unexplained hiccups, nausea or vomiting.
   Acute brainstem syndrome: Brainstem lesions could result in different symptoms and physical signs depending on the location of the lesions.
   Symptomatic narcolepsy or acute diencephalic clinical syndrome with NMOSD-typical diencephalic MRI lesions.
   Symptomatic cerebral syndrome with NMOSD-typical brain lesions.
   
   NOTE: According to the international consensus diagnostic criteria for NMOSD⁵, a diagnosis of NMOSD can be made if the patient is serum anti-AQP4 IgG-positive and has at least one of the core clinical characteristics mentioned above. However, we do not recommend testing of anti-AQP4 IgG in patients with optic neuritis only.
2. Blood sample collection
   ​NOTE: Patients do not need to be fasting.
   1. Draw 2-3 mL of venous blood in a 4 mL vacuum blood collection tube.
   2. Allow the serum to clot for 30 min at room temperature (RT).
      NOTE: Prolonged clotting can increase serum levels due to leakage from platelets.
   3. Centrifuge at 2100 x g for 5 min. Carefully transfer the serum to a new tube.
   4. Analyze the serum immediately or aliquot and store at -20 or -80 °C.
      NOTE: Storage at -20 °C is for storage of months, while -80 °C is for storage of years.

2. Anti-AQP4 Antibody Detection

CAUTION: Patient samples and used kit reagents should be considered infectious materials. Sodium azide-containing reagents in the kit are toxic. A flow chart of the protocol is provided in Figure 1.

1. Preparation
   1. Bring the kit to RT before use.
      NOTE: Store the kit at 2-8 °C.
   2. Prepare at least 200 mL of PBS wash buffer containing 0.2% Tween 20. Pour 100 mL of wash buffer into a 500 mL beaker.
   3. Dilute the serum samples 10x with wash buffer. Prepare the positive and negative controls according to the instructions of the distributor.
2. Sample incubation
   1. Add 30 µL of the pre-diluted samples, positive control, or negative control to the reaction fields of the reagent tray (Figure 2).
      NOTE: Avoid air bubbles.
   2. Remove the protective cover on the biochip slides.
      NOTE: Do not touch the biochips to avoid the detachment and contamination of fixed cells. Hold the slide side-by-side before removing the cover.
   3. Apply the biochip slide to the reagent tray (Figure 2). Start a 30 min incubation at RT.
      NOTE: Every sample should be an individual droplet connected to the corresponding biochip without mixing with other droplets.
   4. Gently rinse the biochip slide once with wash buffer. Immerse the biochip slide into the 500 mL beaker filled with 100 mL of wash buffer for at least 5 min. Place the beaker on a shaker if possible.
   5. Take out the biochip slide from the wash buffer. Carefully wipe away the residual wash buffer outside of the reaction fields with a paper towel. Expose the biochip slide in open air for 1-2 min to evaporate the residual wash buffer on the reaction field.
      NOTE: Do not dry out the reaction fields. Do not touch the biochips with paper towel.
3. Secondary antibody incubation
   1. Prepare the secondary antibody solution according to the instructions of the distributor.
   2. Add 25 µL of fluorescent secondary antibody to the reaction fields of a clean reagent tray.
   3. Apply the biochip slide to the reagent tray loaded with secondary antibody. Incubate for 30 min at RT.
4. Mounting
   1. Pour out the used wash buffer and add 100 mL of fresh wash buffer into the beaker. Repeat the washing as described in steps 2.2.4 and 2.2.5.
   2. Carefully add embedding medium to the reaction fields on a biochip slide (one drop per reaction field). Seal the biochip slide with one piece of cover glass. Avoid air bubbles.
5. Fluorescence detection
   1. Turn on the fluorescent lamp of microscope and pre-heat for 15 min. Choose a 488 nm filter.
   2. Open photograph software. Take pictures from both transfected and untransfected areas of all reaction fields with different magnifications. First, take one picture with 4x magnification for an overview, then take more pictures with 10x and 20x magnifications.
      NOTE: The detailed protocol is shown in Table 1. Interpretation of the results is discussed in the representative results section.

3. Diagnosis of Patients

1. If the patient is serum anti-AQP4 IgG-positive and shows at least one core clinical characteristic of NMOSD (see discussion section), diagnose the patient with NMSOD⁵. However, if the patient is anti-AQP4 IgG-negative, a diagnosis of NMOSD may not be excluded.

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

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