An Assay to Detect Serum Anti-aquaporin-4 Immunoglobulin G

Overview

The video demonstrates a cell-based assay for detecting anti-aquaporin-4 immunoglobulin G using a biochip containing untransfected cells and transfected cells expressing aquaporin-4. This method has applications in diagnosing neuromyelitis optica spectrum disorders, confirmed by strong green fluorescence on the biochip.

Protocol

All procedures involving human participants have been performed in compliance with the institutional, national, and international guidelines for human welfare and have been reviewed by the local institutional review board.

1. Patient Enrollment and Blood Sample Collection

1. Apply the laboratory detection of serum anti-aquaporin-4 immunoglobulin G (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 neuromyelitis optica spectrum disorders (NMOSD)-typical diencephalic MRI lesions. 
   Symptomatic cerebral syndrome with NMOSD-typical brain lesions.
   NOTE: According to the international consensus diagnostic criteria for NMOSD, 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 phosphate-buffered saline (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 the 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 a 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 the 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.

Representative Results

Figure 1: Flow chart of anti-AQP4 IgG detection protocol. AQP4-M1-transfected or untransfected EU 90 cells are fixed on the biochips. When adding serum to biochips, anti-AQP4 IgG in serum is captured by the fixed transfected cells. Then, fluorescein-labelled secondary antibody is applied to detect anti-AQP4 IgG. The fluorescence can be visualized by microscopy with various magnifications.

Figure 2: Sample incubation. (A) Top view of reagent tray. Every reagent tray contains five individual reaction fields. The positive control, samples, and negative control should be added to the separate reaction fields. (B) Top view of biochip slide. Every biochip slide contains five reaction fields, which have two subsections. The transfected subsection contains fixed AQP4-M1-transfected cells, while the untransfected subsection contains untransfected cells. (C) Front view of reagent tray and biochip slide. Reaction fields on the reagent tray and biochip slide are paired with each other. The sample added to the reaction field on reagent tray should be connected to the corresponding reaction field on the biochip slide.

Table 1: Fluorescence microscopy. It is recommended to use 4x, 10x, and 20x objectives for imaging of transfected and untransfected areas.

Magnification	Transfected area	Untransfected area
4x	1 picture	1 picture
10x	4 pictures	1 picture
20x	5 pictures	1 picture

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