A Simple Cell-based Immunofluorescence Assay to Detect Autoantibody Against the N-Methyl-D-Aspartate (NMDA) Receptor in Blood

Summary

We ectopically expressed NR1 subunit of NMDA receptor tagged with green fluorescent protein in human embryonic cells (HEK293) as antigen to detect autoantibodies against NMDA receptor in the blood of patients suspected with autoimmune encephalitis. This simple method may be suitable for screening purposes in clinical settings.

Abstract

The presence of anti-NMDA receptor autoantibody can cause various neuropsychiatric symptoms in the affected patients, termed anti-NMDA receptor autoimmune encephalitis. Detection of the specific autoantibody against the NMDA receptor in the blood or cerebrospinal fluid (CSF) is essential for the accurate diagnosis of this condition. The NMDA receptor is an ion channel protein complex that contains four subunits, including two mandatory NMDA receptor subunit 1 (NR1) and one or two NMDA receptor subunit 2A (NR2A), NMDA receptor subunit 2B (NR2B), NMDA receptor subunit 2C (NR2C), or NMDA receptor subunit 2D (NR2D). The epitope of anti-NMDA receptor autoantibody was reported to be present at the extracellular N-terminal domain of the NR1 subunit of the NMDA receptor. The goal of this study is to develop a simple cell-based immunofluorescence assay that can be used as a screening test to detect the presence of autoantibodies against NR1 subunit of the NMDA receptor in the blood to facilitate the clinical and basic research of anti-NMDA receptor autoimmune encephalitis.

Introduction

Anti-NMDA receptor autoimmune encephalitis is a newly recognized disease entity that can occur in patients of all ages, and affects predominantly female patients^1,2. It is one of the most frequently diagnosed encephalitis among patients with initial unknown etiology of encephalitis³. Patients affected with anti-NMDA receptor encephalitis usually have prodromal symptoms of a headache or fever, followed by the quick development of consciousness level change and a variety of acute neuropsychiatric symptoms, including agitation, irritability, anxiety, insomnia, hallucinations, delusions, aggression, bizarre behaviors, movement abnormalities, autonomic dysregulation, and seizure attacks^4,5. Early recognition of this condition and timely treatment with immunotherapy are important for a better outcome and even full recovery in affected patients⁶. Hence, it is suggested that anti-NMDA receptor autoimmune encephalitis should be considered as an important differential diagnosis of patients presenting with acute or new-onset psychotic features^7,8.

Besides clinical features, detection of autoantibody against the NMDA receptor in the blood or CSF is essential for the accurate diagnosis of anti-NMDA receptor autoimmune encephalitis⁹. Most of the immunological tests to detect the anti-NMDA receptor autoantibody are available in a few research laboratories^10,11, and there is only one commercially available cell-based immunofluorescence assay for screening the anti-NMDA receptor autoantibodies¹². The goal of this study is to develop a simple in-house cell-based immunofluorescence assay that can be conveniently used in the laboratory to screen the presence of anti-NMDA receptor autoantibodies to facilitate the clinical research of anti-NMDA receptor autoimmune encephalitis. The NMDA receptor is a heterotetramer ion channel protein complex specifically expressed in the brain. It is made of two compulsory NR1 subunits, and the combination of one or two subunits of NR2A, NR2B, NR2C, or NR2D¹³. A previous study reported that the main epitope targeted by the antibodies was at the extracellular N-terminal domain of the NR1 subunit⁵. Hence, in this protocol, we express the human recombinant NR1-subunit protein of NMDA receptor tagged with green fluorescent protein (GFP) in the human embryonic kidney epithelial cell line (HEK293), and develop a cell-based immunofluorescence assay to detect the IgG class of anti-NMDA receptor autoantibodies in the blood.

Protocol

The study was approved by the Institutional Review Board of Chang Gung Memorial Hospital at Linkuo, Taoyuan, Taiwan (102-2577A3).

1. Preparation of the NR1-GFP Expression Plasmid

1. Mix 10 ng of NR1-GFP plasmid with 100 µL of Escherichia coli competent cells strain DH5α in a sterile 1.5 mL centrifuge tube, pipet the mixture gently up and down 4-6 times, and incubate the tube on ice for 20 min.
2. Incubate the tube at 42 °C for 1 min in a water bath, remove the tube from the water bath, add 1 mL lysogeny broth (LB) medium into the tube, and incubate the tube at 37 °C in an incubator with shaking for 1 h.
3. Spread 50 µL of the mixture onto an LB agar plate containing ampicillin (0.1 mg/mL), and incubate the LB agar plate at 37 °C in an incubator overnight.
4. Pick a single colony from the overnight LB agar plate using a sterile tip, and swirl the tip in a in a bacterial culture tube containing 5 mL of LB medium and ampicillin (0.1 mg/mL). Incubate the tube at 37 °C in an incubator with shaking overnight.
5. Aliquot 3 mL of the overnight bacterial culture into a 500 mL flask that contains 250 mL sterile LB medium and ampicillin (0.1 mg/mL). Incubate the flask at 37 °C with shaking. Check regularly the optic density (OD) of the bacterial culture at wavelength of 600 nm using a spectrometer until the OD600 reaches 0.6-1.0.
   NOTE: Mix well 800 µL of the overnight bacterial culture with 200 µL glycerol to prepare the glycerol stock, and store the glycerol stock under -80 °C for future use.
6. Prepare NR1-GFP expression plasmid from the 250 mL bacterial culture
   1. Collect the cells by centrifugation at 6,000 x g for 15 min at 4 °C.
   2. Resuspend the bacteria pellet in 10 mL resuspension buffer containing RNase A; vortex thoroughly until no clump is seen.
   3. Add 10 mL lysis buffer to the bacterial suspension, gently invert the mixture 4-6 times, and incubate the lysate at room temperature (RT) for 5 min.
   4. Add 10 mL pre-chilled precipitation buffer to the lysate, gently invert the mixture 4-6 times.
   5. Pour the lysate into the barrel of a filter cartridge with the cap on; wait for 10 min at RT.
   6. Remove the cap from the cartridge outlet nozzle, insert a plunger into the cartridge, and gently press the plunger. Collect the filtered lysate into a 50 mL tube.
   7. Add 2.5 mL proprietary buffer from the manufacturer to the filtered lysate, mix the mixture by inverting the tube approximately 10 times, and incubate the mixture on ice for 30 min.
   8. Apply the filtered lysate mixture to a filter column that was pre-equilibrated with 10 mL equilibration buffer. Allow the column to drain by gravity.
   9. Wash the column with 30 mL wash buffer twice, then elute DNA from the column using 15 mL elution buffer.
   10. Add 10.5 mL (0.7 volumes) isopropanol to the eluted DNA buffer, mix well, and centrifuge the mixture at 20,000 x g for 30 min at 4 °C.
   11. Decant the supernatant carefully, wash the DNA pellet with 5 mL endotoxin-free 70% ethanol once, and centrifuge at 20,000 x g for 10 min.
   12. Decant the supernatant, dry the pellet for 5-10 min in a chemical hood, and dissolve the pellet in 300-500 µL of endotoxin-free buffer.
   13. Determine the plasmid concentration by measuring the absorption of the solution at UV 260/280 nm using a spectrophotometer.
       NOTE: Prepare the expression plasmid GFP using the same protocol.

2. Transfection of HEK293 Cells with NR1-GFP Expression Plasmid

1. Aliquot 200 µL of 2% gelatin solution to each well of a 48-well culture plate and incubate the plate at 37 °C for at least 30 min.
2. Aspirate the gelatin solution from the well, and seed 5 x 10⁴ HEK293 cells in 200 µL of cell culture medium containing 10% fetal bovine serum (FBS) in each well. Incubate the plate at 37 °C in a humidified incubator supplied with 5% CO₂ overnight.
   NOTE: Cells were counted using a hemocytometer.
3. On the next day, replace the spent culture medium with 200 µL of fresh culture medium containing 10% FBS per well.
4. For each single well, prepare solution A by mixing 100 ng of NR1-tGFP plasmid with 20 µL culture medium, and solution B by mixing 0.8 µL transfection reagent with 20 µL culture medium. Multiply the number of wells needed to prepare the total volume for each experiment.
5. Prepare solution C by mixing solution A and solution B, and incubate the mixture at RT for 20-30 min.
6. Aliquot 40 µL of solution C to each well containing the HEK293 cells, swirl the plate gently, and incubate the plate at 37 °C in a humidified incubator supplied with 5% CO₂ overnight.
7. Check the expression of NR1-GFP recombinant protein in the host cells under fluorescent microscope with 40X-200X magnification (excitation/emission: 482/502 nm), and proceed to the cell-based immunofluorescence assay.
   NOTE: Transfect the expression plasmid GFP into the HEK293 cells using the same protocol.

3. Cell-based Immunofluorescence Assay

1. Aspirate the spent medium from the wells, then wash each well with 200 µL of phosphate buffered saline (PBS) three times.
2. Add 200 µL of 4% paraformaldehyde solution to each well; incubate the plate at RT for 15 min.
3. Aspirate the paraformaldehyde solution from the wells, and wash each well with 200 µL of PBS three times.
4. Add 200 µL of 10% skim milk in PBST (0.1% Tween-20 in PBS) solution to each well, and incubate the plate at RT for 1 h with gentle shaking.
5. Aspirate the 10% skim milk in PBST solution from the well, then wash the wells with 200 µL PBST once.
6. Incubate the wells with diluted plasma (1:100 in PBST) as the primary antibody with gentle shaking at RT for 1 h.
   NOTE: Plasma is obtained from the blood of patients suspected to have autoimmune encephalitis.
7. Aspirate the diluted plasma from the wells, and wash each well with 200 µL of PBST three times.
8. Add 200 µL of goat anti-human IgG conjugated with Alexa Fluor 594 (1:1,000 dilution in PBST) to each well, and incubate the culture plate at RT with gentle shaking for 1 h.
9. Aspirate the goat anti-human IgG conjugated with Alexa Fluor 594 solution, and wash each well with 200 µL of PBST three times.
10. Add 100 µL glycerol solution (50% glycerol in PBS and 1:100,000 of 4',6-diamidino-2-phenylindole (DAPI)) to each well.
11. Observe and image the cells under a fluorescent microscope using a 10X eyepiece, and 4X, 10X, and 20X objectives.
    NOTE: Use the correct filter for each dye: for NR1-GFP (excitation/emission = 482/502 nm), for Alexa Fluor 594 (excitation/emission = 561/594 nm), for DAPI (excitation/emission = 358/461 nm). Conduct the negative control using the HEK cells expressing GFP in the same way.

Results

On average, we could obtain 200-300 µg endotoxin-free expression plasmid NR1-GFP and GFP from 250 mL bacterial culture following the procedures described in section 1 of the protocol. An amount of 100 ng/well of the expression plasmid was used for the transfection of the HEK293 cells cultured in the 48-well plate as described in the section 2 of the protocol. 24-30 h after transfection, the cells expressed the NR1-GFP, and GFP recombinant proteins could be detected under fluorescent micro...

Discussion

There are several cell-based immunological assays to screen the presence of autoantibodies against NMDA receptor reported in the literature, including live cell-based immunofluorescence assay¹¹, fixed cell-based immunofluorescence assay⁹, and flow cytometry-based assay¹⁴. The live cell-based immunofluorescence assay should be performed shortly after the preparation of cells ectopically expressing the NR1 protein, while the flow cytometry-based assay ...

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
GRIN1 (GFP-tagged) - Human glutamate receptor, ionotropic, N-methyl D-aspartate 1 (GRIN1), transcript variant NR1-1	Origene (Rockville, MD, USA)	RG219368	NR1-cDNA clone tagged with C-terminal tGFP sequences
pCMV6-AC-GFP	Origene (Rockville, MD, USA)	PS100010	mammalian vector with C-terminal tGFP tag
EndoFree Plasmid Maxi Kit	Qiagen (Hilden Germany)	12362
Lipofectamine 2000 Transfection Reagent	Invitrogen (Carlsbad, CA, USA)	11668-019
Opti-MEM I Reduced Serum Medium	Thermo Fisher	31985-070
DMEM, High Glucose, Pyruvate	Thermo Fisher	11995-065
Characterized Fetal Bovine Serum, US Origin	GE Healthcare Life Sciences	SH30071.01
Goat anti-Human IgG (H+L) Secondary Antibody, Alexa Fluor 594 conjugate	Thermo Fisher	A-11014
Positive control: anti-glutamate receptor (type NMDA)	Euroimmun AG, Lübeck, Germany	CA 112d-0101
Euroimmun Assay Kit