High-throughput Flow Cytometry Cell-based Assay to Detect Antibodies to N-Methyl-D-aspartate Receptor or Dopamine-2 Receptor in Human Serum

Summary

Over the recent years, live cell-based assays have been used successfully to detect antibodies against surface and conformational antigens. Here, we describe a method using high-throughput flow cytometry enabling the analysis of large cohorts of patients. Detection of novel antibodies will improve diagnosis and treatment of immune-mediated disorders.

Abstract

Over the recent years, antibodies against surface and conformational proteins involved in neurotransmission have been detected in autoimmune CNS diseases in children and adults. These antibodies have been used to guide diagnosis and treatment. Cell-based assays have improved the detection of antibodies in patient serum. They are based on the surface expression of brain antigens on eukaryotic cells, which are then incubated with diluted patient sera followed by fluorochrome-conjugated secondary antibodies. After washing, secondary antibody binding is then analyzed by flow cytometry. Our group has developed a high-throughput flow cytometry live cell-based assay to reliably detect antibodies against specific neurotransmitter receptors. This flow cytometry method is straight forward, quantitative, efficient, and the use of a high-throughput sampler system allows for large patient cohorts to be easily assayed in a short space of time. Additionally, this cell-based assay can be easily adapted to detect antibodies to many different antigenic targets, both from the central nervous system and periphery. Discovering additional novel antibody biomarkers will enable prompt and accurate diagnosis and improve treatment of immune-mediated disorders.

Introduction

Over recent years, autoimmune forms of central nervous system (CNS) diseases have been identified. It has been shown that these diseases are associated and defined by the presence of autoantibodies. These antibodies bind to neuronal receptors or synaptic proteins involved in neurotransmission^1,2. Different antigens have been detected, such as N-methyl-D-aspartate receptor (NMDAR)^3-5, γ-aminobutyric acid B receptor (GABA_B) receptor⁶, α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) receptor⁷, voltage-gated potassium channel (VGKC) associated proteins: leucine-rich glioma-activated 1 protein (LGL-1) and contactin-associated protein 2 (capsr2)^8,9, glutamate receptor^5,10, and dopamine-2 receptor (D2R)¹¹. In the past, these autoimmune CNS disorders (mainly called encephalitis) were often undiagnosed and untreated. These novel antibody biomarkers, e.g. NMDAR antibody or D2R antibody, have substantially improved diagnosis and awareness, and have opened treatment options for patients. Indeed, early treatment with immunotherapies is associated with improved outcome^11,12.

Traditional methods to detect antibodies, such as enzyme-linked immunosorbent assay (ELISA) and western blot have been used for detection of antibodies in sera. However, they do not readily enable recognition of extracellular or surface epitopes and, rather, may reveal immunoreactivity towards an intracellular epitope. Furthermore, antibodies that bind to the extracellular domain of important proteins involved in neurotransmission are likely to be pathogenic^2,3,7,13,14. Therefore, the development of accurate and sensitive assays to discover relevant cell surface or extracellular antibody targets in patients is paramount. The gold standard method in the field is based on the use of live cells, in so-called “cell-based assays”. This method involves the expression of an antigen at the surface of mammalian cells (most often human embryonic kidney 293 (HEK293) cells) in its native form by transfection of vectors containing the complete cDNA sequence of the antigen of interest. Live nonpermeabilized cells are then incubated with diluted patient serum and followed by fluorochrome-conjugated anti-human immunoglobulin (Ig) secondary antibody. The intensity or level of fluorescence is then detected and is associated to the level of autoantibody binding. This technique is specific, as only one antigen is overexpressed in cells. The most widely used “read-out” has been confocal microscopy analysis after immunocytochemistry^4,5,8-10. However, flow cytometry cell-based assays have been successfully used to detect antibodies in patients with demyelinating diseases^15-17. In particular, Waters et al.¹⁵ compared the detection of antibody using a panel of antibody detection techniques including cell-based assays followed by microscopy or flow cytometry analyses, and has shown flow cytometry cell-based assay to be the most sensitive, accurate, and reliable method. Therefore, flow cytometry cell-based assay is advantageous as it is quantitative, not investigator-dependent, and does not involve any use of radioactive material. It is also convenient as it allows large patient cohorts to be assayed in a short amount of time.

More recently, we have optimized a high-throughput flow cytometry cell-based assay to detect NMDAR antibody and D2R antibody in patients with autoimmune CNS diseases¹¹. Our group recently detected NMDAR antibody in patient sera using flow cytometry cell-based assay. These NMDAR antibody-positive sera were previously analyzed using confocal microscopy and were also found to be positive¹¹. This protocol outlines a flow cytometry cell-based assay for the detection of conformation-sensitive CNS antibody in patient serum utilizing an automated high-throughput sampler.

Protocol

1. Subcloning Strategy to Construct pIRES2-EGFP Vector Encoding D2R or NMDAR

1. Obtain full-length cDNA clone of human D2R or NMDAR subunit 1 (NR1).
2. Choose an expression vector, such as pIRES2-EGFP, which is suitable for expression of transmembrane proteins with an enhanced green fluorescent protein (GFP) reporter under control of an internal ribosome entry site (IRES), enabling both antigens and GFP to be coexpressed in cells separately.
3. Subclone human cDNA within pIRES2-EGFP vector.
   1. To subclone cDNA, use appropriate restriction enzymes (for example NheI and XhoI for human D2R cDNA).
   2. Ligate cut cDNA insert within restricted pIRES2-EGFP vector.
   3. Sequence ligated pIRES2-EGFP D2R or NMDAR vector to check whether the vector contains the correct sequence.
   4. Purify and amplify pIRES2-EGFP D2R or NMDAR vector for later use in transfection.

2. HEK293 Cell Transfection for Expression of Neuronal Antigen

1. Culture HEK293 cells in tissue culture flasks with fresh Dulbecco’s Modified Eagle Medium (1x) (DMEM) complete with 4.5 g/L D-glucose, L-glutamine and 110 mg/L sodium pyruvate supplemented with 10% fetal bovine serum (FBS), 2 mM GlutaMAX, and 50 μg/ml Gentamicin.
2. Detach HEK293 cells using trypsin and transfer to a conical tube.
   1. Wash by centrifuging cells at 250 x g for 6 min and resuspend the cell pellet in fresh complete DMEM.
3. Count the cells and seed 6 well plates at around 8 x 10⁵ cells/well and culture overnight in 2ml DMEM at 37 °C and 5% CO₂ in an incubator.
4. Once they have reached around 70% confluency, transfect the HEK293 cells with pIRES2-EGFP NR1 (HEK293^NMDAR+ cells), pIRES2-EGFP D2R (HEK293^D2R+ cells), and pIRES2-EGFP (HEK293^CTL cells; vector control) as follows. Keep some untransfected HEK293 cells for compensation later on.
   1. Prepare the required volume of transfection mix comprising 2.5 μg DNA, 200 μl 0.9% sodium chloride and 4 μg/ml polyethylenimine/well (each containing 2 ml fresh complete DMEM).
   2. Immediately vortex the mix for 10 sec and incubate at room temperature (RT) for 10 min, before adding volume of transfection mix to the appropriate wells.
   3. Cover the plate, wrap the sides with Parafilm, and centrifuge at 280 x g for 5 min to aid cell transfection.
   4. Remove the Parafilm and then place in the incubator to culture at 37 °C.
   5. Replace culture media 18 hr later with fresh complete DMEM, and maintain in culture for another 72 hr.
   6. Optional: 72 hr post-transfection, transfected cells can be selected by culture in complete DMEM supplemented with 250 μg/ml Geneticin, in order to obtain a polyclonal stable transfectant.

3. Flow Cytometry Cell-based Assay for Detection of Antibody to Surface Neuronal Antigens

1. Detach HEK293^NMDAR+, HEK293^D2R+, and HEK293^CTL cells by incubation with 500 μl/well Versene for approximately 5 min at 37 °C.
   1. Resuspend in 2ml/well of PBS (-Ca²⁺/-Mg²⁺) supplemented with 2% FBS (PBS/FBS) and transfer to a 15 ml or 50 ml conical tube.
   2. Wash cells by centrifuging at 250 x g for 6 min and resuspend the cell pellet in 15-50 ml PBS/FBS. Repeat wash 2x.
   3. Count the cells and then resuspend in PBS/FBS at 1 x 10⁶ cells/ml.
2. Design the 96-well template for flow cytometry acquisition, considering that HEK293^D2R+ or HEK293^NMDAR+ cells, as well as HEK293^CTL cells will have to be incubated with each patient sample or primary antibody.
3. Seed cells in a V-bottom 96-well plate at 50,000 cells/well according to the flow cytometry acquisition template.
4. Pellet the cells by centrifuging the 96-well plate at 450 x g for 5 min and gently remove the supernatant using an electronic or manual multichannel pipette, tilting the plate on an angle and taking care not to aspirate the cell pellet.
5. Incubate HEK293^D2R+, HEK293^NMDAR+, and HEK293^CTL cells for 1 hr at RT in the dark with:
   1. Serial dilutions of primary antibody in order to assess antigen surface expression.
   2. Patient samples in order to detect antibody.
6. Use appropriate flow cytometry compensation controls, e.g. unstained untransfected HEK293 cells, GFP⁺ HEK293 cells (AF647^-), and untransfected AF647⁺ HEK293 cells (GFP).
7. Wash cells by centrifuging at 450 x g for 5 min and resuspend the cell pellet in 170 μl PBS/FBS. Repeat wash step two more times.
8. Incubate cells for 1 hr at RT in the dark with 1:100 dilution of AF647-conjugated secondary antibody (anti-human IgG for patient serum and anti-mouse IgG for anti-NR1 or anti-D2R primary antibodies).
9. Wash the cells three times, as in step 3.7, and resuspend in 35 μl PBS/FBS for flow cytometry cell acquisition.
10. Set up the high-throughput sampler (HTS) on the flow cytometer (BDLSRII).
11. Acquire 10,000 events/well according to the flow cytometry acquisition template.
12. Export and then analyze data for analysis using a flow cytometry software package, such as FlowJo v7.5:
    1. First, gate the live HEK293 cells based on forward (size) and side (granularity) scatters.
    2. Further gate live HEK293 cells based on high GFP-positivity to analyse only the transfected cells.
    3. Determine the mean fluorescence intensity (MFI) of the AF647 channel within the live GFP-positive cells.
    4. Export data into Excel or Prism for analysis:
       1. Plot concentrations of primary antibody (anti-NMDAR or anti-D2R antibody) versus the MFI obtained from the cells incubated with these antibodies.
       2. For each patient and control sample, determine the ΔMFI by subtracting the MFI of the HEK293^CTL cells from the MFI of the HEK293^D2R+ or HEK293^NMDAR+ cells, respectively.
       3. Calculate the threshold of antibody positivity by adding three standard deviations above the mean ΔMFI of the control cohort.
       4. Plot individual samples grouped according to their serum type on a graph and represent the threshold as a line.

Results

Live HEK293^D2R+ and HEK293^CTL cells were acquired at the flow cytometer using a high-throughput sampler. During analysis, cells were gated based on forward scatter (size) and side scatter (granularity) parameters (Figures 1A and 1D). Transfected HEK293 cells expressed the reporter molecule, GFP, in the cytoplasm, and untransfected cells were excluded from analysis (Figures 1B and 1E). Within the GFP⁺ gate, the M...

Discussion

This paper describes a novel application of flow cytometry live cell-based assay to detect antibodies targeting specific cell surface neuronal proteins using a high-throughput sampler. Using this technique, we report that a subgroup of patients affected with autoimmune CNS diseases have serum antibodies to surface NMDAR or to surface D2R.

Essential steps for optimal antibody detection using this high-throughput flow cytometry live cell-based assay include 1) obtaining a high yield of healthy t...

Materials

Name	Company	Catalog Number	Comments
pIRES2-EGFP vector	Clontech	6029-1
Human HA-Dopamine-2 receptor (D2R) cDNA	Missouri S&T cDNA Resource Centre	DRD020TN00
Human subunit 1 of NMDAR (NR1) cDNA			Gift from Prof A. Vincent  (Oxford, UK)
Monoclonal purified mouse anti-human NR1 antibody (clone: 54.1)	BD Pharmingen	556308
Mouse purified monoclonal anti-DRD2 IgG (clone: 1B11)	Sigma-Aldrich	WH0001813M1-50UG
Alexa Fluor 647 donkey anti-mouse IgG (H + L)	Invitrogen	A31571
Alexa Fluor 647 goat anti-human IgG (H + L)	Invitrogen	A21445
Dulbecco’s Modified Eagle Medium (1x) 4.5 g/L D-glucose, L-Glutamine and 110 mg/L sodium pyruvate	Invitrogen	11995-065
Foetal bovine serum	Invitrogen	10099-141
Gentamicin	Invitrogen	15710-072
GlutaMAX	Invitrogen	35050-061
Penicillin-streptomycin	Invitrogen	15140-122
Geneticin	Invitrogen	10131-035
Dulbecco's Phosphate-Buffered Saline (1x) (-Ca2+/-Mg2+)	Invitrogen	14190-144
TrypLE Express (1x) with Phenol Red	Invitrogen	12605-028
0.9% Sodium chloride	Baxter	AHF7975
Polyethylenimine	Polysciences Inc	9002-98-6
Versene	Invitrogen	15040-066
XhoI	Roche	10899194001
NheI	Roche	10885843001
PureLink PCR Purification Kit	Invitrogen	K3100-01
JetQuick Gel Extraction Spin Kit	Genomed	420050
T4 DNA Ligase	Roche	10481220001
Plasmid Plus Maxi Kit	Qiagen	12963
Name of Equipment/Software	Company	Catalog Number	Model/Version
Flow cytometer with high-throughput sampler system	BD Biosciences		BDLSRII with HTS
Inverted microscope	Olympus		CKX41 (with mercury lamp and U-RFLT50 power supply)
Electronic multichannel pipette (15-300 μl)	Eppendorf	613-2240P	12-channel Xplorer Plus
Excel	Microsoft		2010
Prism	GraphPad Software, Inc.		v4
FlowJo	Treestar		v7.5
50 ml polypropylene conical tubes	BD Biosciences	352070
6-well plate	BD Biosciences	353046
Tissue culture flask (T75)	BD Biosciences	353136
Parafilm	Pechiney Plastic Packaging	PM-992
V-bottom 96-well plate	Corning	651180