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 (LG....

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 cDN....

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 MFI associa.......

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

List of materials used in this article

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