Name: high-throughput flow cytometry cell-based assay to detect antibodies to n-methyl-d-aspartate receptor or dopamine-2 receptor in human serum
Uploaded: 2013-11-23T14:00:00
Description: Watch this Scientific Journal Video about High-throughput Flow Cytometry Cell-based Assay to Detect Antibodies to N-Methyl-D-aspartate Receptor or Dopamine-2 Receptor in Human Serum at JoVE.com

Dette arbejde blev støttet af den australske National Sundhed og Medical Research Council, Star Scientific Foundation (Australien), Tourettes syndrom Association (USA), The Trish multipel sklerose Research Foundation og multipel sklerose Research Australien, Petre Foundation (Australien), Rebecca L. Cooper Medical Research Foundation (Australien). Vi takker alle de patienter og familiemedlemmer, der leveres prøver for vores undersøgelse.

<html> 1.. Subkloning strategi til at konstruere pIRES2-EGFP Vector Encoding D2R eller NMDAR <ol><li> Opnå fuld længde cDNA klon af menneskelig D2R eller NMDAR subunit 1 (NR1). </li><li> Vælg en ekspressionsvektor, såsom pIRES2-EGFP, som er egnet til ekspression af transmembrane proteiner med en forstærket grønt fluorescerende protein (GFP) reporter under kontrol af en intern ribosombindingssite (IRES), hvor både antigener og GFP skal co-udtrykte i celler separat. </li><li> Subklone humant cDNA under pIRES2-EGFP...

<ol>
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Dette papir beskriver en hidtil ukendt anvendelse af flowcytometri levende cellebaseret assay til påvisning af antistoffer rettet mod specifikke celleoverflade neuronale proteiner ved anvendelse af en high-throughput sampler. Ved at bruge denne teknik, rapporterer vi, at en undergruppe af patienter, der lider med autoimmune CNS-sygdomme har serumantistoffer til overflade NMDAR eller til overfladen D2R. Væsentlige skridt til optimal antistofpåvisning ved hjælp af denne high-throughput flo...

Over de senere år har autoimmune former for sygdomme i centralnervesystemet (CNS) sygdomme blevet identificeret. Det er blevet vist, at disse sygdomme er forbundet og defineret ved tilstedeværelsen af ​​autoantistoffer. Disse antistoffer binder til neuronale receptorer eller synaptiske proteiner involveret i neurotransmission 1,2. Er blevet opdaget forskellige antigener, såsom N-methyl-D-aspartat-receptor (NMDAR) 3-5, γ-aminosmørsyre B receptor (GABAB) receptor 6, α-amino-3-hydroxy-5-methyl-4- isoxazolpropionsyre (AMPA)-receptor 7 spændingsstyret kaliumkanal (VGKC) associerede proteiner: leucin-rige gliom-aktiverede 1 protein (LGL-1) og contactin-associeret protein 2 (capsr2) 8,9, glutamatreceptor 5,10 og dopamin-2-receptor (D2R) 11. I fortiden, disse autoimmune CNS sygdomme (hovedsagelig kaldet encephalitis) var ofte udiagnosticeret og ubehandlet. Disse nye antistof biomarkører, fxNMDAR antistof eller D2R antistof, væsentligt har forbedret diagnose og bevidsthed, og har åbnet behandlingsmuligheder for patienterne. Faktisk er tidlig behandling med immunterapi er forbundet med forbedret resultat 11,12. Traditionelle metoder til påvisning af antistoffer, såsom enzymkoblet immunosorbent assay (ELISA) og Western blot er blevet anvendt til påvisning af antistoffer i sera. Men de ikke umiddelbart muliggøre anerkendelse af ekstracellulære eller overfladeepitoper og snarere kan afsløre immunoreaktivitet mod en intracellulær epitop. Antistoffer, som binder til det ekstracellulære domæne af vigtige proteiner involveret i neurotransmission sandsynligvis være patogene 2,3,7,13,14. Derfor er udviklingen af ​​præcise og følsomme assays til at opdage relevante celleoverfladen eller ekstracellulære antistof mål i patienter er altafgørende. Guldstandarden metode i feltet er baseret på brug af levende celler, i såkaldt &quot;celle-baSED assays &quot;. Denne fremgangsmåde involverer ekspressionen af ​​et antigen på overfladen af ​​pattedyrceller (oftest humane embryonale nyre 293 (HEK293) celler) i sin native form ved transfektion af vektorer indeholdende den komplette cDNA-sekvens for antigenet af interesse. Levende nonpermeabilized celler inkuberes derefter med fortyndet patientserum og efterfølges af fluorokrom-konjugeret anti-humant immunglobulin (Ig) sekundært antistof. Intensiteten eller niveauet af fluorescens påvises derefter, og er knyttet til niveauet af autoantistof binding. Denne teknik er specifik, da kun et antigen overudtrykkes i celler. Den mest udbredte &quot;read-out&quot; har været konfokal mikroskopi analyse efter immuncytokemisk 4,5,8-10. Men flowcytometri cellebaserede assays er blevet anvendt med succes til påvisning af antistoffer hos patienter med demyeliniserende 15-17. Især Waters et al. 15. sammenlignet påvisning af antistof ved hjælp af en grydeel af antistof detektionsteknikker, herunder celle-baserede assays efterfulgt af mikroskopi eller flowcytometri analyser og har vist flowcytometri cellebaserede analyse at være den mest følsomme, nøjagtig og pålidelig metode. Derfor flowcytometri cellebaserede analyse er fordelagtig, da det er kvantitativ, ikke investigator-afhængige, og ikke indebærer nogen anvendelse af radioaktivt materiale. Det er også praktisk, da det giver store patientkohorter der skal analyseres i en kort tid. For nylig har vi optimeret en high-throughput flowcytometri cellebaseret assay til påvisning NMDAR antistof og D2R antistof hos patienter med autoimmune sygdomme i centralnervesystemet 11. Vores gruppe nylig opdaget NMDAR antistof i patientens sera hjælp flowcytometri cellebaserede assay. Disse NMDAR antistof-positive sera tidligere analyseret ved hjælp af konfokal mikroskopi og den fandtes også at være positiv 11. Denne protokol beskrives en flowcytometri cellebaseret assay til påvisning af conformation-sensitive CNS antistof i patientens serum ved anvendelse af en automatiseret høj-throughput sampler.

<table border="0" cellpadding="0" cellspacing="0" width="751">
	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:255px;">pIRES2-EGFP vector</td>
			<td style="width:187px;">Clontech</td>
			<td style="width:136px;">6029-1</td>
			<td style="width:173px;"></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:255px;">Human HA-Dopamine-2 receptor (D2R) cDNA</td>
			<td style="width:187px;">Missouri S&#38;T cDNA Resource Centre</td>
			<td style="width:136px;">DRD020TN00</td>
			<td style="width:173px;"></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:255px;">Human subunit 1 of NMDAR (NR1) cDNA</td>
			<td style="width:187px;"></td>
			<td style="width:136px;"></td>
			<td style="width:173px;">Gift from Prof A. Vincent&#160; (Oxford, UK)</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:255px;">Monoclonal purified mouse anti-human NR1 antibody (clone: 54.1)</td>
			<td style="width:187px;">BD Pharmingen</td>
			<td style="width:136px;">556308</td>
			<td style="width:173px;"></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:255px;">Mouse purified monoclonal anti-DRD2 IgG (clone: 1B11)</td>
			<td style="width:187px;">Sigma-Aldrich</td>
			<td style="width:136px;">WH0001813M1-50UG</td>
			<td style="width:173px;"></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:255px;">Alexa Fluor 647 donkey anti-mouse IgG (H + L)</td>
			<td style="width:187px;">Invitrogen</td>
			<td style="width:136px;">A31571</td>
			<td style="width:173px;"></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:255px;">Alexa Fluor 647 goat anti-human IgG (H + L)</td>
			<td style="width:187px;">Invitrogen</td>
			<td style="width:136px;">A21445</td>
			<td style="width:173px;"></td>
		</tr>
		<tr height="63">
			<td height="63" style="height:63px;width:255px;">Dulbecco&#8217;s Modified Eagle Medium (1x) 4.5 g/L D-glucose, L-Glutamine and 110 mg/L sodium pyruvate</td>
			<td style="width:187px;">Invitrogen</td>
			<td style="width:136px;">11995-065</td>
			<td style="width:173px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:255px;">Foetal bovine serum</td>
			<td style="width:187px;">Invitrogen</td>
			<td style="width:136px;">10099-141</td>
			<td style="width:173px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:255px;">Gentamicin</td>
			<td style="width:187px;">Invitrogen</td>
			<td style="width:136px;">15710-072</td>
			<td style="width:173px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:255px;">GlutaMAX</td>
			<td style="width:187px;">Invitrogen</td>
			<td style="width:136px;">35050-061</td>
			<td style="width:173px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:255px;">Penicillin-streptomycin</td>
			<td style="width:187px;">Invitrogen</td>
			<td style="width:136px;">15140-122</td>
			<td style="width:173px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:255px;">Geneticin</td>
			<td style="width:187px;">Invitrogen</td>
			<td style="width:136px;">10131-035</td>
			<td style="width:173px;"></td>
		</tr>
		<tr height="45">
			<td height="45" style="height:45px;width:255px;">Dulbecco&#39;s Phosphate-Buffered Saline (1x) (-Ca2+/-Mg2+)</td>
			<td style="width:187px;">Invitrogen</td>
			<td style="width:136px;">14190-144</td>
			<td style="width:173px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:255px;">TrypLE Express (1x) with Phenol Red</td>
			<td style="width:187px;">Invitrogen</td>
			<td style="width:136px;">12605-028</td>
			<td style="width:173px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:255px;">0.9% Sodium chloride</td>
			<td style="width:187px;">Baxter</td>
			<td style="width:136px;">AHF7975</td>
			<td style="width:173px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:255px;">Polyethylenimine</td>
			<td style="width:187px;">Polysciences Inc</td>
			<td style="width:136px;">9002-98-6</td>
			<td style="width:173px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:255px;">Versene</td>
			<td style="width:187px;">Invitrogen</td>
			<td style="width:136px;">15040-066</td>
			<td style="width:173px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:255px;">XhoI</td>
			<td style="width:187px;">Roche</td>
			<td style="width:136px;">10899194001</td>
			<td style="width:173px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:255px;">NheI</td>
			<td style="width:187px;">Roche</td>
			<td style="width:136px;">10885843001</td>
			<td style="width:173px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:255px;">PureLink PCR Purification Kit</td>
			<td style="width:187px;">Invitrogen</td>
			<td style="width:136px;">K3100-01</td>
			<td style="width:173px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:255px;">JetQuick Gel Extraction Spin Kit</td>
			<td style="width:187px;">Genomed</td>
			<td style="width:136px;">420050</td>
			<td style="width:173px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:255px;">T4 DNA Ligase</td>
			<td style="width:187px;">Roche</td>
			<td style="width:136px;">10481220001</td>
			<td style="width:173px;"></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:255px;">Plasmid&#160;Plus&#160;Maxi Kit</td>
			<td style="width:187px;">Qiagen</td>
			<td style="width:136px;">12963</td>
			<td style="width:173px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:255px;"></td>
			<td style="width:187px;"></td>
			<td style="width:136px;"></td>
			<td style="width:173px;"></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:255px;">Name of Equipment/Software</td>
			<td style="width:187px;">Company</td>
			<td style="width:136px;">Catalog Number</td>
			<td style="width:173px;">Model/Version</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:255px;">Flow cytometer with high-throughput sampler system</td>
			<td style="width:187px;">BD Biosciences</td>
			<td style="width:136px;"></td>
			<td style="width:173px;">BDLSRII with HTS</td>
		</tr>
		<tr height="63">
			<td height="63" style="height:63px;width:255px;">Inverted microscope</td>
			<td style="width:187px;">Olympus</td>
			<td style="width:136px;"></td>
			<td style="width:173px;">CKX41 (with mercury lamp and U-RFLT50 power supply)</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:255px;">Electronic multichannel pipette (15-300 &#956;l)</td>
			<td style="width:187px;">Eppendorf</td>
			<td style="width:136px;">613-2240P</td>
			<td style="width:173px;">12-channel Xplorer Plus</td>
		</tr>
		<tr height="56">
			<td height="56" style="height:56px;width:255px;">Excel</td>
			<td style="width:187px;">Microsoft</td>
			<td style="width:136px;"></td>
			<td style="width:173px;">2010</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:255px;">Prism</td>
			<td style="width:187px;">GraphPad Software, Inc.</td>
			<td style="width:136px;"></td>
			<td style="width:173px;">v4</td>
		</tr>
		<tr height="27">
			<td height="27" style="height:27px;width:255px;">FlowJo</td>
			<td style="width:187px;">Treestar</td>
			<td style="width:136px;"></td>
			<td style="width:173px;">v7.5</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:255px;">50 ml polypropylene conical tubes</td>
			<td style="width:187px;">BD Biosciences</td>
			<td style="width:136px;">352070</td>
			<td style="width:173px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:255px;">6-well plate</td>
			<td style="width:187px;">BD Biosciences</td>
			<td style="width:136px;">353046</td>
			<td style="width:173px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:255px;">Tissue culture flask (T75)</td>
			<td style="width:187px;">BD Biosciences</td>
			<td style="width:136px;">353136</td>
			<td style="width:173px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:255px;">Parafilm</td>
			<td style="width:187px;">Pechiney Plastic Packaging</td>
			<td style="width:136px;">PM-992</td>
			<td style="width:173px;"></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:255px;">V-bottom 96-well plate</td>
			<td style="width:187px;">Corning</td>
			<td style="width:136px;">651180</td>
			<td style="width:173px;"></td>
		</tr>
	</tbody>
</table>

high-throughput flow cytometry cell-based assay to detect antibodies to n-methyl-d-aspartate receptor or dopamine-2 receptor in human serum

I de senere år har antistoffer mod overflade og konformationelle proteiner involveret i neurotransmission blevet detekteret i autoimmune CNS-sygdomme hos børn og voksne. Disse antistoffer er blevet benyttet til at lede diagnose og behandling. Cellebaserede assays har forbedret påvisning af antistoffer i patientens serum. De er baseret på overflade-ekspression i hjernen antigener på eukaryote celler, som derefter inkuberes med fortyndet patientsera efterfulgt af fluorochrom-konjugerede sekundære antistoffer. Efter vask sekundært antistof binder derefter analyseret ved flowcytometri. Vores gruppe har udviklet et high-throughput flowcytometri levende celle-baserede assay til pålideligt antistoffer mod specifikke neurotransmitterreceptorer. Denne flowcytometri fremgangsmåde er ligetil, kvantitativ, effektiv og anvendelse af en high-throughput sampler system giver mulighed for store patientkohorter at være let analyseres på kort tid. Derudover denne celle-baseredesiger, kan let tilpasses til påvisning af antistoffer mod mange forskellige antigene mål, både fra centralnervesystemet og periferien. Opdage yderligere roman antistof biomarkører vil muliggøre hurtig og præcis diagnose og forbedre behandlingen af ​​immun-medierede sygdomme.

Lev HEK293 D2R + og HEK293 CTL celler blev erhvervet på flowcytometeret bruge en high-throughput sampler. Under analysen blev cellerne gated baseret på forward scatter (størrelse) og sidespredning (granularitetskravene) parametre (figur 1A og 1D). Transficerede HEK293 celler udtrykte reportermolekylet GFP, i cytoplasmaet, og transficerede celler blev udelukket fra analysen (figur 1B og 1E). Inden GFP + gate blev MFI f...

Watch this Scientific Journal Video about High-throughput Flow Cytometry Cell-based Assay to Detect Antibodies to N-Methyl-D-aspartate Receptor or Dopamine-2 Receptor in Human Serum at JoVE.com

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

I de senere år har levende cellebaserede assays med held blevet anvendt til påvisning af antistoffer mod overflade og konformationelle antigener. Her beskriver vi en fremgangsmåde, ved hjælp af høj-throughput flowcytometri muliggør analyse af store grupper af patienter. Påvisning af nye antistoffer vil forbedre diagnosticering og behandling af immun-medierede sygdomme.

High-throughput Flowcytometri cellebaseret assay til påvisning af antistoffer til N-methyl-D-aspartat-receptor eller dopamin-2-receptoren i humant serum

Over the recent years, antibodies against surface and conformational proteins involved in neurotransmission have been detected in autoimmune CNS diseases ...

Research

JoVE Journal

Medicine

The Measurement and Treatment of Suppression in Amblyopia

Måling og behandling af Suppression i Amblyopi

Amblyopia, a developmental disorder of the visual cortex, is one of the leading causes of visual dysfunction in the working age population. Current ...

Stereological and Flow Cytometry Characterization of Leukocyte Subpopulations in Models of Transient or Permanent Cerebral Ischemia

Stereologisk og flowcytometri karakterisering af leukocytsubpopulationer i modeller af Forbigående eller permanent cerebral iskæmi

Microglia activation, as well as extravasation of haematogenous macrophages and neutrophils, is believed to play a pivotal role in brain injury after ...

Molecular Probe Optimization to Determine Cell Mortality in a Photosynthetic Organism (Microcystis aeruginosa) Using Flow Cytometry

Molecular Probe Optimization to Determine Cell Mortality in a Photosynthetic Organism Microcystis aeruginosa Using Flow Cytometry

Molekylær Probe Optimering konstateres Cell Dødelighed i en Fotosyntetisk Organisme (<em&gt; Microcystis aeruginosa</em&gt;) Under anvendelse af flowcytometri

Microbial subpopulations in field and laboratory studies have been shown to display high heterogeneity in morphological and physiological parameters. ...

Systems Biology of Metabolic Regulation by Estrogen Receptor Signaling in Breast Cancer

Systembiologi i Metabolic forordning ved østrogen-receptor signalering i Breast Cancer

With the advent of the -omics approaches our understanding of the chronic diseases like cancer and metabolic syndrome has improved. However, effective ...

A High Throughput, Multiplexed and Targeted Proteomic CSF Assay to Quantify Neurodegenerative Biomarkers and Apolipoprotein E Isoforms Status

En højt gennemløb, Multipleksede og målrettet proteomiske CSF Assay at kvantificere neurodegenerative Biomarkører og apolipoprotein E Isoformer status

Many neurodegenerative diseases are&#160;still lacking effective treatments. Reliable biomarkers for identifying and classifying these diseases will be ...

A Kinetic Fluorescence-based Ca2+ Mobilization Assay to Identify G Protein-coupled Receptor Agonists, Antagonists, and Allosteric Modulators

A Kinetic Fluorescence-based Ca2+ Mobilization Assay to Identify G Protein-coupled Receptor Agonists, Antagonists, and Allosteric Modulators

En kinetisk fluorescens-baserede Ca2 + mobilisering Assay at identificere G Protein-koblet Receptor agonister og antagonister allosteriske modulatorer

G protein-coupled receptors (GPCRs) are of great importance to the pharmaceutical industry as they are involved in many human diseases and include ...

Characterization of Immune Cells in Human Adipose Tissue by Using Flow Cytometry

Karakterisering af immunceller i menneskers fedtvæv ved hjælp af flowcytometri

Infiltration of immune cells in the subcutaneous and visceral adipose tissue (AT) deposits leads to a low-grade inflammation contributing to the ...

High-throughput Nitrobenzoxadiazole-labeled Cholesterol Efflux Assay

Høj overførselshastighed Nitrobenzoxadiazole-mærket Cholesterol Efflux Assay

Atherosclerosis leads to cardiovascular disease (CVD). It is still unclear whether cholesterol-HDL (cHDL) concentration plays a causal role in ...

Application of Biochip Microfluidic Technology to Detect Serum Allergen-specific Immunoglobulin E (sIgE)

Application of Biochip Microfluidic Technology to Detect Serum Allergen-specific Immunoglobulin E sIgE

Anvendelse af Biochip mikrofluid teknologi at detektere Serum Allergen-specifikt immunglobulin E (sIgE)

Allergic disease is common in both adults and children. Identification of the causative allergens is significant in disease management and prevention. ...

Improved Renal Denervation Mitigated Hypertension Induced by Angiotensin II Infusion

Forbedret nyredenervation afbødet hypertension induceret af Angiotensin II infusion

The benefits of renal sympathetic denervation (RDN) on blood pressure have been proved in a large number of clinical trials in recent years. However, the ...