Name: a high throughput mhc ii binding assay for quantitative analysis of peptide epitopes
Uploaded: 2014-03-25T13:45:00
Description: Watch this Scientific Journal Video about A High Throughput MHC II Binding Assay for Quantitative Analysis of Peptide Epitopes at JoVE.com

Dette arbejde blev støttet af tilskud fra NIH R01-GM-098.977 og R21-AI-098.122 til CBK og KEG. RSS blev støttet delvist af en Luce Foundation Fellowship og delvist af en Thayer Innovation Program Fellowship fra Thayer School of Engineering.

Fire store aktiviteter omfatter bindende peptid-MHC II-analysen: 1-Binding: Test peptider konkurrere med mærkede kontrol peptider til opløsning fase binding af opløselige MHC II-proteiner. Binding måles over en bred vifte af test-peptidkoncentrationer. 2-Capture: Efter bindingsreaktionen nærmer ligevægt, er peptid-MHC II-komplekserne indfanges og adskilt fra ubundet peptid og protein ved konformationsafhængige anerkendelse med et immobiliseret antistof. 3-Detection: Captured kontrol peptid kvantitativt påvises v...

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Bioterapeutiske har etableret sig som en hjørnesten i moderne medicin, der repræsenterer fire af de fem bedste sælgende lægemidler i 2012 32. Den Biopharmaceutics sektor har vist en vedvarende vækst i flere år 6, og den igangværende udvikling af nye stoffer, samt fremkomsten af biosimilars har udvidet biofarmaceutiske rørledninger. Et blik på fremtiden, vurdere og afbøde immunogeniciteten af ​​terapeutiske proteiner vil blive en integreret del af tidlig fase bioterapeutisk udvikling. ...

Proteiner er den hurtigst voksende klasse af terapeutiske midler 6, og den hurtige ekspansion af bioterapeutiske rørledninger har fokuseret stigende opmærksomhed på de udfordringer, der er forbundet med udvikling og anvendelse af proteinbaserede lægemidler. En unik overvejelse stammer fra det faktum, at i et sundt og fungerende immunsystem, er alle ekstracellulære proteiner udskilt af antigenpræsenterende celler (APC&#39;er). Når internaliseret af APC&#39;er, er et protein, spaltes i små peptidfragmenter, og formodede immunogene segmenter er lagt ind i rillen af ​​klasse II store histokompatibilitetskompleks proteiner (MHC II). Peptid-MHC II-komplekser vises derefter på APC-overfladen og sande immunogene peptider, betegnet T-celleepitoper, danner ternære MHC II-peptid-T-celle-receptor-komplekser med kognate CD4 T-celleoverfladereceptorer 7. Denne kritiske molekylær genkendelse begivenhed starter en kompleks signaleringskaskade, hvilket resulterer i T-celle-aktivering, frigivelse af cytokins, CD4 T-cellemedieret B-celle modning og i sidste ende produktion af cirkulerende IgG-antistoffer, som binder til og rydde ulovlige exogent protein. Således kan immunogene proteiner deimmunized ved at identificere udgør T-celleepitoper og mutere nøglegrupper er ansvarlige for MHC II-komplekset formation. Det bærer imidlertid bemærker, at T-celle-epitoper kan være mange og bredt fordelt i hele immunogene proteiner, og størstedelen af ​​epitop-slette mutationer kan forårsage en utilsigtet tab af protein funktion eller stabilitet. Derfor teknik deimmunized bioterapier kan være en kompliceret og teknisk udfordrende mål, men der findes flere eksempler på vellykkede T-celle epitop sletning projekter 3,5,8-12. I modsætning til podning-baserede &quot;humanisering&quot;, som stort set begrænset til antistoflægemidler, kan anvendes epitop sletning til stort set alle protein-target uanset sekvens, struktur, funktion eller tilgængeligheden af ​​homologoos menneskelige stillads. Det første skridt til at gennemføre en sådan tilgang er at identificere de vigtigste peptidepitoper indlejret i målproteinsekvensen. High throughput biokemiske analyser ved hjælp af syntetiske peptider og rekombinante humane MHC II-molekyler kan give hurtige foreløbige indsigt i identifikation og afhjælpning 1,3-5 epitop. Disse ELISA-assays kan være et effektivt supplement til andre protein / vaccine design og udviklingsværktøjer. For eksempel er en veletableret eksperimenterende tilgang til epitopkortlægning bygger på tid, arbejdskraft og ressourcekrævende ex vivo celleproliferationsassays 15. Kort fortalt er den primære sekvens af et målprotein først opdelt i et panel af overlappende peptider overlapper ofte 15-merer med 12 rester mellem tilstødende peptider. Peptidet panel kemisk syntetiseret og immunogeniciteten af ​​hvert peptid afprøves i en af ​​flere forskellige immunoassays, der anvender perifere blood mononukleære celler (PBMC) isoleret fra humane donorer 13,14. At give større tillid til resultaterne, peptider typisk testet i replikat med PBMC fra 50 eller flere forskellige donorer. I tilfælde, hvor deimmunization er det ultimative mål, er det arbejde, forværres yderligere af behovet for at producere yderligere paneler af muterede peptider og afprøve de nye peptid-paneler i PBMC analyser, før der indføres nogen deimmunizing mutationer i proteinet i fuld længde for efterfølgende funktionel analyse 10. Mens disse cellulære assays fortsat guldstandarden for vurdering immunogen potentiale i menneskelige patienter, kan effektiviteten af ​​en sådan udtømmende tilgang forbedres ved forfiltrering formodede immunogene epitoper ved hjælp af en hurtig og høj MHC II-peptid-binding assay. Ligeledes kan biokemiske peptid-MHC II bindingsassays kombineres med prædiktiv in silico metoder til at radikalt fremskynde epitop identifikationsprocessen.Der findes en række beregningsværktøjer til T-celle epitop forudsigelse; eksempler ProPred 16 MHCPred 17. SVRMHC 18, ARB 19, SMM-tilpasse 20 NetMHCIIpan 21 samt proprietære værktøjer såsom EpiMatrix af EpiVax 22. Ligeledes har epitop prædiktorer for nylig blevet kombineret med andre bioinformatik og molekylære modelleringsværktøjer til afkaster integrerede protein deimmunization algoritmer designet til at mindske risikoen for, at deimmunizing mutationer kan forstyrre proteiners struktur og funktion 23-26. Mens flere epitop prædiktorer har vist sig at være rimeligt nøjagtige 27,28, beregningsmæssige resultater uvægerligt kræve eksperimentel validering. Hurtig og høj kapacitet, og omkostningseffektive eksperimentelle metoder er bedst egnet som en foreløbig filter for in silico epitop forudsigelser. I en lignende åre kan epitop prædiktorer drive antigen udvælgelse til omvendt vaccinology 29,30. For eksempel har fremskridt i bioinformatik gav hele genomet skærme, der hurtigt identificerer vaccinekandidater i form af hele proteiner eller peptidepitoper udvundet patogen proteomer. Mens dette nødvendig teknologi er omformningen opdagelse og udvikling af beskyttende vacciner, det indfører en ny udfordring i form af intractably store lister af immunogene vaccine kandidater. High throughput peptid-MHC II bindingsassays kan vejlede epitop udvælgelse ved at kvantificere peptid bindingsaffinitet og bindende promiskuitet blandt flere MHC II alleler. Som med protein deimmunization er sådanne eksperimentelle metoder i sidste ende forpligtet til at validere beregningsmæssige forudsigelse af lovende vaccine kundeemner. Her er en bindende peptid-MHC II-analysen skaleret til 384-brønds format beskrevet. Protokollen er meget paralleliseret og reducerer reagensomkostninger med 75% i forhold til tidligere beskrevne 96-brønds plade formater 1,3-5. Hjælp af en enkelt liquid håndteringsrobot, denne metode giver en forsker for nemt at analysere ca ninety testpeptider i tre eksemplarer over en række på otte koncentrationer og fire MHC II allel typer i mindre end 48 timer. Denne artikel beskriver opsætningen af ​​en 384-brønds ELISA-plade til analyse af syv eksperimentelle peptider mod et MHC II-allel, men regneark regnemaskiner leveres som supplerende materiale så nemt skalere eksperimentet til et vilkårligt antal af ønskede peptider og / eller MHC II-molekyler.

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			<td height="35" style="height:35px;width:587px;">Sodium Tetraborate Decahydrate</td>
			<td style="width:357px;">Sigma</td>
			<td style="width:217px;">221732</td>
			<td></td>
		</tr>
		<tr height="35">
			<td height="35" style="height:35px;">Citric Acid</td>
			<td>Sigma</td>
			<td>C1901</td>
			<td></td>
		</tr>
		<tr height="35">
			<td height="35" style="height:35px;">Dibasic Sodium Phosphate</td>
			<td>Sigma</td>
			<td>S7907</td>
			<td></td>
		</tr>
		<tr height="35">
			<td height="35" style="height:35px;">Trizma HCl</td>
			<td>OmniPur</td>
			<td>9310</td>
			<td></td>
		</tr>
		<tr height="35">
			<td height="35" style="height:35px;">Tween-20</td>
			<td>Sigma</td>
			<td>P7949</td>
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			<td height="35" style="height:35px;">100% DMSO</td>
			<td>Sigma</td>
			<td>D8418</td>
			<td></td>
		</tr>
		<tr height="35">
			<td height="35" style="height:35px;">Octyl-&#946;-D-Glucopyranaside</td>
			<td>Fischer</td>
			<td>29836-26-8</td>
			<td></td>
		</tr>
		<tr height="35">
			<td height="35" style="height:35px;">Pefa Bloc SCF</td>
			<td>Roche</td>
			<td>1158591600</td>
			<td></td>
		</tr>
		<tr height="35">
			<td height="35" style="height:35px;">Dulbecco&#8217;s 10X Phosphate Buffered Saline</td>
			<td>Invitrogen</td>
			<td>14200-166</td>
			<td></td>
		</tr>
		<tr height="35">
			<td height="35" style="height:35px;">DELFIA Assay Buffer</td>
			<td>Perkin Elmer</td>
			<td>4002-0010</td>
			<td></td>
		</tr>
		<tr height="35">
			<td height="35" style="height:35px;">DELFIA Enhancement Buffer</td>
			<td>Perkin Elmer</td>
			<td>4001-0010</td>
			<td></td>
		</tr>
		<tr height="35">
			<td height="35" style="height:35px;">Europium Labelled Streptavidin</td>
			<td>Perkin Elmer</td>
			<td>1244-360</td>
			<td></td>
		</tr>
		<tr height="35">
			<td height="35" style="height:35px;">L243 anti-HLA-DR antibody</td>
			<td>Biolegend</td>
			<td>307602</td>
			<td></td>
		</tr>
		<tr height="35">
			<td height="35" style="height:35px;">Biotinylated tracer peptides</td>
			<td>21st Century Biochemicals</td>
			<td>Custom Order</td>
			<td></td>
		</tr>
		<tr height="35">
			<td height="35" style="height:35px;">Test peptides (1-4mg, 85% purity)</td>
			<td>Genscript</td>
			<td>Custom Order</td>
			<td></td>
		</tr>
		<tr height="35">
			<td height="35" style="height:35px;">Purified HLA-DRB1 monomers (non-biotinylated)</td>
			<td>Benaroya Research Institute*</td>
			<td>Custom Order</td>
			<td></td>
		</tr>
		<tr height="35">
			<td height="35" style="height:35px;">384-well white EIA/RIA plate</td>
			<td>Thermo</td>
			<td>460372</td>
			<td></td>
		</tr>
		<tr height="35">
			<td height="35" style="height:35px;">Polypropylene 384-well plate</td>
			<td>Costar</td>
			<td>3656</td>
			<td></td>
		</tr>
		<tr height="35">
			<td height="35" style="height:35px;">Film, AxySeal, 80 &#181;m, ELISA Applicator&#160;</td>
			<td>Axygen Asicentific</td>
			<td>PCR-SP</td>
			<td></td>
		</tr>
		<tr height="35">
			<td height="35" style="height:35px;">MilliQ Water</td>
			<td>N/A</td>
			<td>N/A</td>
			<td></td>
		</tr>
		<tr height="35">
			<td height="35" style="height:35px;">Epimotion Liquid Handler (or similar)</td>
			<td>Eppendorf</td>
			<td>5075</td>
			<td></td>
		</tr>
		<tr height="35">
			<td height="35" style="height:35px;">Select TS Plate Washer (or similar)</td>
			<td>BioTek</td>
			<td>405</td>
			<td></td>
		</tr>
		<tr height="35">
			<td height="35" style="height:35px;">SpectraMax Gemini Microplate Reader (or similar)</td>
			<td>Molecular Devices</td>
			<td>N/A</td>
			<td></td>
		</tr>
		<tr>
			<td colspan="4" height="98" style="height:99px;width:1161px;">*Recombinant human MHC II molecules can be obtained from the Benaroya Tetramer Core Laboratory. See: https://www.benaroyaresearch.org/our-research/core-resources/tetramer-core-laboratory</td>
		</tr>
	</tbody>
</table>

a high throughput mhc ii binding assay for quantitative analysis of peptide epitopes

Biokemiske analyser med rekombinant humant MHC II-molekyler kan give hurtige, kvantitative indsigt i immunogen epitop identifikation, sletning eller design 1,2. Her er en bindende peptid-MHC II-analysen skaleret til 384-godt-format. Den skaleret ned protokol reducerer reagensomkostninger med 75%, og er højere gennemløb end tidligere beskrevet 96-brønds protokoller 1,3-5. Specifikt eksperimentelle design tillader robust og reproducerbar analyse af op til 15 peptider mod et MHC II allel pr 384-brønds ELISA-pladen. Ved hjælp af en enkelt væskehåndtering robot, giver denne metode en forsker for at analysere ca ninety testpeptider i tre eksemplarer over et område af otte koncentrationer og fire MHC II allel typer i mindre end 48 timer. Andre, der arbejder inden for protein deimmunization eller vaccine design og udvikling kan finde protokollen til at være nyttige i at fremme deres eget arbejde. Især de trin-for-trin instruktioner og visuelle formataf JOVE bør give andre brugere mulighed for hurtigt og nemt at etablere denne metode i deres egne laboratorier.

Den modne peptidsekvens af Enterobacter cloacae P99 beta-lactamase-(BLA) (Genbank ID # X07274.1) blev analyseret med ProPred 16 for formodede peptid bindemidler til MHC II allelen DRB1 * 1501 (Tabel 4). ProPred identificeret 117 nonamere peptider med en obligatorisk P1 ankerenhed (dvs. M, L, I, V, F, Y eller W i position 1, som er nødvendig for MHC-II-bindende 31). Ved en tærskel på 5% peptider kun med scoringer større end eller lig med 2,6 er sandsynlige bind...

Watch this Scientific Journal Video about A High Throughput MHC II Binding Assay for Quantitative Analysis of Peptide Epitopes at JoVE.com

A High Throughput MHC II Binding Assay for Quantitative Analysis of Peptide Epitopes

Biokemiske analyser med rekombinant humant MHC II-molekyler kan give hurtige, kvantitative indsigt i immunogen epitop identifikation, sletning eller design. Her er en bindende peptid-MHC II-analysen skaleret til 384-brønds plader beskrevet. Denne omkostningseffektive format skulle vise sig nyttig i de områder af protein deimmunization og vaccine design og udvikling.

En High Throughput MHC II Bindende Assay til kvantitativ analyse af peptidepitoper

Biochemical assays with recombinant human MHC II molecules can provide rapid, quantitative insights into immunogenic epitope identification, deletion, or ...

Research

JoVE Journal

Biology

Visualizing Antigen Specific CD4+ T Cells using MHC Class II Tetramers

Major histocompatibility complex (MHC) class II tetramers allow the direct visualization of antigen specific CD4+ T cells by flow cytometry.  This method ...

A Neuronal and Astrocyte Co-Culture Assay for High Content Analysis of Neurotoxicity

High Content Analysis (HCA) assays combine cells and detection reagents with automated imaging and powerful image analysis algorithms, allowing ...

Mutagenesis and Functional Analysis of Ion Channels Heterologously Expressed in Mammalian Cells

We will demonstrate how to study the functional effects of introducing a point mutation in an ion channel. We study G protein-gated inwardly rectifying ...

High-throughput Saccharification Assay for Lignocellulosic Materials

Polysaccharides that make up plant lignocellulosic biomass can be broken down to produce a range of sugars that subsequently can be 
used in establishing ...

A Quantitative Fitness Analysis Workflow

En kvantitativ Fitness Analyse Workflow

Quantitative Fitness Analysis (QFA) is an experimental and computational workflow for comparing fitnesses of microbial cultures grown in parallel1,2,3,4. ...

High-throughput Functional Screening using a Homemade Dual-glow Luciferase Assay

High-throughput Funktionel Screening ved hjælp af en hjemmelavet Dual-glød Luciferase Assay

We present a rapid and inexpensive high-throughput screening protocol to identify transcriptional regulators of alpha-synuclein, a gene associated with ...

Flow Cytometric Analysis of Bimolecular Fluorescence Complementation: A High Throughput Quantitative Method to Study Protein-protein Interaction

Flow cytometri af bimolekylær Fluorescens Complementation: En High Throughput Kvantitativ metode til at studere protein-protein interaktion

Among methods to study protein-protein interaction inside cells, Bimolecular Fluorescence Complementation (BiFC) is relatively simple and sensitive. BiFC ...

High Throughput Microinjections of Sea Urchin Zygotes

High Throughput Microinjections af søpindsvin Zygoter

Microinjection into cells and embryos is a common technique that is used to study a wide range of biological processes. In this method a small amount of ...

High-throughput Screening for Chemical Modulators of Post-transcriptionally Regulated Genes

High-throughput screening for kemiske modulatorer af post-transkriptionelt regulerede gener

Both transcriptional and post-transcriptional regulation have a profound impact on genes expression. However, commonly adopted cell-based screening assays ...

qPCRTag Analysis - A High Throughput, Real Time PCR Assay for Sc2.0 Genotyping

qPCRTag Analyse - En High Throughput, Real Time PCR-analysen for Sc2.0 Genotypebestemmelse

The Synthetic Yeast Genome Project (Sc2.0) aims to build 16 designer yeast chromosomes and combine them into a single yeast cell. To date one synthetic ...