Name: peptide scanning-assisted identification of a monoclonal antibody-recognized linear b-cell epitope
Uploaded: 2017-03-24T14:00:00
Description: Watch this Scientific Journal Video about Peptide Scanning-assisted Identification of a Monoclonal Antibody-recognized Linear B-cell Epitope at JoVE.com

The authors thank Miss Ching-Chun Lin and Miss Diana Lin of the Core Facility of the Institute of Cellular and Organismic Biology (ICOB) of Academia Sinica for offering their expertise on peptide synthesis and DNA sequencing, respectively. This study was supported by Academia Sinica.

1. Framställning av monoklonal antikropp <ol><li> Kultur de RG-M56 musmonoklonala hybridomceller 2 i serumfritt medium i 175T-kolvar vid 37 ° C med 5% CO2 tillägg. Samla supernatanten när färgen på mediet blir gult efter fem dagars inkubation. OBS: Hybridomceller odlades i serumfritt medium för att undvika antikropps kontamination från fetalt bovint serum. </li><li> Centrifugera supernatanten vid 4500 xg under 30 min vid 4 ° C och kasta celldebris pellet. </li><li> Ti...

<ol>
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C., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Crystal+Structures+of+a+Piscine+Betanodavirus:+Mechanisms+of+Capsid+Assembly+and+Viral+Infection.">Crystal Structures of a Piscine Betanodavirus: Mechanisms of Capsid Assembly and Viral Infection.</a> PLoS Pathog. 11 (10), e1005203 (2015).</li><li>Badani, H., Garry, R. F., Wimley, W. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Peptide+entry+inhibitors+of+enveloped+viruses:+The+importance+of+interfacial+hydrophobicity.">Peptide entry inhibitors of enveloped viruses: The importance of interfacial hydrophobicity.</a> Biochim Biophys Acta. , (2014).</li><li>Qureshi, N. M., Coy, D. H., Garry, R. F., Henderson, L. 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Detta protokoll ger en snabb och enkel teknik för att identifiera en mAb erkända linjär epitop. Med hänsyn till kostnaden för peptidsyntes och produktionseffektiviteten för syntetisera peptider, var den antigena regionen av virushöljeproteinet reduceras genom att uttrycka seriellt stympade rekombinanta proteiner före peptidavsökningsanalys. Som sådan var den pålitliga och effektiva E.coli pET-expressionssystem som används för att producera dessa seriellt stympade rekombinanta proteiner, såsom reko...

I immunsystemet, tillåter rekombinationen av V-, D-, och J-segment för antikroppar för att skapa enorma variationer av komplementaritetsbestämmande regioner (CDR) för bindning till olika antigener för att skydda värden från patogen infektion. Den neutraliserande försvar av antikroppar mot antigener beror på den rumsliga komplementaritet mellan de CDR av antikropparna och de epitoper av antigenerna. Därför kommer en förståelse av denna molekylär interaktion hjälpa profylaktiskt vaccin design och terapeutiska peptiden läkemedelsutveckling. Emellertid kan denna neutralisation växelverkan påverkas både genom multipla antigena domäner från en enda antigen och genom multipla CDRs av antikroppar, vilket följaktligen gör epitopen bestämningsprocessen mer komplex. Lyckligtvis möjliggör en ständigt dividera sats av celler till sek utveckling av hybridom-teknik, som säkringar individuella antikroppsproducerande celler med myelomceller,rete en specifik antikropp, känd som en monoklonal antikropp (mAb) 1. Hybridomceller producerar dessa rena, med hög affinitet mAbs att binda till ett enda antigent domänen av ett specifikt antigen. I förhållandet mellan antigen-antikropps etablerad, flera tillvägagångssätt, inklusive peptid skanning, kan användas för att bestämma epitopen av ett antigen med användning av dess motsvarande mAb. Den senaste utvecklingen inom syntetisk peptid teknik har gjort peptid scanning tekniken mer tillgänglig och enklare att utföra. I korthet är en uppsättning av överlappande syntetiska peptider som framställts i enlighet med en målantigen-sekvens och är associerade till en fast-uppburen membran för mAb hybridisering. Peptid scanning erbjuder inte bara ett enkelt sätt att kartlägga den antikroppsbindande regionen, utan underlättar också aminosyra (aa) mutagenes genom rest scanning eller substitution för att utvärdera bindningsväxelverkan mellan varje aa rest av epitopen peptiden och CDR av antikroppen. <p class = &quot;jove_content&quot;&gt; Här beskriver föreliggande studie ett protokoll för effektiv identifiering av den linjära epitopen av den gula grouper nervösa necrosis virus (YGNNV) höljesprotein med användning av en neutraliserande mAb 2, 3, 4. Protokollet innehåller mAb förberedelse, konstruktion och uttryck av serie stympade rekombinanta proteiner, syntetiska överlappande peptid design, dot-blot-hybridisering, alaninavsökning och substitutionsmutagenes. Med tanke på de höga kostnaderna för peptidsyntes, var steget att seriellt trunkera de rekombinanta proteiner av ett önskat målprotein modifieras och den antigena regionen minskat ner till omkring 100 till 200 aa-rester innan den syntetiska peptiden array dot-blot-analys utfördes.

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>Hybrid-SFM medium</td>
			<td>Gibco</td>
			<td>12045-076</td>
			<td></td>
		</tr>
		<tr>
			<td>Dulbeccos&#39;s Phophate-Buffered Saline (PBS)</td>
			<td>Gibco</td>
			<td>21600-069</td>
			<td></td>
		</tr>
		<tr>
			<td>Pfu DNA Polymerase&#160;</td>
			<td>Thermo Scientific&#160;</td>
			<td>EP0502&#160;</td>
			<td>Including buffers</td>
		</tr>
		<tr>
			<td>T4 DNA Ligase</td>
			<td>Roche</td>
			<td>10799009001</td>
			<td>Including buffers</td>
		</tr>
		<tr>
			<td>NdeI&#160;</td>
			<td>New England Biolabs</td>
			<td>R0111S</td>
			<td>Including buffers</td>
		</tr>
		<tr>
			<td>XhoI&#160;</td>
			<td>New England Biolabs</td>
			<td>R0146S</td>
			<td>Including buffers</td>
		</tr>
		<tr>
			<td>pET-20b(+) vector</td>
			<td>Novagen, Merck Millipore</td>
			<td>69739</td>
			<td></td>
		</tr>
		<tr>
			<td>E.coli DH-5&#945; competent cell</td>
			<td>RBC Bioscience</td>
			<td>RH617</td>
			<td></td>
		</tr>
		<tr>
			<td>E.coli BL-21(DE3) competent cell</td>
			<td>RBC Bioscience</td>
			<td>RH217</td>
			<td></td>
		</tr>
		<tr>
			<td>Ampicillin</td>
			<td>Amresco</td>
			<td>0339-25G</td>
			<td></td>
		</tr>
		<tr>
			<td>LB broth&#160;</td>
			<td>Invitrongen</td>
			<td>12780-052</td>
			<td></td>
		</tr>
		<tr>
			<td>Isopropylthio-&#946;-D-thiogalactoside (IPTG)</td>
			<td>MDBio, Inc.</td>
			<td>101-367-93-1</td>
			<td></td>
		</tr>
		<tr>
			<td>Methanol</td>
			<td>Merck Millipore</td>
			<td>106009</td>
			<td></td>
		</tr>
		<tr>
			<td>Polyoxyethylene 20 Sorbitan Monolaurate (Tween-20)</td>
			<td>J.T.Baker</td>
			<td>X251-07</td>
			<td></td>
		</tr>
		<tr>
			<td>Dimethyl sulfoxide (DMSO)</td>
			<td>Sigma</td>
			<td>D2650</td>
			<td></td>
		</tr>
		<tr>
			<td>Glycine</td>
			<td>Amresco</td>
			<td>0167-5KG</td>
			<td></td>
		</tr>
		<tr>
			<td>Tris</td>
			<td>Affymetrix, USB</td>
			<td>75825</td>
			<td></td>
		</tr>
		<tr>
			<td>NaCl</td>
			<td>Amresco</td>
			<td>0241-1KG</td>
			<td></td>
		</tr>
		<tr>
			<td>EDTA</td>
			<td>Amresco</td>
			<td>0105-1KG</td>
			<td></td>
		</tr>
		<tr>
			<td>Glycerol</td>
			<td>Amresco</td>
			<td>0854-1L</td>
			<td></td>
		</tr>
		<tr>
			<td>NaN3</td>
			<td>Sigma</td>
			<td>S2002-500G</td>
			<td></td>
		</tr>
		<tr>
			<td>BCIP/NBT</td>
			<td>PerkinElmer</td>
			<td>NEL937001PK</td>
			<td></td>
		</tr>
		<tr>
			<td>Goat Anti-Mouse IgG, Fc fragment antibody</td>
			<td>Jackson ImmunoResearch</td>
			<td>115-055-008</td>
			<td></td>
		</tr>
		<tr>
			<td>Immobilon-P (Polyvinylidene fluoride, PVDF)</td>
			<td>Merck Millipore</td>
			<td>IPVH00010</td>
			<td></td>
		</tr>
		<tr>
			<td>Protein G Agarose Fast Flow</td>
			<td>Merck Millipore</td>
			<td>16-266</td>
			<td></td>
		</tr>
		<tr>
			<td>QIAquick PCR Purification kit</td>
			<td>Qiagen</td>
			<td>28106</td>
			<td></td>
		</tr>
		<tr>
			<td>UVP BioSpectrum 600 Image System</td>
			<td>UVP</td>
			<td>n/a</td>
			<td></td>
		</tr>
		<tr>
			<td>VisionWorks LS Analysis Software Ver 6.8</td>
			<td>UVP</td>
			<td>n/a</td>
			<td></td>
		</tr>
		<tr>
			<td>MyCycler thermal cycler</td>
			<td>BioRad</td>
			<td>1709713</td>
			<td></td>
		</tr>
	</tbody>
</table>

peptide scanning-assisted identification of a monoclonal antibody-recognized linear b-cell epitope

Identifieringen av en antigenepitop av immunsystemet gör det möjligt för förståelsen av skyddsmekanism av neutraliserande antikroppar som kan underlätta utvecklingen av vacciner och peptidläkemedel. Peptid scanning är en enkel och effektiv metod att rättframt kartor den linjära epitop som igenkännes av en monoklonal antikropp (mAb). Här presenterar författarna en epitop bestämningsmetod som involverar serie stympade rekombinanta proteiner, syntetisk peptid design och dot-blot-hybridisering för antigen erkännande av nervös necrosis virus coat protein med användning av en neutraliserande mAb. Denna teknik förlitar sig på dot-blot-hybridisering av syntetiska peptider och mAbs på en polyvinylidenfluorid (PVDF) membran. Den minsta antigena regionen av ett viralt höljeprotein som känns igen av den RG-M56 mAb kan inskränkas för steg-för-steg trimmas peptidkartläggning på en 6-mer-peptid-epitopen. Dessutom, mutagenes och återstoden alaninscanning subkonstitution kan utföras för att karaktärisera bindningen betydelsen av varje aminosyrarest som utgör epitopen. Resterna flankerar epitopen platsen konstaterades att spela kritiska roller i peptidkonforma reglering. Den identifierade epitopen peptiden kan användas för att bilda kristaller av epitoppeptiden-antikroppskomplex för en röntgendiffraktion studien och funktionell konkurrens, eller för terapeutika.

Målet med detta experiment var att identifiera en epitop genom punktblotting med hjälp av mAb. Att snabbt och effektivt begränsa den antigena regionen som känns igen av mAb, var av full längd och i serie stympade YGNNV rekombinant höljesproteiner med en 6xHis-fusions tagg vid C-terminus som uttrycks från en E. coli-PET-expressionssystemet 10 (Figur 1A). De resulterande rekombinanta proteiner sågs på PVDF-membranet med hjälp av R...

Watch this Scientific Journal Video about Peptide Scanning-assisted Identification of a Monoclonal Antibody-recognized Linear B-cell Epitope at JoVE.com

Peptide Scanning-assisted Identification of a Monoclonal Antibody-recognized Linear B-cell Epitope

Here, the authors present a simple and efficient protocol to define a linear antigenic epitope using a purified monoclonal antibody and peptide scanning through dot-blot hybridization. The identified epitope can then be used in therapeutic and diagnostic applications.

Peptid Scanning-assisterad Identifiering av en monoklonal antikropp-erkända Linear B-cell-epitop

The identification of an antigenic epitope by the immune system allows for the understanding of the protective mechanism of neutralizing antibodies that ...

The overall goal of this protocol is to identify a monoclonal antibody-recognized linear B-cell epitope using serially truncated recombinant proteins and peptide scanning by dot-blot hybridization. This method can help answer key questions concerning the molecule interaction between antigen and the antibody. The main advantages of this technique are its simplicity and efficiency.The identifying epitope can then be used in fuller investigations such as in therapeutical and diagnostic applications. Demonstrating the procedure will be Chien-Wen Chen, a post doctorate from my laboratory. To begin, grow RGM56 mouse monoclonal hybridoma cells in a hybrid serum free medium.Maintain cell cultures in 175 T flasks at 37 degrees Celsius 5%carbon dioxide. After five days of incubation, collect the culture medium after it has turned yellow and centrifuge the supernatant at 4, 500 times G for 30 minutes at four degrees Celsius. Then, discard the pelleted cell debris to obtain the antibody in solution.Next, add two milliliters of 50%protein G agro slurry to a five milliliter column and equilibrate the resin with 10 milliliters of ice cold PBS, equaling 10 volumes of preloaded resin. Load 200 milliliters of the previously prepared antibody solution onto the equilibrated column and discard the fraction that passes through the column. Using 10 milliliters of ice cold PBS, wash the column twice.Finally, to loot the antibody from the resin, add 10 milliliters of millimolar glycerin solution at pH 2.7 to the column. Collect 900 microliter fractions in microcentrifuge tubes previously filled with 100 microliters of 10X neutralization buffer. Store the purified antibody in 50%glycerol solution supplemented with sodium azide at minus 20 degrees Celsius.Prepare the constructs and transform E.coli BL21 cells as described in the text protocol. Then, transfer a single colony into a 15 milliliter tube containing three milliliters of LB broth supplemented with 100 micrograms per milliliter ampicillin and loosely cap the tube. Incubate the culture at 37 degrees Celsius with 150 RPM shaking.Monitor the optical density of the culture at 600 nanometers. When it reaches about 0.6, cool the culture to 25 degrees Celsius. Then, add IPTG at a final concentration of 0.4 millimolar to induce the expression of the recombinant protein.Incubate the culture for another four hours at 25 degrees Celsius with 200 RPM shaking. After incubation, transfer one milliliter of the bacterial culture into a microcentrifuge tube. Centrifuge the cells at 12, 000 times G for one minute and discard the supernatant.Pipetting up and down with a micropipette, resuspend the cell pellet in 100 microliters of denaturation buffer. And mix the resulting suspension by vigorous vortexing to obtain a ready to use recombinant protein sample. To proceed with dot-blot hybridization, dissolve each synthetic peptide in DMSO to obtain 10 milligram per milliliter solutions.Then, activate the PVDF membrane in methanol for two minutes and equilibrate it with modified Towbin buffer for another two minutes. Rinse a piece of chromatography paper with modified Towbin buffer and lay the equilibrated PVDF membrane on it. Let the membrane sit until the buffer disappears from its surface.Using a 10 microliter tip, add two microliters of each peptide or recombinant protein sample onto the membrane and let it air dry for 10 minutes. Spotting a symbol onto the membrane is the most critical step in this protocol. Loading a precise amount of a sample onto a small area of the PVDF membrane needs careful attention.Next, block the membrane in TBST buffer supplemented with 5%nonfat milk for 30 minutes at room temperature with gentle shaking. Dilute the RGM56 monoclonal antibody in TBST buffer supplemented with 5%nonfat milk. And add the antibody solution to the membrane.Incubate the membrane at 37 degrees Celsius for one hour with gentle shaking. After incubation, remove the antibody solution. And wash the membrane twice in TBST buffer for five minutes with gentle shaking.Then, add to the membrane the solution of the secondary antibody diluted in TBST buffer with 5%nonfat milk. Incubate the membrane for one hour at 37 degrees Celsius with gentle shaking. Upon incubation, discard the antibody solution and wash the membrane in TBST buffer twice for five minutes with gentle shaking.Then, develop the membrane with the substrate solution at room temperature for 15 minutes in the dark. When the signal appears, stop the reaction by washing the membrane with doubly distilled water. After air drying the membrane, capture the dot-blot image using an imaging system.Finally, using image analysis software measure the intensity of each dot-blot. Presented here is the full length nervous necrosis virus code protein comprising amino acids one through 338 along with its serially truncated variants. To confirm the expression of the proteins in the transformed bacterial cells, dot-blot analysis using anti-6XHis antibody was performed showing the presence of all the proteins under investigation.Then, the recombinant proteins were analyzed by dot-blot hybridization in which the RG-M56 monoclonal antibody was used. The analysis revealed that the protein epitope recognized by the antibody is found in peptide 195 to 338. Finally, an epitope consisting of 20 or eight amino acid residues was identified by peptide scanning.Alanine scanning showed that substitutions of valine residues at positions 197 and 199 as well as a cystine at 201 abolished the epitope's binding capability and dot-blot hybridization. Similarly, the decreased binding affinity was observed in binding intensity analysis confirming that the identified residues are essential for the epitope's binding to the RGM56 monoclonal antibody. This peptide identification technique can be used to develop therapeutical peptide drugs or peptide medicines.

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Immunology and Infection

Peptide:MHC Tetramer-based Enrichment of Epitope-specific T cells

Peptid: MHC Tetramer-baserad Anrikning av Epitop-specifika T-celler

A  basic necessity for researchers studying adaptive immunity with in vivo experimental models is an  ability to identify T cells based on their T cell ...

Peptide-based Identification of Functional Motifs and their Binding Partners

Peptid-baserad identifiering av funktionella motiv och deras bindningspartners

Specific short  peptides derived from motifs found in full-length proteins, in our case HIV-1  Nef, not only retain their biological function, but can ...

Isolation of Precursor B-cell Subsets from Umbilical Cord Blood

Isolering av prekursor B-cell-underuppsättningar från navelsträngsblod

Umbilical cord blood is highly enriched for hematopoietic progenitor cells at different lineage commitment stages. We have developed a protocol for ...

Isolation, Identification, and Purification of Murine Thymic Epithelial Cells

Isolering, identifiering och rening av murina Thymic epitelceller

The thymus is a vital organ for T lymphocyte development. Of thymic stromal cells, thymic epithelial cells (TECs) are particularly crucial at multiple ...

Generation of Murine Monoclonal Antibodies by Hybridoma Technology

Generering av murina monoklonala antikroppar genom hybridomteknik

Monoclonal antibodies are universal binding molecules and are widely used in biomedicine and research. Nevertheless, the generation of these binding ...

Analysis of Simian Immunodeficiency Virus-specific CD8+ T-cells in Rhesus Macaques by Peptide-MHC-I Tetramer Staining

Analysis of Simian Immunodeficiency Virus-specific CD8+ T-cells in Rhesus Macaques by Peptide-MHC-I Tetramer Staining

Analys av Simian Immunodeficiency Virus-specifika CD8<sup&gt; +</sup&gt; T-celler i rhesusmakaker av peptid-MHC-I tetramer färgning

Peptide-major histocompatibility complex class I (pMHC-I) tetramers have been an invaluable tool to study CD8+ T-cell responses. Because these reagents ...

Generation of Escape Variants of Neutralizing Influenza Virus Monoclonal Antibodies

Generation av Escape varianter av neutraliserande influensa Virus monoklonala antikroppar

Influenza viruses exhibit a remarkable ability to adapt and evade the host immune response. One way is through antigenic changes that occur on the surface ...

Purification of Viral DNA for the Identification of Associated Viral and Cellular Proteins

Rening av virus-DNA för identifiering av associerade Viral och cellulära proteiner

The goal of this protocol is to isolate herpes simplex virus type 1 (HSV-1) DNA from infected cells for the identification of associated viral and ...

Overlapping Peptide Library to Map Qa-1 Epitopes in a Protein

Överlappande peptid-bibliotek för att mappa Qa-1 epitoper i ett Protein

Qa-1 (HLA-E in human) belongs to a group of non-classical major histocompatibility complex 1b (MHC-Ib) molecules. Recent data suggest that Qa-1 molecules ...

Zika Virus Specific Diagnostic Epitope Discovery

Zikaviruset specifika diagnostiska epitop Discovery

High-density peptide microarrays allow screening of more than six thousand peptides on a single standard microscopy slide. This method can be applied for ...