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. Fremstilling av monoklonalt antistoff <ol><li> Kultur RG-M56 musmonoklonale hybridomceller 2 i serumfritt medium i 175T kolber ved 37 ºC med 5% CO 2 supplement. Samle supernatanten når fargen av mediet blir gul etter fem dagers inkubasjon. MERK: Hybridomceller ble dyrket i serumfritt medium for å unngå forurensning antistoff fra føtalt bovint serum. </li><li> Sentrifuger supernatanten ved 4500 xg i 30 min ved 4 ° C og kast celleavfall pellet. </li><li> Tilsett 2 ml pr...

<ol>
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Denne protokollen gir en hurtig og enkel teknikk for å identifisere en mAb-gjenkjent lineær epitop. Tatt i betraktning kostnadene for peptidsyntese og produksjonseffektivitet til å syntetisere peptider, ble den antigene region av viruskappeproteinet redusert ved å uttrykke i serie trunkerte rekombinante proteiner før peptidet scanning-analyse. Som sådan, er en pålitelig og effektiv E. coli pET-ekspresjonssystemet anvendt for å fremstille disse serielt trunkerte rekombinante proteiner som rekombinante pr...

I immunsystemet, det rekombinasjon av V, D og J segmentene gjør det mulig for antistoffer til å skape store variasjoner av komplementaritetsbestemmende regioner (CDR) for binding til forskjellige antigener for å beskytte verten fra sykdomsfremkallende infeksjon. Den nøytraliserende forsvar av antistoffer mot antigener som er avhengig av den romlige komplementaritet mellom CDR&#39;ene fra antistoffene og epitoper av antigenene. Derfor vil en forståelse av denne molekylær interaksjon hjelpe profylaktisk vaksine utforming og terapeutiske peptidlegemiddel-utvikling. Dette kan imidlertid nøytralisering interaksjonen bli påvirket både av flere antigene domener fra ett enkelt antigen, og av flere CDR fra antistoffer, som dermed gjør det epitop bestemmelse prosessen mer kompleks. Heldigvis utvikling av hybridoma-teknologi, som sikringer individuelle antistoffproduserende celler med myelomceller, gjør det mulig for en kontinuerlig delende gruppe med celler for å sekrete en spesifikt antistoff, kjent som et monoklonalt antistoff (mAb) 1. Hybridomceller som produserer slike ren, høy-affinitets mAb til å bindes til en enkelt antigenisk område av et spesifikt antigen. Med forholdet mellom antigen-antistoff-etablert, flere fremgangsmåter, inkludert peptid skanning, kan brukes til å bestemme den epitop av et antigen ved hjelp av tilsvarende mAb. Den siste utviklingen i syntetisk peptid teknologi har gjort peptid skanning teknikk mer tilgjengelig og mer praktisk å utføre. I korthet er et sett av overlappende syntetiske peptider fremstilt i henhold til et mål-antigen sekvens og er forbundet til en fast støttede membran for mAb hybridisering. Peptid skanning ikke bare gir en enkel måte for å kartlegge antistoffbindingsregion, men letter også aminosyre (aa) mutagenese gjennom rest skanning eller substitusjon for å evaluere bindingsinteraksjonen mellom hver aa rest av epitopen peptidet og CDR&#39;ene i antistoffet. <p class = &quot;jove_content&quot;&gt; Her, beskriver den foreliggende studien en protokoll for effektiv identifikasjon av den lineære epitop av gule havabbor nervøs nekrose virus (YGNNV) kappeproteinet ved hjelp av en nøytraliserende mAb to, tre, fire. Protokollen omfatter mAb forberedelse, konstruksjon og ekspresjon av serielt trunkerte rekombinante proteiner, syntetiske overlappende peptid utforming, dot-blot-hybridisering, alanin-skanning, og substitusjon mutagenese. Tatt i betraktning den høye kostnaden for peptid-syntese, ble det trinn å serielt å avkorte de rekombinante proteiner fra et ønsket mål-protein modifiseres, og det antigeniske region ble redusert til rundt 100 til 200 aa rester før det syntetiske peptid matrisen dot-blot-analyse ble utført.

<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

Identifiseringen av en antigen epitop av immunsystemet åpner for forståelsen av den beskyttende mekanisme av nøytraliserende antistoffer som kan fremme utvikling av vaksiner og peptid narkotika. Peptid-skanning er en enkel og effektiv metode som oversiktlig kartlegger lineær epitop som gjenkjennes av et monoklonalt antistoff (mAb). Her forfatterne presenterer en epitop besluttsomhet metodikk som involverer serielt avkortede rekombinante proteiner, syntetisk peptid design, og dot-blot hybridisering for antigen anerkjennelse av nervøs nekrose virus kappeproteinet ved hjelp av en nøytraliserende mAb. Denne teknikk er avhengig av dot-blot-hybridisering av syntetiske peptider og mAb&#39;er på en polyvinylidenfluorid (PVDF) membran. Den minimale antigene region av et viralt kappeprotein som gjenkjennes av RG-M56 mAb kan begrenses ved steg-for-steg trimmet peptidkartlegging på en 6-mer peptid-epitop. I tillegg, alanin mutagenese og residuet ble underGrunnloven kan bli utført for å karakterisere bindingen betydningen av hver aminosyrerest som utgjør epitopen. Restene flankerer epitop nettstedet ble funnet å spille viktige roller i peptid konformasjon regulering. Den identifiserte epitop peptidet kan bli anvendt for å danne krystaller av epitop peptid-antistoff-komplekser for en røntgendiffraksjon studium og funksjonell konkurranse, eller for terapeutiske midler.

Målet med dette forsøket var å identifisere en epitop gjennom dot blotting ved hjelp av mAb. Til hurtig og effektivt begrense det antigene området gjenkjent av mAb, ble full-lengde og avkortede serielt YGNNV rekombinante kappeproteiner med en 6xHis fusjon kode ved C-terminus uttrykt fra en E. coli-ekspresjonssystem PET 10 (figur 1A). De resulterende rekombinante proteiner ble flekket ut på PVDF-membran ved hjelp av RG-M56 mAb og anti...

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 Skanning-assistert Identifisering av et monoklonalt antistoff-gjenkjent Linear B-celle-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.

Research

JoVE Journal

Immunology and Infection

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

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

Peptide-baserte Identifikasjon av Funksjonelle Motifs og deres Binding Partnere

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 forløper B-celle undergrupper fra navlestrengsblod

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

Isolasjon, Identifikasjon, og rensing av Muse thymisk 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 monoklonale antistoffer fra mus ved Hybridoma Technology

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

Analyse av Simian Immunodeficiency Virus-spesifikke CD8<sup&gt; +</sup&gt; T-celler i rhesusape av Peptide-MHC-I Tetramer Farging

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

Generasjon av Escape varianter av nøytralisere influensa Virus monoklonale antistoffer

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

Rensing av Viral DNA for identifikasjon av tilknyttet Viral og cellulær 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

Overlappende peptid biblioteket tilordne Qa-1 Epitopes i et 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

Zika Virus spesifikke diagnostiske Epitope oppdagelsen

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