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 af monoklonalt antistof <ol><li> Kultur de RG-M56 muse monoklonale hybridomaceller 2 i serum-frit medium i 175T kolber ved 37 ºC med 5% CO2 supplement. Opsaml supernatanten når farven af ​​mediet bliver gult efter fem dages inkubering. BEMÆRK: Hybridomceller blev dyrket i serumfrit medium for at undgå antistof forurening fra føtalt bovint serum. </li><li> Centrifugeres supernatanten ved 4.500 xg i 30 min ved 4 * C og kassér celledebris pellet. </li><l...

<ol>
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Denne protokol giver en enkel og hurtig teknik til at identificere et mAb-anerkendt lineær epitop. Under hensyntagen til omkostningerne ved peptidsyntese og produktionseffektiviteten til syntetisering peptider, blev den antigene region af viruscoat-proteinet reduceres ved at udtrykke serielt trunkerede rekombinante proteiner før peptidscanning analyse. Som sådan blev pålidelig og effektiv E. coli pET udtryk, der anvendes til at producere disse serielt trunkerede rekombinante proteiner, som rekombinante prot...

I immunsystemet, rekombination af V, D og J-segmenter giver mulighed for antistoffer mod skabe enorme variationer af komplementaritetsbestemmende regioner (CDR&#39;er) for binding til forskellige antigener til at beskytte værten mod patogen infektion. Den neutraliserende forsvar af antistoffer mod antigener afhænger af den rumlige komplementaritet mellem CDR&#39;erne af antistofferne og epitoperne af antigenerne. Derfor vil en forståelse af denne molekylære interaktion bistå profylaktisk vaccine design og terapeutisk peptid lægemiddeludvikling. Dette kan dog neutralisering interaktion påvirkes både af flere antigene domæner fra et enkelt antigen og ved multiple CDR&#39;er af antistoffer, som derfor gør epitop bestemmelsesproces mere kompleks. Heldigvis udviklingen af ​​hybridomteknologi, som sammensmelter individuelle antistofproducerende celler med myelomaceller, giver mulighed for en konstant dividere batch af celler til secrete én specifikt antistof, er kendt som et monoklonalt antistof (mAb) 1. Hybridomaceller producerer disse rene, høj affinitet mAb&#39;er til at binde til et enkelt antigent domæne af et specifikt antigen. Med forholdet af antigen-antistof etableret, flere tilgange, herunder peptid scanning, kan anvendes til at bestemme epitopen af ​​et antigen ved hjælp af dens tilsvarende mAb. Den seneste udvikling i syntetisk peptid-teknologi har gjort peptidet skanningsteknik mere tilgængelig og mere bekvemt at udføre. Kort fortalt et sæt af overlappende syntetiske peptider fremstillet ifølge et målantigen sekvens og er knyttet til en fast-understøttet membran til mAb hybridisering. Peptidscanning ikke kun tilbyder en enkel måde at kortlægge antistofbindende region, men også letter aminosyre (aa) mutagenese gennem rest scanning eller substitution at evaluere bindingsinteraktionen mellem hver aa rest af epitopen peptidet og CDR&#39;erne i antistoffet. <p class = &quot;jove_content&quot;&gt; Her den foreliggende undersøgelse beskriver en protokol for en effektiv identifikation af den lineære epitop af det gule grouper nervøs nekrose-virus (YGNNV) kappeprotein under anvendelse af et neutraliserende mAb 2, 3, 4. Protokollen indeholder mAb forberedelse, konstruktion og udtryk for serielt trunkerede rekombinante proteiner, syntetisk overlappende peptid design, dot-blot hybridisering, alaninscanning og substitution mutagenese. I betragtning af den høje pris for peptidsyntese, blev trinet med serielt trunkering af rekombinante proteiner med en ønsket målprotein modificeret, og den antigene region blev indsnævret til omkring 100 til 200 aa rester før det syntetiske peptid-array dot-blot-analyse blev udført.

<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

Identifikationen af ​​en antigen epitop af immunsystemet giver mulighed for at forstå den beskyttende mekanisme af neutraliserende antistoffer, som kan fremme udviklingen af ​​vacciner og peptidlægemidler. Peptid scanning er en enkel og effektiv metode, ligefremt kortlægger den lineære epitop genkendes af et monoklonalt antistof (mAb). Her, forfatterne præsenterer en epitop bestemmelse metode involverer serielt trunkerede rekombinante proteiner, syntetisk peptid design og dot-blot hybridisering for den antigene anerkendelse af nervøs nekrose viruscoat-protein under anvendelse af et neutraliserende mAb. Denne teknik bygger på dot-blot-hybridisering af syntetiske peptider og mAb&#39;er på en polyvinylidenfluorid (PVDF) membran. Den mindste antigene region af et viralt kappeprotein genkendes af RG-M56 mAb kan indsnævres ved trin-for-trin trimmet peptidkortlægning på en 6-mer peptid epitop. Desuden alaninscanningsmutagenese og restprodukt substitution kan udføres for at karakterisere bindingen betydning af hver aminosyrerest, der udgør epitopen. Resterne flankerer epitop stedet viste sig at spille kritiske roller i peptid kropsbygning regulering. Den identificerede epitop peptid kan anvendes til at danne krystaller af epitop peptid-antistof komplekser til en x-ray diffraktion undersøgelse og funktionel konkurrence eller til terapeutiske midler.

Målet med dette forsøg var at identificere en epitop gennem dot blotting under anvendelse af mAb. Hurtigt og effektivt at indsnævre den antigene region genkendes af mAb blev fuldlængde og serielt trunkerede YGNNV rekombinante kappeproteiner med en 6xHis fusion tag ved C-terminus udtrykt fra en E. coli-PET ekspressionssystem 10 (figur 1A). De resulterende rekombinante proteiner blev spottet på PVDF-membran under anvendelse af RG-M56 m...

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-assisteret Identifikation af et monoklonalt antistof-anerkendt Linear B-celleepitop

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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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

Peptid-baserede identifikation af funktionskravene motiver og deres bindingspartnere

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 af precursor 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

Isolering, identifikation og oprensning af murine thymusepitelmonolag Celler

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

Frembringelse af murine monoklonale antistoffer ved hybridomteknologi

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 af Simian Immunodeficiency Virus-specifikke CD8<sup&gt; +</sup&gt; T-celler i makakaber efter Peptid-MHC-I tetramerfarvning

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 af undslippe varianter af neutraliserende Influenza 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

Rensning af Viral DNA til identifikation af tilknyttede 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 Bibliotek kortlægge Qa-1 epitoper 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 specifikke diagnostiske epitop opdagelse

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