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. Herstellung des monoklonalen Antikörpers <ol><li> Kultur die RG-M56 monoklonalen Maus - Hybridomzellen 2 in Serum-freiem Medium in 175T - Kolben bei 37 ° C mit 5% CO 2 zu ergänzen. Die überstehende Flüssigkeit, wenn die Farbe des Mediums nach fünf Tagen Inkubation gelb wird. HINWEIS: Hybridoma-Zellen wurden in serumfreiem Medium Antikörper Kontamination aus fötalem Rinderserum zu vermeiden. </li><li> Zentrifugieren Sie den Überstand bei 4.500 × g für 30 min bei 4...

<ol>
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Dieses Protokoll bietet eine schnelle und einfache Technik, um ein mAb anerkannte lineares Epitop zu identifizieren. Unter Berücksichtigung der Kosten für die Peptidsynthese und die Produktionseffizienz der Synthese von Peptiden wurde die antigene Region des Virus-Hüllproteins reduziert durch serielles trunkierten rekombinanten Proteinen vor Peptid-Scanning-Analyse exprimieren. Als solche wurde die zuverlässig und effizient E. coli pET Expressionssystem diese seriell trunkierten rekombinanten Proteine als r...

Im Immunsystem, die Rekombination von V-, D- und J - Segmente können für Antikörper für die Bindung an verschiedene Antigene enorme Variationen von komplementaritätsbestimmenden Regionen (CDRs) zu erzeugen , die Host von pathogenen Infektion zu schützen. Die neutralisierende Abwehr von Antikörpern gegen Antigene hängt von der räumlichen Komplementarität zwischen den CDRs der Antikörper und die Epitope der Antigene. Daher wird ein Verständnis dieser molekularen Wechselwirkung prophylaktischen Impfstoff-Design und therapeutischen Peptid Arzneimittelentwicklung unterstützen. Jedoch kann diese Neutralisation Wechselwirkung sowohl aus einem einzigen Antigen und durch mehrere CDRs von Antikörpern durch multiple antigene Domänen beeinflußt werden, die somit das Epitop Bestimmungsprozess komplexer machen. Glücklicherweise ist die Entwicklung der Hybridom-Technologie, die einzelnen Antikörper-produzierenden Zellen mit Myelomzellen verschmilzt, ermöglicht eine ständig Dividieren Charge von Zellen secrete einem spezifischen Antikörper, bekannt als ein monoklonaler Antikörper (mAb) 1. Hybridomzellen produzieren diese reine, hochaffine mAb an einem einzigen Antigen-Domäne eines spezifischen Antigens zu binden. Mit der Beziehung des Antigen-Antikörper festgestellt, mehrere Ansätze, einschließlich Peptid-Scanning, verwendet werden, um das Epitop eines Antigens zu bestimmen seinen entsprechenden mAb verwendet. Die jüngsten Entwicklungen in der synthetischen Peptid-Technologie haben die Peptid-Scanning-Technik zugänglicher und bequemer gemacht auszuführen. Kurz gesagt, werden gemäß einer Zielantigensequenz einen Satz von überlappenden synthetischen Peptiden hergestellt und zu einem Feststoff-Trägermembran für mAb Hybridisierung verbunden. Peptid-Scanning bietet nicht nur eine einfache Möglichkeit, die Antikörper-Bindungsregion auf der Karte, sondern erleichtert auch Aminosäure (aa) Mutagenese durch Rückstands Scannen oder Substitution die Bindungswechselwirkung zwischen jedem aa-Rest des Epitop-Peptid und die CDRs des Antikörpers zu bewerten. <p class = &quot;jove_content&quot;&gt; Hier beschreibt die vorliegende Studie ein Protokoll für die effiziente Identifikation des linearen Epitop des gelben Grouper nervös Nekrose - Virus (YGNNV) Hüllprotein mit einem neutralisierenden mAb 2, 3, 4. Das Protokoll enthält mAb Vorbereitung, Konstruktion und Expression von seriell verkürzten rekombinanten Proteine, synthetische überlappende Peptid Design, Dot-Blot-Hybridisierung, Alanin-Scanning und Substitutionsmutagenese. die hohen Kosten für die Peptidsynthese unter Berücksichtigung, wurde der Schritt des seriell die rekombinanten Proteine ​​eines gewünschten Zielproteins Verkürzen modifiziert und die antigene Region wurde auf etwa 100 bis 200 aa Reste, bevor der synthetische Peptidarray Dot-Blot-Analyse durchgeführt wurde eingeengt.

<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

Die Identifizierung eines antigenes Epitop von dem Immunsystem ermöglicht das Verständnis des Schutzmechanismus von neutralisierenden Antikörpern, die die Entwicklung von Impfstoffen und Peptid-Medikamente können erleichtern. Peptid-Scanning ist eine einfache und effiziente Methode, die ohne weiteres das lineare Epitop durch einen monoklonalen Antikörper (mAb) erkannt abbildet. Hier präsentieren die Autoren ein Epitop Bestimmung Methodik seriell verkürzten rekombinanten Proteinen, synthetischen Peptid-Design und Dot-Blot-Hybridisierung für die Antigen-Erkennung von Nerven Nekrose-Virus-Hüllprotein mit einem neutralisierenden mAb beteiligt sind. Diese Technik beruht auf dem Dot-Blot-Hybridisierung von synthetischen Peptiden und mAbs auf einem Polyvinylidenfluorid (PVDF) -Membran. Die minimale antigen Bereich eines viralen Hüllprotein vom RG-M56 mAb erkannt durch die Schritt-für-Schritt-getrimmten Peptidkartierung auf ein 6-mer-Peptid-Epitops eingegrenzt werden. Zusätzlich Alanin-Scanning-Mutagenese und Rückstands substitution kann die Bindungs ​​Bedeutung jeder Aminosäurerest, aus denen das Epitop charakterisieren, durchgeführt werden. Die Reste, die die Epitop-Website flankieren, wurden gefunden kritische Rollen in Peptidkonformation Regulation zu spielen. Das identifizierte Epitop-Peptid kann verwendet werden, um Kristalle von Epitop-Peptid-Antikörper-Komplexe für eine Röntgenbeugungsuntersuchung zu bilden und funktionellen Wettbewerb oder für Therapeutika.

Das Ziel dieses Experimentes war es ein Epitop durch Dot-Blotting unter Verwendung von mAb zu identifizieren. Um schnell und effizient verengen die antigene Region anerkannt durch mAb, die in voller Länge und seriell abgeschnitten YGNNV rekombinanten Hüllproteine mit einem 6xHis Fusions - Tag am C-Terminus wurden aus einem E. coli - PET - Expressionssystem 10 (1A) ausgedrückt. Die resultierenden rekombinanten Proteine ​​wurden auf ...

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-gestützte Identifizierung eines monoklonalen Antikörpers anerkannte Linear B-Zell-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

Peptid: MHC-Tetramer-basierte Anreicherung von Epitop-spezifischer T-Zellen

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 ...

Forschung

Immunologie und Infektion

Peptide-based Identification of Functional Motifs and their Binding Partners

Peptid-basierte Identifizierung von Functional Motive und ihre Bindungspartner

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

Isolierung von Precursor B-Zell-Subsets aus Nabelschnurblut

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

Isolierung, Identifizierung und Reinigung von Murine Thymusepithelzellen

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

Generation of Murine Monoclonal Antibodies by Hybridomtechnologie

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

Analysis of Simian Immunodeficiency Virus-spezifischen CD8<sup&gt; +</sup&gt; T-Zellen in Rhesusaffen von Peptid-MHC-I Tetramerfärbung

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 von Escape Varianten des Influenza-Virus Monoclonal Antikörper neutralisieren

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

Reinigung der viralen DNA für die Identifizierung von assoziierten viralen und zellulären Proteinen

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

Überlappende Peptid-Bibliothek zuordnen Qa-1 Epitope in einem 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 spezifische diagnostische Epitop Entdeckung

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