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. Préparation de l&#39;anticorps monoclonal <ol><li> Culture les RG-M56 cellules hybridomes monoclonaux de souris 2 dans un milieu sans sérum dans des flacons de 175T à 37 ° C avec 5% de CO 2 supplément. Recueillir le surnageant lorsque la couleur du milieu devient jaune après cinq jours d&#39;incubation. REMARQUE: Les cellules d&#39;hybridome sont cultivées dans un milieu exempt de sérum pour éviter la contamination anticorps à partir du sérum de foetus bovin. </l...

<ol>
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Ce protocole offre une technique simple et rapide pour identifier un épitope linéaire mAb reconnu. Prenant en considération le coût de la synthèse peptidique et l&#39;efficacité des peptides de synthèse de la production, de la région antigénique de la protéine d&#39;enveloppe du virus a été réduite par l&#39;expression de protéines recombinantes tronquées en série avant l&#39;analyse de balayage de peptides. En tant que tel, le système d&#39;expression pET de E. coli fiable et efficace a été ...

Dans le système immunitaire, la recombinaison V, D et J pour les anticorps segments permet de créer des variations considérables des régions déterminant la complémentarité (CDR) pour la liaison à des antigènes différents pour protéger l&#39;hôte contre une infection pathogène. La défense de neutralisation des anticorps dirigés contre des antigènes dépend de la complémentarité spatiale entre les CDRs de l&#39;anticorps et les épitopes des antigènes. Par conséquent, une compréhension de cette interaction moléculaire facilitera la conception d&#39;un vaccin prophylactique et le développement peptide médicament thérapeutique. Cependant, cette interaction de neutralisation peut être influencée à la fois par de multiples domaines antigéniques à partir d&#39;un seul antigène et par plusieurs CDR d&#39;anticorps, ce qui rend par conséquent le procédé de détermination de l&#39;épitope plus complexe. Heureusement, le développement de la technologie des hybridomes, qui fusionne les cellules productrices d&#39;anticorps individuelles avec des cellules de myélome, permet de diviser un lot de cellules en permanence à secrete un anticorps spécifique, connu comme un anticorps monoclonal (mAb) 1. Les cellules d&#39;hybridome produisent ces mAb, à haute affinité pures pour se lier à un domaine antigénique unique d&#39;un antigène spécifique. La relation de l&#39;antigène-anticorps mis en place, plusieurs approches, y compris le balayage des peptides, peut être utilisé pour déterminer l&#39;épitope d&#39;un antigène en utilisant son anticorps monoclonal correspondant. Les développements récents dans la technologie de peptide synthétique ont fait la technique de balayage de peptide plus accessible et plus commode d&#39;exécuter. En bref, un ensemble de peptides synthétiques se chevauchant sont produites selon une séquence de l&#39;antigène cible et sont associés à une membrane supporté sur un solide pour mAb hybridation. balayage Peptide non seulement offre un moyen simple pour cartographier la région de liaison de l&#39;anticorps, mais facilite également l&#39;acide aminé (aa) la mutagenèse par balayage de résidus ou de substitution pour évaluer l&#39;interaction de liaison entre chaque résidu aa du peptide d&#39;épitope et les CDR de l&#39;anticorps. <p class = &quot;jove_content&quot;&gt; Ici, la présente étude décrit un protocole pour l&#39;identification efficace de l&#39;épitope linéaire du mérou jaune virus de la nécrose nerveuse (YGNNV) protéine d&#39;enveloppe en utilisant une neutralisation mAb 2, 3, 4. Le protocole comprend mAb préparation, la construction et l&#39;expression de protéines recombinantes tronquées en série, la conception de peptides se chevauchant de synthèse, hybridation dot-blot, le balayage d&#39;alanine et la mutagenèse par substitution. Compte tenu du coût élevé de la synthèse peptidique, l&#39;étape consistant à tronquer en série les protéines recombinantes d&#39;une protéine cible souhaitée a été modifiée, et la région antigénique a été réduit à environ 100 à 200 résidus aa avant la synthèse réseau peptidique analyse dot-blot a été réalisée.

<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

L&#39;identification d&#39;un epitope antigénique par le système immunitaire permet la compréhension du mécanisme de protection d&#39;anticorps neutralisants qui peuvent faciliter le développement de vaccins et de médicaments peptidiques. balayage peptidique est une méthode simple et efficace qui mappe carrément l&#39;épitope linéaire reconnu par un anticorps monoclonal (mAb). Ici, les auteurs présentent une méthodologie de détermination de l&#39;épitope impliquant des protéines recombinantes en série tronquées, la conception de peptides synthétiques et hybridation dot-blot pour la reconnaissance antigénique du virus de la nécrose nerveuse protéine d&#39;enveloppe en utilisant un mAb neutralisant. Cette technique repose sur l&#39;hybridation dot-blot de peptides synthétiques et des mAb sur un fluorure de polyvinylidène (PVDF). La région antigénique minimum d&#39;une protéine d&#39;enveloppe virale reconnue par le RG-M56 mAb peut être réduite par l&#39;étape par étape taillée cartographie peptidique sur un épitope de peptide 6-mer. En outre, le balayage d&#39;alanine mutagenèse et les résidus sousconstitution peut être effectuée pour caractériser l&#39;importance de liaison de chaque résidu d&#39;acides aminés constituant l&#39;épitope. Les résidus flanquant le site d&#39;épitope ont été trouvés à jouer un rôle critique dans la régulation peptide de conformation. Le peptide d&#39;épitope identifié peut être utilisé pour former des cristaux de complexes peptide-anticorps épitope pour une étude de diffraction des rayons X et de la compétition fonctionnelle ou pour la thérapeutique.

Le but de cette expérience était d&#39;identifier un épitope par dot blot en utilisant mAb. Pour affiner rapidement et efficacement en bas de la région antigénique reconnu par mAb, la pleine longueur et YGNNV protéines d&#39;enveloppe recombinante en série tronquée avec une fusion tag 6xHis à l&#39;extrémité C-terminale ont été exprimées à partir d&#39; un système d&#39;expression E. coli PET 10 (figure 1A). Les protéines...

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.

L&#39;identification d&#39;un épitope linéaire des cellules B monoclonales d&#39;anticorps reconnus par le peptide à balayage assistée

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

Peptide: CMH tétramère basée sur l&#39;enrichissement des cellules T spécifiques d&#39;épitopes

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 ...

Recherche

Immunologie et infection

Peptide-based Identification of Functional Motifs and their Binding Partners

Identification de Motifs Fonctionnelles et leurs PARTENAIRES DE LIAISON Peptide-basée sur

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

Isolement des précurseurs des cellules B des sous-ensembles à partir de sang de cordon ombilical

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

Isolement, identification et la purification de cellules murines thymique épithéliales

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

La génération d&#39;anticorps monoclonaux murins par 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 de l&#39;immunodéficience simienne CD8 spécifiques Virus<sup&gt; +</sup&gt; T-cellules dans rhésus macaques par Peptide-CMH-I tétramère Coloration

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

Génération des variantes d’évasion de neutraliser les anticorps monoclonaux Influenza Virus

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

Purification de l’ADN Viral pour l’Identification des protéines virus et cellulaires associées

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

Bibliothèque de Peptide qui se chevauchent pour mapper les épitopes de Qa-1 dans une protéine

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 spécifique Diagnostic épitope découverte

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