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. Preparation of Monoclonal Antibody
<ol>
	<li>Culture the RG-M56 mouse monoclonal hybridoma cells2 in serum-free medium in 175T flasks at 37 &#186;C with 5% CO2 supplement. Collect the supernatant when the color of the medium turns yellow after five days of incubation. 
	NOTE: Hybridoma cells were cultured in serum-free medium to avoid antibody contamination from fetal bovine serum.</li>
	<li>Centrifuge the supernatant at 4,500 x g for 30 min at 4 &#186;C and discard the cell d...

<ol>
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Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Expression+of+ayu+(Plecoglossus+altivelis)+Pit-1+in+Escherichia+coli:+its+purification+and+immunohistochemical+detection+using+monoclonal+antibody.">Expression of ayu (Plecoglossus altivelis) Pit-1 in Escherichia coli: its purification and immunohistochemical detection using monoclonal antibody.</a> Protein Expr Purif. 24 (2), 292-301 (2002).</li><li>Atassi, M. Z. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Antigenic+structures+of+proteins.+Their+determination+has+revealed+important+aspects+of+immune+recognition+and+generated+strategies+for+synthetic+mimicking+of+protein+binding+sites.">Antigenic structures of proteins. Their determination has revealed important aspects of immune recognition and generated strategies for synthetic mimicking of protein binding sites.</a> Eur J Biochem. 145 (1), 1-20 (1984).</li><li>Opuni, K. F., 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=Mass+spectrometric+epitope+mapping.">Mass spectrometric epitope mapping.</a> Mass Spectrom Rev. , (2016).</li><li>Chang, C. Y., Chiu, C. C., Christopher John, J. A., Liao, I. C., Lea&#241;o, E. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> The Aquaculture of Groupers. , 207-224 (2008).</li><li>Conti-Tronconi, B. M., 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=Alpha-bungarotoxin+and+the+competing+antibody+WF6+interact+with+different+amino+acids+within+the+same+cholinergic+subsite.">Alpha-bungarotoxin and the competing antibody WF6 interact with different amino acids within the same cholinergic subsite.</a> Biochemistry. 30 (10), 2575-2584 (1991).</li><li>Briggs, S., Price, M. R., Tendler, S. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Fine+specificity+of+antibody+recognition+of+carcinoma-associated+epithelial+mucins:+antibody+binding+to+synthetic+peptide+epitopes.">Fine specificity of antibody recognition of carcinoma-associated epithelial mucins: antibody binding to synthetic peptide epitopes.</a> Eur J Cancer. 29 (2), 230-237 (1993).</li><li>Chen, N. 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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This protocol offers a rapid and straightforward technique to identify a mAb-recognized linear epitope. Taking into consideration the cost of peptide synthesis and the production efficiency of synthesizing peptides, the antigenic region of the virus coat protein was reduced by expressing serially truncated recombinant proteins before peptide scanning analysis. As such, the reliable and efficient E. coli pET expression system was used to produce these serially truncated recombinant proteins, as recombinant protei...

In the immune system, the recombination of V, D, and J segments allows for antibodies to create tremendous variations of complementarity determining regions (CDRs) for binding to various antigens to protect the host from pathogenic infection. The neutralizing defense of antibodies against antigens depends on the spatial complementarity between the CDRs of the antibodies and the epitopes of the antigens. Therefore, an understanding of this molecular interaction will assist prophylactic vaccine design and therapeutic peptide drug development. However, this neutralization interaction may be influenced both by multiple antigenic domains from one single antigen and by multiple CDRs of antibodies, which consequently make the epitope determination process more complex. Fortunately, the development of hybridoma technology, which fuses individual antibody-producing cells with myeloma cells, allows for a constantly dividing batch of cells to secrete one specific antibody, known as a monoclonal antibody (mAb)1. Hybridoma cells produce these pure, high-affinity mAbs to bind to a single antigenic domain of a specific antigen. With the relationship of the antigen-antibody established, several approaches, including peptide scanning, can be used to determine the epitope of an antigen using its corresponding mAb. Recent developments in synthetic peptide technology have made the peptide scanning technique more accessible and more convenient to perform. Briefly, a set of overlapping synthetic peptides are produced according to a target antigen sequence and are associated to a solid-supported membrane for mAb hybridization. Peptide scanning not only offers a simple way to map the antibody binding region, but also facilitates amino acid (aa) mutagenesis through residue scanning or substitution to evaluate the binding interaction between each aa residue of the epitope peptide and the CDRs of the antibody.
Here, the present study describes a protocol for the efficient identification of the linear epitope of the yellow grouper nervous necrosis virus (YGNNV) coat protein using a neutralizing mAb2,3,4. The protocol includes mAb preparation, construction and expression of serially truncated recombinant proteins, synthetic overlapping peptide design, dot-blot hybridization, alanine scanning, and substitution mutagenesis. Considering the high cost of peptide synthesis, the step of serially truncating the recombinant proteins of a desired target protein was modified, and the antigenic region was narrowed down to around 100 to 200 aa residues before the synthetic peptide array dot-blot analysis was performed.

<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

The identification of an antigenic epitope by the immune system allows for the understanding of the protective mechanism of neutralizing antibodies that may facilitate the development of vaccines and peptide drugs. Peptide scanning is a simple and efficient method that straightforwardly maps the linear epitope recognized by a monoclonal antibody (mAb). Here, the authors present an epitope determination methodology involving serially truncated recombinant proteins, synthetic peptide design, and dot-blot hybridization for the antigenic recognition of nervous necrosis virus coat protein using a neutralizing mAb. This technique relies on the dot-blot hybridization of synthetic peptides and mAbs on a polyvinylidene fluoride (PVDF) membrane. The minimum antigenic region of a viral coat protein recognized by the RG-M56 mAb can be narrowed down by step-by-step trimmed peptide mapping onto a 6-mer peptide epitope. In addition, alanine scanning mutagenesis and residue substitution can be performed to characterize the binding significance of each amino acid residue making up the epitope. The residues flanking the epitope site were found to play critical roles in peptide conformation regulation. The identified epitope peptide may be used to form crystals of epitope peptide-antibody complexes for an x-ray diffraction study and functional competition, or for therapeutics.

The goal of this experiment was to identify an epitope through dot blotting using mAb. To rapidly and efficiently narrow down the antigenic region recognized by mAb, the full-length and serially truncated YGNNV recombinant coat proteins with a 6xHis fusion tag at the C-terminus were expressed from an E. coli PET expression system10 (Figure 1A). The resulting recombinant proteins were spotted onto the PVDF membrane using RG-M56 mAb and 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.

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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