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 debris pellet.</li>
	<li>Add 2 mL of protein G agarose (supplied as a 50% slurry) to a 5 mL column and equilibrate with 10 resin volumes (10 mL) of ice-cold PBS.</li>
	<li>Load 200 mL of the antibody supernatant (step 1.2) onto the column and discard the pass-through.</li>
	<li>Add 10 mL of ice-cold PBS to the column to wash it. Repeat twice.</li>
	<li>Add 10 mL of 50 mM glycine, pH 2.7 to the column to elute the protein G-associated antibody. Collect 900 &#181;L fractions in a microcentrifuge tube containing 100 &#181;L of 10x neutralization buffer (1 M Tris, 1.5 M NaCl, and 1 mM EDTA, pH 8.0).</li>
	<li>Store the purified antibody in 50% glycerol with 0.03% NaN3 at -20 &#186;C.</li>
</ol>
2. Construction and Expression of Serially Truncated Recombinant Proteins
<ol>
	<li>Prepare a PCR reaction mixture: 5 &#181;L of 10x Pfu buffer, 0.2 mM of each dNTP, 0.2 &#181;M forward primer3, 0.2 &#181;M reverse primer3, 2 mM MgSO4, 1 ng of pET20b-1A593 plasmid DNA, and 2.5 U (unit) of Pfu DNA polymerase; add ddH2O to a final volume of 50 &#181;L.
	<ol>
		<li>Run samples in an automatic thermal cycler using the following parameters: Cycle 1 (94 &#186;C for 5 min); cycles 2-36 (94 &#186;C for 30 s, 63 &#186;C for 30 s, and 72 &#186;C for 60 s); and cycle 37 (72 &#186;C for 7 min).</li>
	</ol>
	</li>
	<li>Extract the polymerase chain reaction (PCR) products by using a PCR purification kit5 to facilitate the following restriction enzyme digestion.</li>
	<li>Digest the PCR-amplified DNA fragments with NdeI and XhoI restriction enzymes and ligate each of these DNA fragments into NdeI and XhoI enzyme-cleaved pET-20b(+) vector. Transform the constructs into Escherichia coli DH-5&#945;-competent cells6.
	<ol>
		<li>Prepare the digestion mixture in digestion buffer (20 mM Tris-acetate, 10 mM Mg(CH3COO)2, 50 mM KCH3COO, and 1 mM DTT, pH 7.9) with 1 &#181;g of PCR-amplified DNA or pET-20b(+) vector DNA, and 2 U of NdeI and XhoI restriction enzymes in a final volume of 20 &#181;L.</li>
		<li>Mix the digestion mixture gently and quickly spin down. Incubate at 37 &#186;C for 2 h in a dry bath to ensure the complete cutting of the restriction sites.</li>
		<li>Extract the restriction enzyme-digested DNA fragments using a PCR purification kit5 to facilitate the following plasmid construction.</li>
		<li>Prepare the ligation mixture in ligation buffer (66 mM Tris, 5 mM MgCl2, 1 mM ATP, and 5 mM DTT, pH 7.5) with 100 ng of predigested, PCR-amplified DNA, 10 ng of predigested pET-20b(+) vector DNA, and 5 U of T4 DNA ligase in a final volume of 10 &#181;L.</li>
		<li>Mix the ligation mixture gently and quickly spin down. Incubate at 16 &#186;C for 18 h in a water bath.</li>
		<li>Put 10 &#181;L of the ligation samples into 100 &#181;L of the DH-5&#945;-competent cells and mix gently before placing the microcentrifuge tube on ice for 30 min. Put the microcentrifuge tube in a dry bath at 42 &#186;C for 90 s to induce heat shock7. Immediately transfer the tube onto ice for 2 min.</li>
		<li>Add 900 &#181;L of Luria-Bertani (LB) broth (1% Bacto tryptone, 0.5% Bacto yeast extract, and 0.5% NaCl, pH 7.0) to the tube. Incubate at 37 &#186;C with 150 rpm shaking for 45 min. Pellet the cells by centrifugation at 4,000 x&#160;g for 10 min and discard the supernatant.</li>
		<li>Resuspend the pellet with 50 &#181;L of LB broth and spread each transformation onto pre-warmed LB plates containing 100 &#181;g/mL ampicillin. Incubate the plates at 37 &#186;C for 16 h.</li>
		<li>Pick up a single colony using a 200 &#181;L tip and place it into 3 mL of LB broth containing 100 &#181;g/mL ampicillin in a loosely capped 15 mL tube. Incubate at 37 &#186;C with 150 rpm shaking for 12 h.</li>
		<li>Extract the plasmid DNA8 from each culture and sequence it using T7 promoter and T7 terminator primers to confirm the sequence9.</li>
	</ol>
	</li>
	<li>After sequence confirmation, transform 10 ng of DNA from these pET-20b(+) plasmids with varying lengths of YGNNV coat protein gene into the BL-21 (DE3) strain of E. coli by using the heat-shock method7. Follow steps 2.3.6-2.3.8 to perform the transformation.</li>
	<li>Transfer a single colony from each transformed E. coli BL-21 cell into 3 mL of LB broth containing 100 &#181;g/mL ampicillin in a loosely capped 15 mL tube. Incubate the culture at 37 &#186;C with 150 rpm shaking.</li>
	<li>Cool the culture to 25 &#186;C when the OD600 of the culture is about 0.6 and add IPTG to a final concentration of 0.4 mM to induce the expression of the recombinant protein. Incubate the culture for an extra 4 h at 25 &#186;C with 200 rpm shaking.</li>
	<li>Transfer 1 mL of the culture into a microcentrifuge tube. Pellet the cells by centrifugation at 12,000 x g for 1 min and discard the supernatant.</li>
	<li>Resuspend the cell pellet in 100 &#181;L of denaturation buffer (8 M urea, 20 mM sodium phosphate, and 0.5 M NaCl, pH 7.4) by pipetting up and down with a micropipette. Mix by vigorous vortexing. 
	NOTE: The sample solutions should now be semi-transparent, a little sticky, and ready for the dot-blot hybridization assay.</li>
</ol>
3. Design and Synthesis of Overlapping Peptides
<ol>
	<li>Design and synthesize3 serial 20-mer peptides that each overlap with its successor by 10 aa residues from the 195-338 aa region of the YGNNV coat protein to narrow down the epitope region of RG-M56 mAb by dot blotting.</li>
	<li>Design and synthesize3 three 8-mer peptides (195VNVSVLCR202, 197VSVLCRWS204, and 199VLCRWSVR206) with an overlap of 6 aa residues onto the next synthetic peptide to narrow down the epitope region of 195-206 aa by dot blotting.</li>
	<li>Design and synthesize3 7-mer (196NVSVLCR202 and 195VNVSVLC201), 6-mer (195VNVSVL200, 196NVSVLC201, and 197VSVLCR202), and 5-mer peptides (195VNVSV199, 196NVSVL200, 197VSVLC201, and 198SVLCR202) with an overlap of 6, 5, and 4 aa residues, respectively, onto their neighboring peptides to minimize the epitope region by dot blotting.</li>
</ol>
4. Dot-blot Hybridization
<ol>
	<li>Dissolve each synthesized peptide in dimethyl sulfoxide (DMSO) to a final concentration of 10 mg/mL. 
	NOTE: To overcome the varied solubility of synthetic peptides, all synthetic peptides should be dissolved in DMSO. DMSO is a good solvent to completely dissolve hydrophobic or hydrophilic peptides.</li>
	<li>Soak the polyvinylidene fluoride (PVDF) membrane with methanol for 2 min. 
	NOTE: PVDF membrane is up to 100% resistant to DMSO; others may not be so.</li>
	<li>Equilibrate the PVDF membrane with modified Towbin buffer (25 mM Tris, 192 mM glycine, and 0.1% SDS, pH 8.3) for 2 min. 
	NOTE: 10-20% (v/v) of methanol can be added to modified Towbin buffer to improve the transfer results.</li>
	<li>Rinse a piece of chromatography paper with the modified Towbin buffer. Place the PVDF membrane onto the chromatography paper. Wait until the modified Towbin buffer has disappeared from the PVDF membrane surface before continuing to the next step.</li>
	<li>Add 2 &#181;L of each peptide sample to the membrane with a 10 &#181;L tip. Air-dry the PVDF membrane on the chromatography paper for 10 min. Add each peptide sample slowly and gradually onto the membrane to avoid too much diffusion.</li>
	<li>Block the membrane in TBST buffer (0.05% (v/v) Tween-20, 20 mM Tris, and 150 mM NaCl, pH 7.4) with 5% nonfat milk for 30 min at room temperature with gentle shaking.</li>
	<li>Add RG-M56 mAb at a final dilution of 1:1,000 in TBST buffer with 5% nonfat milk to the membrane. Incubate the membrane at 37 &#186;C for 1 h with gentle shaking.</li>
	<li>Remove the antibody solution. Wash the membrane in TBST buffer for 5 min with gentle shaking. Repeat twice.</li>
	<li>Add the secondary antibody (goat anti-mouse IgG, Fc, conjugated alkaline phosphatase) at a final dilution of 1:5,000 in TBST buffer with 5% nonfat milk to the membrane. Incubate the membrane at 37 &#186;C for 1 h with gentle shaking.</li>
	<li>Discard the antibody solution. Wash the membrane in TBST buffer for 5 min with gentle shaking. Repeat the wash step twice.</li>
	<li>Develop the membrane with BCIP/NBT substrate solution at room temperature for 15 min in the dark. Stop the developing by washing the membrane with ddH2O when the signal appears.</li>
	<li>Air-dry the membrane and capture the dot-blot image using an image system.</li>
	<li>Measure the intensity of each dot blot using image analysis software3.</li>
</ol>
5. Alanine Scanning and Substitution
<ol>
	<li>Design and synthesize3 alanine and methionine substitution peptides. Replace each aa residue with alanine for the 8-mer peptide 195VNVSVLCR202 and synthesize peptides 195ANVSVLCR202, 195VAVSVLCR202, 195VNASVLCR202, 195VNVAVLCR202, 195VNVSALCR202, 195VNVSVACR202, 195VNVSVLAR202, and 195VNVSVLCA202. Replace aa residue leucine 200 to methionine to create the SJNNV genotype epitope peptide 195VNVSVMCR202.</li>
	<li>Follow Section 4 to perform alanine scanning and substitution mutagenesis dot blotting.</li>
</ol>

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The authors have no conflicts of interest related to this report.

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>
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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 ...

peptide-scanning-assisted-identification-monoclonal-antibody

Research

JoVE Journal

Immunology and Infection

9.3K Views.  Academia Sinica. 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.

Video: Peptide Scanning-assisted Identification of a Monoclonal Antibody-recognized Linear B-cell Epitope

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 antigen receptor (TCR) specificity. Many indirect methods are available in which a bulk population of T cells is stimulated in vitro with a specific antigen and epitope-specific T cells are identified through the measurement of a functional response such as proliferation, cytokine production, or expression of activation markers1. However, these methods only identify epitope-specific T cells exhibiting one of many possible functions, and they are not sensitive enough to detect epitope-specific T cells at naive precursor frequencies. A popular alternative is the TCR transgenic adoptive transfer model, in which monoclonal T cells from a TCR transgenic mouse are seeded into histocompatible hosts to create a large precursor population of epitope-specific T cells that can be easily tracked with the use of a congenic marker antibody2,3. While powerful, this method suffers from experimental artifacts associated with the unphysiological frequency of T cells with specificity for a single epitope4,5. Moreover, this system cannot be used to investigate the functional heterogeneity of epitope-specific T cell clones within a polyclonal population.
	The ideal way to study adaptive immunity should involve the direct detection of epitope-specific T cells from the endogenous T cell repertoire using a method that distinguishes TCR specificity solely by its binding to cognate peptide:MHC (pMHC) complexes. The use of pMHC tetramers and flow cytometry accomplishes this6, but is limited to the detection of high frequency populations of epitope-specific T cells only found following antigen-induced clonal expansion. In this protocol, we describe a method that coordinates the use of pMHC tetramers and magnetic cell enrichment technology to enable detection of extremely low frequency epitope-specific T cells from mouse lymphoid tissues3,7. With this technique, one can comprehensively track entire epitope-specific populations of endogenous T cells in mice at all stages of the immune response.

This protocol describes the use of peptide:MHC tetramers and magnetic microbeads to isolate low frequency populations of epitope-specific T cells and analyze them by flow cytometry. This method enables the direct study of endogenous T cell populations of interest from in vivo experimental systems.

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

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 also competitively inhibit the function of the full-length protein. A set of 20 Nef scanning peptides, 20 amino acids in length with each overlapping 10 amino acids of its neighbor, were used to identify motifs in Nef responsible for its induction of apoptosis. Peptides containing these apoptotic motifs induced apoptosis at levels comparable to the full-length Nef protein. A second peptide, derived from the Secretion Modification Region (SMR) of Nef, retained the ability to interact with cellular proteins involved in Nef's secretion in exosomes (exNef). This SMRwt peptide was used as the "bait" protein in co-immunoprecipitation experiments to isolate cellular proteins that bind specifically to Nef's SMR motif. Protein transfection and antibody inhibition was used to physically disrupt the interaction between Nef and mortalin, one of the isolated SMR-binding proteins, and the effect was measured with a fluorescent-based exNef secretion assay. The SMRwt peptide's ability to outcompete full-length Nef for cellular proteins that bind the SMR motif, make it the first inhibitor of exNef secretion. Thus, by employing the techniques described here, which utilize the unique properties of specific short peptides derived from motifs found in full-length proteins, one may accelerate the identification of functional motifs in proteins and the development of peptide-based inhibitors of pathogenic functions.

Techniques to dissect the mechanisms underlying the secretion of HIV-1 Nef in exosomes are described. Specific short peptides derived from Nef and protein transfection were exploited to determine the structure, function, and binding partners of Nef&#x2019;s Secretion Modification Region. These procedures have general relevance in many mechanistic studies.

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

Umbilical cord blood is highly enriched for hematopoietic progenitor cells at different lineage commitment stages. We have developed a protocol for isolating precursor B-cells at four different stages of differentiation. Because genes are expressed and epigenetic modifications occur in a tissue specific manner, it is vital to discriminate between tissues and cell types in order to be able to identify alterations in the genome and the epigenome that may lead to the development of disease. This method can be adapted to any type of cell present in umbilical cord blood at any stage of differentiation.	
This method comprises 4 main steps. First, mononuclear cells are separated by density centrifugation. Second, B-cells are enriched using biotin conjugated antibodies that recognize and remove non B-cells from the mononuclear cells. Third the B-cells are fluorescently labeled with cell surface protein antibodies specific to individual stages of B-cell development. Finally, the fluorescently labeled cells are sorted and individual populations are recovered. The recovered cells are of sufficient quantity and quality to be utilized in downstream nucleic acid assays.

Here we describe a protocol for isolating subsets of precursor B-cells from umbilical cord blood. A sufficient quantity and quality of nucleic acids may be extracted from the cells and used in subsequent assays utilizing DNA or RNA.

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

The thymus is a vital organ for T lymphocyte development. Of thymic stromal cells, thymic epithelial cells (TECs) are particularly crucial at multiple stages of T cell development: T cell commitment, positive selection and negative selection. However, the function of TECs in the thymus remains incompletely understood. In the article, we provide a method to isolate TEC subsets from fresh mouse thymus using a combination of mechanical disruption and enzymatic digestion. The method allows thymic stromal cells and thymocytes to be efficiently released from cell-cell and cell-extracellular matrix connections and to form a single-cell suspension. Using the isolated cells, multiparameter flow cytometry can be applied to identification and characterization of TECs and dendritic cells. Because TECs are a rare cell population in the thymus, we also describe an effective way to enrich and purify TECs by depleting thymocytes, the most abundant cell type in the thymus. Following the enrichment, cell sorting time can be decreased so that loss of cell viability can be minimized during purification of TECs. Purified cells are suitable for various downstream analyses like Real Time-PCR, Western blot and gene expression profiling. The protocol will promote research of TEC function and as well as the development of in vitro T cell reconstitution.

Here we describe an efficient method for isolation, identification, and purification of mouse thymic epithelial cells (TECs). The protocol can be utilized for studies of thymus function for normal T cell development, thymus dysfunction, and T cell reconstitution.

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

Monoclonal antibodies are universal binding molecules and are widely used in biomedicine and research. Nevertheless, the generation of these binding molecules is time-consuming and laborious due to the complicated handling and lack of alternatives. The aim of this protocol is to provide one standard method for the generation of monoclonal antibodies using hybridoma technology. This technology combines two steps. Step 1 is an appropriate immunization of the animal and step 2 is the fusion of B lymphocytes with immortal myeloma cells in order to generate hybrids possessing both parental functions, such as the production of antibody molecules and immortality. The generated hybridoma cells were then recloned and diluted to obtain stable monoclonal cell cultures secreting the desired monoclonal antibody in the culture supernatant. The supernatants were tested in enzyme-linked immunosorbent assays (ELISA) for antigen specificity. After the selection of appropriate cell clones, the cells were transferred to mass cultivation in order to produce the desired antibody molecule in large amounts. The purification of the antibodies is routinely performed by affinity chromatography. After purification, the antibody molecule can be characterized and validated for the final test application. The whole process takes 8 to 12 months of development, and there is a high risk that the antibody will not work in the desired test system.

An optimized protocol is presented for the generation of monoclonal antibodies based on the hybridoma technology. Mice were immunized with an immunoconjugate. Spleen cells were fused by PEG and an electric impulse with immortal myeloma cells. Antibody-producing hybridoma cells were selected by HAT and antigen-specific ELISA screening.

Monoclonal antibodies are universal binding molecules and are widely used in biomedicine and research. Nevertheless, the generation of these binding ...

Generation of Escape Variants of Neutralizing Influenza Virus Monoclonal Antibodies

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 glycoproteins of the virus. The generation of escape variants is a powerful method in elucidating how viruses escape immune detection and in identifying critical residues required for antibody binding. Here, we describe a protocol on how to generate influenza A virus escape variants by utilizing human or murine monoclonal antibodies (mAbs) directed against the viral hemagglutinin (HA). With the use of our technique, we previously characterized critical residues required for the binding of antibodies targeting either the head or stalk of the novel avian H7N9 HA. The protocol can be easily adapted for other virus systems. Analyses of escape variants are important for modeling antigenic drift, determining single nucleotide polymorphisms (SNPs) conferring resistance and virus fitness, and in the designing of vaccines and/or therapeutics.

We describe a method by which we identify critical residues required for the binding of human or murine monoclonal antibodies that target the viral hemagglutinin of influenza A viruses. The protocol can be adapted to other virus surface glycoproteins and their corresponding neutralizing antibodies.

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

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 cellular proteins by mass spectrometry. Although proteins that interact with viral genomes play major roles in determining the outcome of infection, a comprehensive analysis of viral genome associated proteins was not previously feasible. Here we demonstrate a method that enables the direct purification of HSV-1 genomes from infected cells. Replicating viral DNA is selectively labeled with modified nucleotides that contain an alkyne functional group. Labeled DNA is then specifically and irreversibly tagged via the covalent attachment of biotin azide via a copper(I)-catalyzed azide-alkyne cycloaddition or click reaction. Biotin-tagged DNA is purified on streptavidin-coated beads and associated proteins are eluted and identified by mass spectrometry. This method enables the selective targeting and isolation of HSV-1 replication forks or whole genomes from complex biological environments. Furthermore, adaptation of this approach will allow for the investigation of various aspects of herpesviral infection, as well as the examination of the genomes of other DNA viruses.

The goal of this protocol is to specifically tag and selectively isolate viral DNA from infected cells for the characterization of viral genome associated proteins.

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

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 play important roles in surveying cells for structural and functional integrity, inducing immune regulation, and limiting immune responses to viral infections. Additionally, functional augmentation of Qa-1-restricted CD8+ T cells through epitope immunization has shown therapeutic effects in several autoimmune disease animal models, e.g. experimental allergic encephalomyelitis, collagen-induced arthritis, and non-obese diabetes. Therefore, there is an urgent need for a method that can efficiently and quickly identify functional Qa-1 epitopes in a protein. Here, we describe a protocol that utilizes Qa-1-restricted CD8+ T cell lines specific for an overlapping peptide (OLP) library for determining Qa-1 epitopes in a protein. This OLP library contains 15-mer overlapping peptides that cover the whole length of a protein, and adjacent peptides overlap by 11 amino acids. Using this protocol, we recently identified a 9-mer Qa-1 epitope in myelin oligodendrocyte glycoprotein (MOG). This newly mapped MOG Qa-1 epitope was shown to induce epitope-specific, Qa-1-restricted CD8+ T cells that enhanced myelin-specific immune regulation. Therefore, this protocol is useful for future investigation of novel targets and functions of Qa-1-restricted CD8+ T cells.

Qa-1 (HLA-E in human) belongs to a group of non-classical major histocompatibility complex 1b molecules. Immunization with Qa-1-binding epitopes has been shown to augment tissue-specific immune regulation and ameliorate several autoimmune diseases. Herein we describe an overlapping peptide library strategy for the identification of Qa-1 epitopes in a 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

High-density peptide microarrays allow screening of more than six thousand peptides on a single standard microscopy slide. This method can be applied for drug discovery, therapeutic target identification, and developing of diagnostics. Here, we present a protocol to discover specific Zika virus (ZIKV) diagnostic peptides using a high-density peptide microarray. A human serum sample validated for ZIKV infection was incubated with a high-density peptide microarray containing the entire ZIKV protein translated into 3,423 unique 15 linear amino acid (aa) residues with a 14-aa residue overlap printed in duplicate. Staining with different secondary antibodies within the same array, we detected peptides that bind to Immunoglobulin M (IgM) and Immunoglobulin G (IgG) antibodies present in serum. These peptides were selected for further validation experiments. In this protocol, we describe the strategy followed to design, process, and analyze a high-density peptide microarray.

In this protocol, we describe a technique to discover Zika virus specific diagnostic peptides using a high-density peptide microarray. This protocol can readly be adapted for other emerging infectious diseases.

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

Generation of Monoclonal Antibodies Against Natural Products

The analysis of the bioactive components present in foods and natural products has become a popular area of study in many fields, including traditional Chinese medicine and food safety/toxicology. Many of the classical analysis techniques require expensive equipment and/or expertise. Notably, enzyme-linked immunosorbent assays (ELISAs) have become an emerging method for the analysis of foods and natural products. This method is based on antibody-mediated detection of the target components. However, as many of the bioactive components in natural products are small (&lt;1,000 Da) and do not induce an immune response, creating monoclonal antibodies (mAbs) against them is often difficult. In this protocol, we provide a detailed explanation of the steps required to generate mAbs against target molecules as well as those needed to create the associated indirect competitive (ic)ELISA for the rapid analysis of the compound in multiple samples. The procedure describes the synthesis of the artificial antigen (i.e., the hapten-carrier conjugate), immunization, cell fusion, monoclonal hybridoma preparation, characterization of the mAb, and the ELISA-based application of the mAb. The hapten-carrier conjugate was synthesized by the sodium periodate method and evaluated by MALDI-TOF-MS. After immunization, splenocytes were isolated from the immunized mouse with the highest antibody titer and fused with the hypoxanthine-aminopterin-thymidine (HAT)-sensitive mouse myeloma cell line Sp2/0 -Ag14 using a polyethylene glycol (PEG)-based method. The hybridomas secreting mAbs reactive to the target antigen were screened by icELISA for specificity and cross-reactivity. Furthermore, the limiting dilution method was applied to prepare monoclonal hybridomas. The final mAbs were further characterized by icELISA and then utilized in an ELISA-based application for the rapid and convenient detection of the example hapten (naringin (NAR)) in natural products.

This article provides a detailed protocol for the preparation and evaluation of monoclonal antibodies against natural products for use in various immunoassays. This procedure includes immunization, cell fusion, indirect competitive ELISA for positive clone screening, and monoclonal hybridoma preparation. The specifications for antibody characterization using MALDI-TOF-MS and ELISA analyses are also provided.

The analysis of the bioactive components present in foods and natural products has become a popular area of study in many fields, including traditional ...