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><tbody><tr><td>Hybrid-SFM medium</td><td>Gibco</td><td>12045-076</td><td></td></tr><tr><td>Dulbeccos&#039;s Phophate-Buffered Saline (PBS)</td><td>Gibco</td><td>21600-069</td><td></td></tr><tr><td>Pfu DNA Polymerase&amp;nbsp;</td><td>Thermo Scientific&amp;nbsp;</td><td>EP0502&amp;nbsp;</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&amp;nbsp;</td><td>New England Biolabs</td><td>R0111S</td><td>Including buffers</td></tr><tr><td>XhoI&amp;nbsp;</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>&lt;em&gt;E. coli&lt;/em&gt; DH-5&amp;alpha; competent cell</td><td>RBC Bioscience</td><td>RH617</td><td></td></tr><tr><td>&lt;em&gt;E. coli&lt;/em&gt; 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&amp;nbsp;</td><td>Invitrongen</td><td>12780-052</td><td></td></tr><tr><td>Isopropylthio-&amp;beta;-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>NaN&lt;sub&gt;3&lt;/sub&gt;</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...

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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 ...

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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

Semi-automated Biopanning of Bacterial Display Libraries for Peptide Affinity Reagent Discovery and Analysis of Resulting Isolates

Biopanning bacterial display libraries is a proven technique for peptide affinity reagent discovery for recognition of both biotic and abiotic targets. Peptide affinity reagents can be used for similar applications to antibodies, including sensing and therapeutics, but are more robust and able to perform in more extreme environments. Specific enrichment of peptide capture agents to a protein target of interest is enhanced using semi-automated sorting methods which improve binding and wash steps and therefore decrease the occurrence of false positive binders. A semi-automated sorting method is described herein for use with a commercial automated magnetic-activated cell sorting device with an unconstrained bacterial display sorting library expressing random 15-mer peptides. With slight modifications, these methods are extendable to other automated devices, other sorting libraries, and other organisms. A primary goal of this work is to provide a comprehensive methodology and expound the thought process applied in analyzing and minimizing the resulting pool of candidates. These techniques include analysis of on-cell binding using fluorescence-activated cell sorting (FACS), to assess affinity and specificity during sorting and in comparing individual candidates, and the analysis of peptide sequences to identify trends and consensus sequences for understanding and potentially improving the affinity to and specificity for the target of interest.

Biopanning bacterial display libraries is a proven technique for discovery of peptide affinity reagents, a robust alternative to antibodies. The semi-automated sorting method herein has streamlined biopanning to decrease the occurrence of false positives. Here we illustrate the thought process and techniques applied in evaluating candidates and minimizing downstream analysis.

Biopanning bacterial display libraries is a proven technique for peptide affinity reagent discovery for recognition of both biotic and abiotic targets. ...

Biochemistry

Personalized Peptide Arrays for Detection of HLA Alloantibodies in Organ Transplantation

In organ transplantation, the function and longevity of the graft critically rely on the success of controlling immunological rejection reactivity against human leukocyte antigens (HLA). Histocompatibility guidelines are based on laboratory tests of anti-HLA immunity, which presents either as pre-existing or de novo generated HLA antibodies that constitute a major transplantation barrier. Current tests are built on a single-antigen beads (SAB) platform using a fixed set of ~100 preselected recombinant HLA antigens to probe transplant sera. However, in humans there exist a far greater variety of HLA types, with no two individuals other than identical twins who can share the same combination of HLA sequences. While advanced technologies for HLA typing and direct sequencing can precisely capture any mismatches in DNA sequence between a donor's and recipient's HLA, the SAB assay, due to its limited variety in sequence representation, is unable to precisely detect alloantibodies specifically against the donor HLA mismatches. We sought to develop a complementary method using a different technology to detect and characterize anti-donor HLA antibodies on a personalized basis. The screening tool is a custom peptide array of donor HLA-derived sequences for probing post-transplant sera of the organ recipient to assess the risk for antibody-mediated rejection. On a single array for one donor-recipient pair, up to 600 unique peptides are made based on the donor's HLA protein sequences, each peptide carrying at least one mismatched residue in a 15-amino acid sequence. In our pilot experiments to compare antigen patterns for pre- and post-transplant sera on these arrays, we were able to detect anti-HLA signals with the resolution that also allowed us to pinpoint the immune epitopes involved. These personalized antigen arrays allow high-resolution detection of donor-specific HLA epitopes in organ transplantation.

Mismatches in human leukocyte antigen (HLA) sequences between organ donor and recipient pairs are the major cause of antibody-mediated rejection in organ transplantation. Here we present the use of custom antigen arrays that are based on individual donors' HLA sequences to probe anti-donor HLA alloantibodies in organ recipients.

In organ transplantation, the function and longevity of the graft critically rely on the success of controlling immunological rejection reactivity against ...

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 ...

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 ...

Generation of Discriminative Human Monoclonal Antibodies from Rare Antigen-specific B Cells Circulating in Blood

Monoclonal antibodies (mAbs) are powerful tools useful for both fundamental research and in biomedicine. Their high specificity is indispensable when the antibody needs to distinguish between highly related structures (e.g., a normal protein and a mutated version thereof). The current way of generating such discriminative mAbs involves extensive screening of multiple Ab-producing B cells, which is both costly and time consuming. We propose here a rapid and cost-effective method for the generation of discriminative, fully human mAbs starting from human blood circulating B lymphocytes. The originality of this strategy is due to the selection of specific antigen binding B cells combined with the counter-selection of all other cells, using readily available Peripheral Blood Mononuclear Cells (PBMC). Once specific B cells are isolated, cDNA (complementary deoxyribonucleic acid) sequences coding for the corresponding mAb are obtained using single cell Reverse Transcription-Polymerase Chain Reaction (RT-PCR) technology and subsequently expressed in human cells. Within as little as 1 month, it is possible to produce milligrams of highly discriminative human mAbs directed against virtually any desired antigen naturally detected by the B cell repertoire.

We describe a method for the production of human antibodies specific for an antigen of interest, starting from rare B cells circulating in human blood. Generation of these natural antibodies is efficient and rapid, and the antibodies obtained can discriminate between highly related antigens.

Monoclonal antibodies (mAbs) are powerful tools useful for both fundamental research and in biomedicine. Their high specificity is indispensable when the ...

Tetrameric Fluorescent Antigen Arrays for Single-Step Identification of Antigen-Specific B Cells

Fluorescent antigen production is a critical step in the identification of antigen-specific B cells. Here, we detailed the preparation, purification, and the use of four-arm, fluorescent PEG-antigen conjugates to selectively identify antigen-specific B cells through avid engagement with cognate B cell receptors. Using modular click chemistry and commercially available fluorophore kit chemistries, we demonstrated the versatility of preparing customized fluorescent PEG-conjugates by creating distinct arrays for proteolipid protein (PLP139-151) and insulin, which are important autoantigens in murine models of multiple sclerosis and type 1 diabetes, respectively. Assays were developed for each fluorescent conjugate in its respective disease model using flow cytometry. Antigen arrays were compared to monovalent autoantigen to quantify the benefit of multimerization onto PEG backbones. Finally, we illustrated the utility of this platform by isolating and assessing anti-insulin B cell responses after antigen stimulation ex vivo. Labeling insulin-specific B cells enabled the amplified detection of changes to co-stimulation (CD86) that were otherwise dampened in aggregate B cell analysis. Together, this report enables the production and use of fluorescent antigen arrays as a robust tool for probing B cell populations.

This protocol details the customizable production and use of fluorescent probes for labeling antigen-specific B cells.

Fluorescent antigen production is a critical step in the identification of antigen-specific B cells. Here, we detailed the preparation, purification, and ...

Bioengineering

Single-cell Screening Method for the Selection and Recovery of Antibodies with Desired Specificities from Enriched Human Memory B Cell Populations

The human antibody repertoire represents a largely untapped source of potential therapeutic antibodies and useful biomarkers. While current computational methods, such as next generation sequencing (NGS), yield enormous sets of data on the antibody repertoire at the sequence level, functional data is required to identify which sequences are relevant for a particular antigen or set of antigens. Here, we describe a method to identify and recover individual antigen-specific antibodies from peripheral blood mononuclear cells (PBMCs) from a human blood donor. This method utilizes an initial enrichment of mature B cells and requires a combination of phenotypic cell markers and fluorescently-labeled protein to isolate IgG memory B cells via flow cytometry. The heavy and light chain variable regions are then cloned and re-screened. Although limited to the memory B cell compartment, this method takes advantage of flow cytometry to interrogate millions of B cells and returns paired heavy and light chain sequences from a single cell in a format ready for expression and confirmation of specificity. Antibodies recovered with this method can be considered for therapeutic potential, but can also link specificity and function with bioinformatic approaches to assess the B cell repertoire within individuals.

The BSelex method to identify and recover individual antigen-specific antibodies from human peripheral blood mononuclear cells combines flow cytometry with single cell PCR and cloning.

The human antibody repertoire represents a largely untapped source of potential therapeutic antibodies and useful biomarkers. While current computational ...

Analysis of HBV-Specific CD4 T-cell Responses and Identification of HLA-DR-Restricted CD4 T-Cell Epitopes Based on a Peptide Matrix

CD4 T cells play important roles in the pathogenesis of chronic hepatitis B. As a versatile cell population, CD4 T cells have been classified as distinct functional subsets based on the cytokines they secreted: for example, IFN-&#947; for CD4 T helper 1 cells, IL-4 and IL-13 for CD4 T helper 2 cells, IL-21 for CD4 T follicular helper cells, and IL-17 for CD4 T helper 17 cells. Analysis of hepatitis B virus (HBV)-specific CD4 T cells based on cytokine secretion after HBV-derived peptides stimulation could provide information not only about the magnitude of HBV-specific CD4 T-cell response but also about the functional subsets of HBV-specific CD4 T cells. Novel approaches, such as transcriptomics and metabolomics analysis, could provide more detailed functional information about HBV-specific CD4 T cells. These approaches usually require isolation of viable HBV-specific CD4 T cells based on peptide-major histocompatibility complex-II multimers, while currently the information about HBV-specific CD4 T-cell epitopes is limited. Based on an HBV-derived peptide matrix, a method has been developed to evaluate HBV-specific CD4 T-cell responses and identify HBV-specific CD4 T-cell epitopes simultaneously using peripheral blood mononuclear cells samples from chronic HBV infection patients.

Based on a hepatitis B virus (HBV)-derived peptide matrix, HBV-specific CD4 T-cell responses could be evaluated in parallel with identification of HBV-specific CD4 T-cell epitopes.

CD4 T cells play important roles in the pathogenesis of chronic hepatitis B. As a versatile cell population, CD4 T cells have been classified as distinct ...

A High Throughput MHC II Binding Assay for Quantitative Analysis of Peptide Epitopes

Biochemical assays with recombinant human MHC II molecules can provide rapid, quantitative insights into immunogenic epitope identification, deletion, or design1,2. Here, a peptide-MHC II binding assay is scaled to 384-well format. The scaled down protocol reduces reagent costs by 75% and is higher throughput than previously described 96-well protocols1,3-5. Specifically, the experimental design permits robust and reproducible analysis of up to 15 peptides against one MHC II allele per 384-well ELISA plate. Using a single liquid handling robot, this method allows one researcher to analyze approximately ninety test peptides in triplicate over a range of eight concentrations and four MHC II allele types in less than 48 hr. Others working in the fields of protein deimmunization or vaccine design and development may find the protocol to be useful in facilitating their own work. In particular, the step-by-step instructions and the visual format of JoVE should allow other users to quickly and easily establish this methodology in their own labs.

Biochemical assays with recombinant human MHC II molecules can provide rapid, quantitative insights into immunogenic epitope identification, deletion, or design. &#160;Here, a peptide-MHC II binding assay scaled to 384-well plates is described. This cost effective format should prove useful in the fields of protein deimmunization and vaccine design and development.

Biochemical assays with recombinant human MHC II molecules can provide rapid, quantitative insights into immunogenic epitope identification, deletion, or ...

Biology

Development of a Colloidal Gold-based Immunochromatographic Test Strip for Detection of Cetacean Myoglobin

This protocol describes the development of a colloidal gold immunochromatographic test strip based on the sandwich format that can be used to differentiate the myoglobin (Mb) of cetaceans from that of seals and other animals. The strip provides rapid and on-the-spot screening for cetacean meat, thereby restraining its illegal trade and consumption. Two monoclonal antibodies (mAbs) with reactivity toward the Mb of cetaceans were developed. The amino acid sequences of Mb antigenic reactive regions from various animals were analyzed in order to design two synthetic peptides (a general peptide and a specific peptide) and thereafter raise the mAbs (subclass IgG1). The mAbs were selected from hybridomas screened by indirect ELISA, western blot and dot blot. CGF5H9 was specific to the Mbs of rabbits, dogs, pigs, cows, goats, and cetaceans while it showed weak to no affinity to the Mbs of chickens, tuna and seals. CSF1H13 can bind seals and cetaceans with strong affinity but showed no affinity to other animals. Cetacean samples from four families (Balaenopteridae, Delphinidae, Phocoenidae and Kogiidae) were used in this study, and the results indicated that these two mAbs have broad binding ability to Mbs from different cetaceans. These mAbs were applied on a sandwich-type colloidal gold immunochromatographic test strip. CGF5H9, which recognizes many species, was colloid gold-labeled and used as the detection antibody. CSF1H13, which was coated on the test zone, detected the presence of cetacean and seal Mbs. Muscle samples from tuna, chicken, seal, five species of terrestrial mammals and 15 species of cetaceans were tested in triplicate. All cetacean samples showed positive results and all the other samples showed negative results.

This protocol describes the development of two IgG class monoclonal antibodies (mAbs) strongly reactive to myoglobin of cetaceans. These mAbs are applied on a colloidal gold immunochromatographic test strip based on the sandwich format to differentiate the Mb of cetaceans from seal and other animals.