Name: a high throughput mhc ii binding assay for quantitative analysis of peptide epitopes
Uploaded: 2014-03-25T13:45:00
Description: Watch this Scientific Journal Video about A High Throughput MHC II Binding Assay for Quantitative Analysis of Peptide Epitopes at JoVE.com

This work was supported by NIH grants R01-GM-098977 and R21-AI-098122 to CBK and KEG. RSS was supported in part by a Luce Foundation Fellowship and in part by a Thayer Innovation Program Fellowship from the Thayer School of Engineering.

Four major activities comprise the peptide-MHC II binding assay: 1-Binding: Test peptides compete with labeled control peptides for solution phase binding of soluble MHC II proteins. Binding is measured over a wide range of test peptide concentrations. 2-Capture: After the binding reaction approaches equilibrium, peptide-MHC II complexes are captured and separated from unbound peptide and protein by conformation-dependent recognition with an immobilized antibody. 3-Detection: Captured control peptide is quantitatively de...

<ol>
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Biotherapeutics have established themselves as a cornerstone of modern medicine, representing four of the top five selling drugs in 201232. The biopharmaceutics sector has shown sustained growth for several years6, and the ongoing development of novel agents as well as the emergence of biosimilars has expanded biopharmaceutical pipelines. Looking to the future, assessing and mitigating the immunogenicity of protein therapeutics will become an integral part of early stage biotherapeutic development. ...

An erratum was issued for: <a href="https://www.jove.com/video/51308/a-high-throughput-mhc-ii-binding-assay-for-quantitative-analysis">A High Throughput MHC II Binding Assay for Quantitative Analysis of Peptide Epitopes</a>.&#160; There were typos in the Protocol section and Table 2.
Step 3.1.1 in the Protocol was updated from:
Add 80 &#181;l of 1 mM Pefa Bloc and 60 mg of octyl-&#946;-D-glucopyranaside to 3,920 &#181;l of Citrate Phosphate Buffer. (See supplemental spreadsheet calculator for alternative scaling).
to:
Add 80 &#181;l of 1 mM Pefa Bloc and 60 &#181;g of octyl-&#946;-D-glucopyranaside to 3,920 &#181;l of Citrate Phosphate Buffer. (See supplemental spreadsheet calculator for alternative scaling).
Step 5.2&#160;in the Protocol was updated from:
Further dilute the Control Peptide from step 5.1 (20 mM) 1:100 into the MHC II Master Mix from step 3.2.
to:
Further dilute the Control Peptide from step 5.1 (20 &#181;M) 1:100 into the MHC II Master Mix from step 3.2.
Table 2 was updated from:
<table border="0" cellpadding="0" cellspacing="0" width="376">
	<tbody>
		<tr>
			<td>MHC II Allele DRB1*:</td>
			<td>1501</td>
		</tr>
		<tr>
			<td>MHC II Stock Concentration (mg/ml)</td>
			<td>1.3</td>
		</tr>
		<tr>
			<td>MHC II Stock Concentration (mM)</td>
			<td>20</td>
		</tr>
		<tr>
			<td>Vol. MHC II Stock to Add (ml)</td>
			<td>14.65</td>
		</tr>
		<tr>
			<td>Vol. Reaction Buffer to Add (ml):</td>
			<td>2885.35</td>
		</tr>
		<tr>
			<td>MHC II Master Mix Concentration (nM)</td>
			<td>101</td>
		</tr>
	</tbody>
</table>
to:
<table border="0" cellpadding="0" cellspacing="0" width="376">
	<tbody>
		<tr>
			<td>MHC II Allele DRB1*:</td>
			<td>1501</td>
		</tr>
		<tr>
			<td>MHC II Stock Concentration (mg/ml)</td>
			<td>1.3</td>
		</tr>
		<tr>
			<td>MHC II Stock Concentration (&#956;M)</td>
			<td>20</td>
		</tr>
		<tr>
			<td>Vol. MHC II Stock to Add (&#956;l)</td>
			<td>14.65</td>
		</tr>
		<tr>
			<td>Vol. Reaction Buffer to Add (&#956;l):</td>
			<td>2885.35</td>
		</tr>
		<tr>
			<td>MHC II Master Mix Concentration (nM)</td>
			<td>101</td>
		</tr>
	</tbody>
</table>

An erratum was issued for: <a href="https://www.jove.com/video/51308/a-high-throughput-mhc-ii-binding-assay-for-quantitative-analysis">A High Throughput MHC II Binding Assay for Quantitative Analysis of Peptide Epitopes</a>.&#160; There were typos in the Protocol section and Table 2.

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

Proteins are the fastest growing class of therapeutic agents6, and the rapid expansion of biotherapeutic pipelines has focused increasing attention on the challenges associated with development and use of protein drugs. One unique consideration stems from the fact that, in a healthy and functioning immune system, all extracellular proteins are sampled by antigen presenting cells (APCs). Once internalized by APCs, a protein is cleaved into small peptide fragments, and putative immunogenic segments are loaded into the groove of class II major histocompatibility complex proteins (MHC II). The peptide-MHC II complexes are then displayed on the APC surface, and true immunogenic peptides, termed T cell epitopes, form ternary MHC II-peptide-T cell receptor complexes with cognate CD4 T cell surface receptors7. This critical molecular recognition event initiates a complex signaling cascade, which results in T cell activation, the release of cytokines, CD4 T cell-mediated B cell maturation, and ultimately production of circulating IgG antibodies that bind to and clear the offending exogenous protein. Thus, immunogenic proteins might be deimmunized by identifying constituent T cell epitopes and mutating key residues responsible for MHC II complex formation. It bears noting, however, that T cell epitopes can be numerous and broadly distributed throughout immunogenic proteins, and the majority of epitope-deleting mutations are likely to cause an inadvertent loss of protein function or stability. Therefore, engineering deimmunized biotherapies can be a complex and technically challenging objective, but there exist several examples of successful T cell epitope deletion projects3,5,8-12. Unlike grafting-based &#34;humanization&#34;, which is largely restricted to antibody therapeutics, epitope deletion can be applied to essentially any protein target regardless of sequence, structure, function, or the availability of homologous human scaffolds. The first step to implementing such an approach is identification of key peptide epitopes embedded within the target protein sequence.
High throughput biochemical assays using synthetic peptides and recombinant human MHC II molecules can provide rapid preliminary insights into epitope identification and mitigation1,3-5. These ELISA type assays can be a powerful complement to other protein/vaccine design and development tools. For example, one well established experimental approach to epitope mapping relies on time, labor, and resource intensive ex vivo cell proliferation assays 15. Briefly, the primary sequence of a target protein is first divided into a panel of overlapping peptides, often 15-mers with 12 residues overlap between adjacent peptides. The peptide panel is chemically synthesized and the immunogenicity of each peptide is tested in one of several different immunoassays that employ peripheral blood mononuclear cells (PBMC) isolated from human donors13,14. To provide greater confidence in results, peptides are typically tested in replicate with PBMC from 50 or more different donors. In cases where deimmunization is the ultimate objective, the work is compounded further by the need to produce additional panels of mutated peptides and test the new peptide panels in PBMC assays before introducing any deimmunizing mutations into the full length protein for subsequent functional analysis10. While these cellular assays remain the gold standard for assessing immunogenic potential in human patients, the efficiency of such an exhaustive approach might be improved by prefiltering putative immunogenic epitopes using a rapid and high throughput MHC II-peptide binding assay.
Likewise, biochemical peptide-MHC II binding assays can be combined with predictive in silico methods to radically accelerate the epitope identification process. There exist a variety of computational tools for T cell epitope prediction; examples include ProPred16, MHCPred17, SVRMHC18, ARB19, SMM-align20, NetMHCIIpan21 as well as proprietary tools such as EpiMatrix by EpiVax22. Likewise, epitope predictors have recently been combined with other bioinformatics and molecular modeling tools to yield integrated protein deimmunization algorithms designed to mitigate the risk that deimmunizing mutations might disrupt protein structure and function23-26. While several epitope predictors have proven to be reasonably accurate27,28, computational results invariably require experimental validation. Rapid, high throughput, and cost effective experimental methods are best suited as a preliminary filter for in silico epitope predictions.
In a similar vein, epitope predictors can drive antigen selection for reverse vaccinology29,30. For example, advances in bioinformatics have yielded whole genome screens that rapidly identify vaccine candidates in the form of whole proteins or peptide epitopes extracted from pathogen proteomes. While this enabling technology is reshaping discovery and development of protective vaccines, it introduces a new challenge in the form of intractably large lists of immunogenic vaccine candidates. High throughput peptide-MHC II binding assays can guide epitope selection by quantifying peptide binding affinity and binding promiscuity among multiple MHC II alleles. As with protein deimmunization, such experimental methods are ultimately required to validate computational prediction of promising vaccine leads.
Here, a peptide-MHC II binding assay scaled to 384-well format is described. The protocol is highly parallelized and reduces reagent costs by 75% compared to previously described 96-well plate formats1,3-5. Using a single liquid handling robot, this method allows one researcher to easily analyze approximately ninety test peptides in triplicate over a range of eight concentrations and four MHC II allele types in less than 48 hr. This article describes the setup of one 384-well ELISA plate for analysis of seven experimental peptides against one MHC II allele, but spread sheet calculators are provided as supplemental material so as to easily scale the experiment to any number of desired peptides and/or MHC II molecules.

<table border="0" cellpadding="0" cellspacing="0">
	<colgroup>
		<col />
		<col />
		<col />
	</colgroup>
	<tbody>
		<tr height="35">
			<td height="35" style="height:35px;width:587px;">Sodium Tetraborate Decahydrate</td>
			<td style="width:357px;">Sigma</td>
			<td style="width:217px;">221732</td>
			<td></td>
		</tr>
		<tr height="35">
			<td height="35" style="height:35px;">Citric Acid</td>
			<td>Sigma</td>
			<td>C1901</td>
			<td></td>
		</tr>
		<tr height="35">
			<td height="35" style="height:35px;">Dibasic Sodium Phosphate</td>
			<td>Sigma</td>
			<td>S7907</td>
			<td></td>
		</tr>
		<tr height="35">
			<td height="35" style="height:35px;">Trizma HCl</td>
			<td>OmniPur</td>
			<td>9310</td>
			<td></td>
		</tr>
		<tr height="35">
			<td height="35" style="height:35px;">Tween-20</td>
			<td>Sigma</td>
			<td>P7949</td>
			<td></td>
		</tr>
		<tr height="35">
			<td height="35" style="height:35px;">100% DMSO</td>
			<td>Sigma</td>
			<td>D8418</td>
			<td></td>
		</tr>
		<tr height="35">
			<td height="35" style="height:35px;">Octyl-&#946;-D-Glucopyranaside</td>
			<td>Fischer</td>
			<td>29836-26-8</td>
			<td></td>
		</tr>
		<tr height="35">
			<td height="35" style="height:35px;">Pefa Bloc SCF</td>
			<td>Roche</td>
			<td>1158591600</td>
			<td></td>
		</tr>
		<tr height="35">
			<td height="35" style="height:35px;">Dulbecco&#8217;s 10X Phosphate Buffered Saline</td>
			<td>Invitrogen</td>
			<td>14200-166</td>
			<td></td>
		</tr>
		<tr height="35">
			<td height="35" style="height:35px;">DELFIA Assay Buffer</td>
			<td>Perkin Elmer</td>
			<td>4002-0010</td>
			<td></td>
		</tr>
		<tr height="35">
			<td height="35" style="height:35px;">DELFIA Enhancement Buffer</td>
			<td>Perkin Elmer</td>
			<td>4001-0010</td>
			<td></td>
		</tr>
		<tr height="35">
			<td height="35" style="height:35px;">Europium Labelled Streptavidin</td>
			<td>Perkin Elmer</td>
			<td>1244-360</td>
			<td></td>
		</tr>
		<tr height="35">
			<td height="35" style="height:35px;">L243 anti-HLA-DR antibody</td>
			<td>Biolegend</td>
			<td>307602</td>
			<td></td>
		</tr>
		<tr height="35">
			<td height="35" style="height:35px;">Biotinylated tracer peptides</td>
			<td>21st Century Biochemicals</td>
			<td>Custom Order</td>
			<td></td>
		</tr>
		<tr height="35">
			<td height="35" style="height:35px;">Test peptides (1-4mg, 85% purity)</td>
			<td>Genscript</td>
			<td>Custom Order</td>
			<td></td>
		</tr>
		<tr height="35">
			<td height="35" style="height:35px;">Purified HLA-DRB1 monomers (non-biotinylated)</td>
			<td>Benaroya Research Institute*</td>
			<td>Custom Order</td>
			<td></td>
		</tr>
		<tr height="35">
			<td height="35" style="height:35px;">384-well white EIA/RIA plate</td>
			<td>Thermo</td>
			<td>460372</td>
			<td></td>
		</tr>
		<tr height="35">
			<td height="35" style="height:35px;">Polypropylene 384-well plate</td>
			<td>Costar</td>
			<td>3656</td>
			<td></td>
		</tr>
		<tr height="35">
			<td height="35" style="height:35px;">Film, AxySeal, 80 &#181;m, ELISA Applicator&#160;</td>
			<td>Axygen Asicentific</td>
			<td>PCR-SP</td>
			<td></td>
		</tr>
		<tr height="35">
			<td height="35" style="height:35px;">MilliQ Water</td>
			<td>N/A</td>
			<td>N/A</td>
			<td></td>
		</tr>
		<tr height="35">
			<td height="35" style="height:35px;">Epimotion Liquid Handler (or similar)</td>
			<td>Eppendorf</td>
			<td>5075</td>
			<td></td>
		</tr>
		<tr height="35">
			<td height="35" style="height:35px;">Select TS Plate Washer (or similar)</td>
			<td>BioTek</td>
			<td>405</td>
			<td></td>
		</tr>
		<tr height="35">
			<td height="35" style="height:35px;">SpectraMax Gemini Microplate Reader (or similar)</td>
			<td>Molecular Devices</td>
			<td>N/A</td>
			<td></td>
		</tr>
		<tr>
			<td colspan="4" height="98" style="height:99px;width:1161px;">*Recombinant human MHC II molecules can be obtained from the Benaroya Tetramer Core Laboratory. See: https://www.benaroyaresearch.org/our-research/core-resources/tetramer-core-laboratory</td>
		</tr>
	</tbody>
</table>

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.

The mature peptide sequence of Enterobacter cloacae P99 beta-lactamase (BLA) (Genbank ID# X07274.1) was analyzed with ProPred16 for putative peptide binders to MHC II allele DRB1*1501 (Table 4). ProPred identified 117 nonamer peptides with an obligatory P1 anchor residue (i.e. an M, L, I, V, F, Y, or W at position 1, which is required for MHC II binding31). At a 5% threshold, only peptides with scores greater than or equal to 2.6 are likely binders. Thus, at the 5%...

Watch this Scientific Journal Video about A High Throughput MHC II Binding Assay for Quantitative Analysis of Peptide Epitopes at JoVE.com

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 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 ...

Research

JoVE Journal

Biology

Visualizing Antigen Specific CD4+ T Cells using MHC Class II Tetramers

Major histocompatibility complex (MHC) class II tetramers allow the direct visualization of antigen specific CD4+ T cells by flow cytometry.  This method ...

A Neuronal and Astrocyte Co-Culture Assay for High Content Analysis of Neurotoxicity

High Content Analysis (HCA) assays combine cells and detection reagents with automated imaging and powerful image analysis algorithms, allowing ...

Mutagenesis and Functional Analysis of Ion Channels Heterologously Expressed in Mammalian Cells

We will demonstrate how to study the functional effects of introducing a point mutation in an ion channel. We study G protein-gated inwardly rectifying ...

High-throughput Saccharification Assay for Lignocellulosic Materials

Polysaccharides that make up plant lignocellulosic biomass can be broken down to produce a range of sugars that subsequently can be 
used in establishing ...

A Quantitative Fitness Analysis Workflow

Quantitative Fitness Analysis (QFA) is an experimental and computational workflow for comparing fitnesses of microbial cultures grown in parallel1,2,3,4. ...

High-throughput Functional Screening using a Homemade Dual-glow Luciferase Assay

We present a rapid and inexpensive high-throughput screening protocol to identify transcriptional regulators of alpha-synuclein, a gene associated with ...

Flow Cytometric Analysis of Bimolecular Fluorescence Complementation: A High Throughput Quantitative Method to Study Protein-protein Interaction

Among methods to study protein-protein interaction inside cells, Bimolecular Fluorescence Complementation (BiFC) is relatively simple and sensitive. BiFC ...

High Throughput Microinjections of Sea Urchin Zygotes

Microinjection into cells and embryos is a common technique that is used to study a wide range of biological processes. In this method a small amount of ...

High-throughput Screening for Chemical Modulators of Post-transcriptionally Regulated Genes

Both transcriptional and post-transcriptional regulation have a profound impact on genes expression. However, commonly adopted cell-based screening assays ...

qPCRTag Analysis - A High Throughput, Real Time PCR Assay for Sc2.0 Genotyping

The Synthetic Yeast Genome Project (Sc2.0) aims to build 16 designer yeast chromosomes and combine them into a single yeast cell. To date one synthetic ...