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

Summary

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

Abstract

Biochemical assays with recombinant human MHC II molecules can provide rapid, quantitative insights into immunogenic epitope identification, deletion, or design^1,2. 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 protocols^1,3-5. 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.

Introduction

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 ....

Protocol

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 detected using time-resolved fluorescence. 4-Analysis: Spectroscopic data are processed, plotted and analyzed to ascertain dose-dependent binding proper....

Results

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%.......

Discussion

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. .......

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
Sodium Tetraborate Decahydrate	Sigma	221732
Citric Acid	Sigma	C1901
Dibasic Sodium Phosphate	Sigma	S7907
Trizma HCl	OmniPur	9310
Tween-20	Sigma	P7949
100% DMSO	Sigma	D8418
Octyl-β-D-Glucopyranaside	Fischer	29836-26-8
Pefa Bloc SCF	Roche	1158591600
Dulbecco’s 10x Phosphate Buffered Saline	Invitrogen	14200-166
DELFIA Assay Buffer	Perkin Elmer	4002-0010
DELFIA Enhancement Buffer	Perkin Elmer	4001-0010
Europium Labelled Streptavidin	Perkin Elmer	1244-360
L243 anti-HLA-DR antibody	Biolegend	307602
Biotinylated tracer peptides	21st Century Biochemicals	Custom Order
Test peptides (1-4 mg, 85% purity)	Genscript	Custom Order
Purified HLA-DRB1 monomers (non-biotinylated)	Benaroya Research Institute*	Custom Order
384-well white EIA/RIA plate	Thermo	460372
Polypropylene 384-well plate	Costar	3656
Film, AxySeal, 80 µm, ELISA Applicator	Axygen Ascientific	PCR-SP
MilliQ Water	N/A	N/A
Epimotion Liquid Handler (or similar)	Eppendorf	5075
Select TS Plate Washer (or similar)	BioTek	405
SpectraMax Gemini Microplate Reader (or similar)	Molecular Devices	N/A
*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