MHC-Peptide Binding Assays
out what you need to know with the world's most cited MHC-peptide
binding assay service
MHC peptide-binding assays do what prediction tools can't.
They confirm the physical binding characteristics of actual
peptides, based on their sequence.
the world leader in providing in vitro MHC peptide
binding assays, offering the widest range of MHC alleles and
assay options, from high throughput screening to in-depth
competition assays between candidate peptides. Our specialist
immunology advisory team can help you define your project
needs and quickly tailor the most relevant assay solution.
REVEAL® data published by
researchers at US FDA:
Researchers at the US FDA and colleagues use
ProImmune REVEAL to understand how immunogenicity of therapeutic
Factor VIII protein varies between ethnic groups based on their HLA
et al. PLOS Computational Biology May 2013, Vol. 9 (5) e1003066):
(A) Binding scores of FVIII wild type peptides. Binding scores in a
REVEAL assay were measured for the synthetic wild type peptides
depicted in bold in Table 1. Each row represents a peptide and each
column a MHC-II allele. The peptides are grouped together based on
the position in the FVIII gene where the haplotypes H2 to H6 occur.
Overlapping peptides were used
around the position of each ns-SNP. Positive-control (PC) peptides
are known T-cell epitopes of FVIII and negative control (NC)
peptides are from regions of the FVIII protein where no T-cell
epitopes have been identified. The scale for the binding score is
shown at the top of the figure; dark colors represent peptides that
bind with high affinity to the MHC-II allele. (B) FVIII high
affinity binders. Wild type peptides with binding score >15% are
shown and considered potential T-cell epitopes for each of the six
MHC-II alleles. (C) Binding promiscuity scores for the wild type
FVIII peptides. The binding promiscuity score for each peptide,
defined as the fraction of MHC-II variants the peptide binds with a
score >15%, is shown.
ProImmune REVEAL® is widely applicable across many disease areas including all areas of cancer and infectious diseases.
New CD4+ and CD8+ T cell epitopes identified with the help of this technology can be used
to assess protein antigenicity or as core building blocks for vaccine
development or as targets for new immunotherapy.
2. Example process flows for ProImmune REVEAL assays for class I and
class II MHC.
case peptides are synthesized for the relevant project, based
on ProImmune's versatile thinkpeptides peptide synthesis platform. Peptides used can be in any
relevant form, whether from overlapping sequences or individually
Peptides are then incubated in each case with recombinant MHC molecules
and the stabilization of the MHC peptide complex is measured in an
Assembly and dissociation can be measured in high throughput at
a single time interval in each case, allowing many alleles and
peptides to be screened in parallel and an affordable cost.
Alternatively, or as a follow-up for peptides of particular
interest, more in-depth rate assays can be performed to establish
more detailed on and off rate characteristics.
validation can be carried out either using MHC-multimers, such as ProVE®
Pentamers for MHC class I or ProT2®
Tetramers for MHC class II. As an alternative, or in combination with MHC multimer
technology, functional assays, such as ELISpot, flow cytometry analysis
of intracellular cytokine staining, or proliferation assays can be
performed to obtain functional confirmation for specific peptides.
Praise for ProImmune REVEAL®
Prof. Julio Delgado, University of Utah, USA
advantage of ProImmune’s in vitro system is that we could
characterize binding to each HLA molecule individually and not
have to guess which of the ranIge of HLA molecules expressed by
our patients was responsible for antigen presentation. "
Dr. Gene Olinger, United States Army Medical Research Institute
of Infectious Diseases (USAMRID) "ProImmune accomplished in two months what would have taken
three years in our laboratory".
What will you get?
ProImmune REVAL® data is reported in our
detailed reporting templates for this assay range. Reports present data from the particular assays selected.
REVEAL Class II Example Data
Presentation Binding as a percentage of the positive control is
shown in green. Red overlay: Stability after 24hrs at 37°C.
Available MHC Alleles
Class I HLA
Class I: H-2
Class I: Mamu
Class II: HLA DR
Class II: HLA DQ
Class II: HLA DP
ProImmune REVEAL® data for Remicade®
REVEAL® results showing the population impact of Class II HLA binding
peptides representing part of the mouse variable region of Remicade® (Infliximab).
Remicade, a highly successful drug for the treatment of inflammatory
autoimmune diseases also shows high rates of incidence of immunogenicity.
The high-throughput nature of ProImmune REVEAL® allows researchers to
develop a comprehensive understanding of how entire domains of monoclonal
antibodies or therapeutic proteins interact with HLA molecules from a global
population group based on actual experimental binding studies.
ProImmune REVEAL® Applications
epitope mapping, resolving MHC binding restriction experimentally.
Study of the immunogenicity
of different variants of a protein, e.g. comparison of the variable and
conserved regions of immunogenic proteins in different clades of a virus
Optimization of clinical
monitoring e.g. selection of the optimal Pentamer specificities for
monitoring a target patient group
Identification of cross
presentation of a peptide by several alleles
Improvement in vaccine
design and efficacy.
epitope binding optimization for therapeutic application, such as the
insertion of anchor residues in peptide sequences with only partial or
ProImmune REVEAL® Advantages
Physical assay. Experimental
results overcome hypothetical nature of MHC-peptide binding prediction.
Speed. Get experimental data
Flexibility. Many options exist for
our ProImmune REVEAL® system both for high throughput analysis of many
peptides across a wide range of alleles to in-depth analysis for a
smaller number of peptides on specific MHC types. Our immunology
specialist sales team can help configure the study best suited to your
Affordability. The scalability and
flexibility of ProImmune REVEAL® means that a project can be designed to
suit your budget objective. We can crystalize the most important
questions to be answered and design projects that can be staged to
manage program risks.
Improved Intellectual Property Position
Identification of the individual epitopes of a
therapeutic protein or other protein of interest allows separation of the intellectual property (IP) of
that protein from the IP around the method of delivery or its therapeutic
use. During vaccine development, defining IP for specific epitopes
increases the options for future changes in product design. If the IP
for the specific antigens in a protein of interest is not secured,
competitive organizations may have the opportunity to develop an obstructive
Q: What information is required to get
a quote for a ProImmune REVEAL project?
A: Ideally we need to know the peptide
sequences you are interested along with the MHC alleles you want us to
screen. We can however provide outline quotes for you based on numbers of
peptides and alleles only, which will be a good start for scoping your
Q: How long does this service take?
A: This will very much depend on the project scope,
including the number and specification of the peptides used. Once we know
your project objective we will always provide you with an estimated delivery
time with our quotes.
Q: With all the available options, how do I decide
which one is best for me?
A: Based on the understanding that we develop in
discussing your requirement with you, we will be able to propose the most
suitable project scope for you and discuss how this can fit your broader
study objectives and other assay work.
ProImmune REVEAL® & ProVE® Rapid Epitope Discovery System used to identify
novel T cell epitopes in HIV-1
et al. (2009). "Novel approach to recognition of
predicted HIV-1 Gag B*35:01-restricted CD8 T-cell epitopes by HLA-B*35:01+
patients: Confirmation by quantitative ELISpot analyses and characterisation
using multimers." J Immunol Methods. 341: 76-85.
Figure. Results of the MHC-peptide binding assay for controls and the
12 peptides that were considered potential epitopes; binding signal is shown
as a percentage relative to the pass/fail control; peptide 8, HPVHAGPIA
(red) was further validated by Pentamer staining.
Figure. Pro5® Pentamer staining of live lymphocytes gated on CD3+
cells; the left plot shows staining with an allele mismatched negative
control Pentamer, the right plot shows staining with the HA9 (B*35:01/HPVHAGPIA)
Pentamer. 0.39% of CD3+ live lymphocytes are CD8+/HA Pentamer+ with a
background stain of 0.03%.
The author commented that this study
“confirms the novel ProImmune technology as a useful and superior tool for
revealing potential immunogenic epitopes”, and also highlighted the
importance of validating results in parallel using functional assays, such
as ELISpot and Pro5® Pentamer staining.
See how Baxter (now Baxalta) to have used
to understand the immunogenicity of human Factor VIII treatment:
Steinitz, K. et
al . "CD4+
T-cell epitopes associated with antibody responses after
intravenously and subcutaneously applied human FVIII in
humanized hemophilic E17 HLA-DRB1*15:01 mice." Blood (2012)