Rapid T-Cell epitope discovery

Validate your T cell epitopes



The REVEAL & ProVE® Rapid Epitope Discovery System is a customized modular service that allows screening of any protein for T cell epitopes in weeks instead of months or years. Our service takes away the cost and effort of setting up assays in your laboratory, and gets rapid results with minimal assay variation. The modular system runs according to a well defined schedule and therefore significantly reduces time uncertainty in your epitope discovery project.

Now you can rapidly acquire the final piece of information to complete that long running epitope discovery project.

Our highly sensitive REVEAL™ MHC-Peptide Complete Rate Assays allow you to detect subtle differences in MHC-peptide binding kinetics, which ultimately relates to the ability of that peptide to be a strong T cell epitope.

The rate assays form one module of our REVEAL & ProVE® Rapid Epitope Discovery System. This technology offers the widest commercial range of HLA class I and class II alleles for CD4+ and CD8+ T cell epitope discovery and kinetic rate and stability assays, allowing the user to investigate T cell epitopes across a wide range of patient samples.


REVEAL & ProVE® in more detail



Alleles available for Class I REVEAL & ProVE®

The REVEAL & ProVE® technology is available for the following class I alleles.

Human
A*0101  A*0201  A*0301  A*1101 A*2402 A*2902
B*0702  B*0801  B*1402  B*1501 B*2705   B*3501  B*4001

Mouse
H2Kb  H2Db  H2Kd  H2Dd  H2Ld

Rhesus Macaque
Mamu-A*01  Mamu-A*02

Module 1: PEPscreen®: Custom Peptide Library Synthesis

Objective: Rapid, cost-effective synthesis of peptide libraries

REVEAL & ProVE® Epitope Discovery System comprises of a series of in vitro assay procedures requiring synthetic peptides. ProImmune can provide high throughput synthesis of custom peptides as a PEPscreen®: Custom Peptide Library.  Peptides (6-20 amino acids) are supplied in 0.5-2mg quantities with high average purity.  Quality control by MALDI-TOF Mass Spectrometry is carried out on 100% of samples and the library is supplied in only 10-15 working days.  Alternatively the customer may supply the peptides.

Module 2: REVEAL™ MHC-Peptide Binding Assay

Objective: Quantitative binding of candidate peptides to MHC alleles and determination of their MHC restriction

The high throughput REVEAL™ MHC-peptide binding assay quickly determines the ability of each candidate peptide to bind to one or more MHC allele compared with two control peptides.   The peptide-binding assay is a measure of the ability of each peptide to stabilize the ternary MHC-peptide complex.  Detection is based on the presence or absence of the native conformation of the MHC-peptide complex.

Each peptide is given a score relative to the positive control peptide, which is a known T cell epitope.  In addition results for an intermediate control peptide (another known T cell epitope) are shown for comparison. The score is reported as a percentage of the signal generated by the test peptide versus the control peptide.   The customer may select any or all of the peptides that pass the REVEAL™ assay for ProVE® Pentamer synthesis and/or further REVEAL™ binding assays.


Figure 1:
Illustrates the binding of a peptide (red) to the MHC protein (blue) and beta-2 microglobulin (grey); when the individual components come together in the correct conformation a positive signal is generated.

Module 3: ProVE® Pentamer Library Synthesis

Objective: Produce MHC Class I Pentamers ready for validating T cell epitopes on clinical samples

ProVE® MHC Class I Pentamers are made using a rapid, high-throughput, parallel synthesis method.  They are supplied unlabeled and at a minimum quantity of 20 tests (one test labels 1-2 x 106 cells).  A separate Pro5® Fluorotag is supplied (R-PE or APC-labeled) for two-layer fluorescent staining.  Each Pentamer is QC tested using ELISA and is guaranteed for 3 months when stored at 4oC.  Storage of unlabeled Pentamers at -80oC extends the shelf life.

ProVE® Pentamers are suitable for multiplexed staining of antigen-specific T cells in flow cytometry.  This enables the user to identify and quantitate different populations of single antigen-specific T cells very rapidly and attain conclusive validation of new T cell epitopes.

With REVEAL & ProVE® technology it is possible to start with a large number of peptides in an overlapping library, screen against 6 MHC alleles, identify candidate peptides and provide their corresponding ProVE® Pentamers ready for testing on clinical samples within only ten to fifteen working days.

Module 4: REVEAL™ Rate Assays

Objective: Determine quantitative on and off rates for candidate peptides

At any stage after the initial REVEAL™ binding assay is completed the customer may request additional on- and off-rate assays that will give a quantitative picture of the binding properties of individual peptides.  These assays may be particularly important for intermediate affinity peptides.  Such peptides may exhibit weaker binding to a particular MHC allele than peptides with a fast on-rate.  However, if they have a slow off-rate, once the peptide is bound it could be presented for a considerable period of time, meriting further study as a potential epitope.


Figure 2: On-rate of the test peptide (left) is measured in comparison with the on-rate of the intermediate control peptide (complete rate assay).


Figure 3: Comparison of the off-rate of the test peptide (left) with the intermediate control peptide  (complete rate assay).

There are two options available for Module 4, the Quick Check Off Rate Assay and the Complete Rate Assay. 

Option 1:  Quick Check Off-Rate Assay

The 'Quick Check' option for Module 4 must be ordered with module 3, ProVE® Pentamers.  The off-rates for the MHC-peptide complexes are measured at 0h, 2h and 24h, at 37°C.  Results are supplied graphically and in terms of half-life values.  The Quick Check off-rate assay provides information relating to the likelihood of a peptide being presented long enough for it to be a good T cell epitope.  The results of this analysis can be combined with the results from the REVEAL™ binding assay, which typically reflect the on-rate properties of a peptide more strongly than the stability of assembled complexes.  The results are not as quantitative as the 'Complete Rate Assay' which determines the on-off rates more comprehensively, but the cost of the Quick Check assay is significantly less.  The results can be used as a cost effective way to prioritize which of the ProVE® Pentamers should be used on cellular samples first.  This enables the user to optimize the use of cell samples and the time needed for testing.

Option 2:  Complete Rate Assay

The 'Complete Rate Assay' may be ordered at any point in the REVEAL & ProVE® process.  The on and off-rates for peptides that have passed the REVEAL™ MHC-binding assay are measured at six points over 48 hours (on-rate) and 24 hours (off-rate).  Results are presented graphically and tabulated as half-life values.  Peptides are given a kinetic score based on the results of both rates; a higher score indicates a better candidate T cell epitope.  Finally the 'R' score is defined for each peptide.  This incorporates the kinetic score with the REVEAL™ MHC-peptide binding assay score; the higher the R score, the better the epitope.  Scores from the rate assays are offered as a general guideline; peptide epitopes should be validated using antigen-specific T cell detection with Pentamers.  The Complete Rate Assay module is ideal for studying the peptide presentation with Pentamers in depth and comparing peptide kinetics to other quantitative assays, such as staining with Pentamers and functional assays.

Publications citing REVEAL & ProVE®

Bell, MJ. et al. (2009) The peptide length specificity of some HLA class I alleles is very broad and includes peptides of up to 25 amino acids in length.  Mol Immunol. 46: 1911-1917 [PubMedID: 19157553]

Blancou, P. et al.  (2007).  Immunization of HLA Class I transgenic mice identifies autoantigenic epitopes eliciting dominant responses in type 1 diabetes patients.  J. Immunol. 178: 7458-7466.  [PubMedID: 17513797].

Burrows, J.M. et al.  (2007).  The impact of HLA-B micropolymorphism outside primary peptide anchor pockets on the CTL response to CMV.  Eur. J. Immunol. 37: 946-953.  [PubMedID: 17357107].

Harrop, R. et al. (2008). Vaccination of colorectal cancer patients with TroVax given alongside chemotherapy (5-fluorouracil, leukovorin and irinotecan) is safe and induces potent immune responses. Cancer Immunol. Immunother. 57: 977-86. [PubMedID: 18060404].

Muixi, L. et al (2008). Thyroglobulin peptides associate in vivo to HLA-DR in autoimmune thyroid glands.  J Immunol.  181: 795-807 [PubMedID: 18566446]

Ramaswami, B. et al. (2009) HLA-A01, -A03 and -A024 binding nanomeric epitopes in polyomavirus BK large T-antigen.  Human Immunology. doi: 10.1016/j.humimm.2009.05.003 [PubMedID: 19446588]

Westrop, S. et al. (2009). Novel approach to recognition of predicted HIV-1 Gag B*3501-restricted CD8 T-cell epitopes by B*3501+ patients: Confirmation by quantitative ELISPOT analyses and characterisation using multimers.  J Immunol. Methods. 341: 76-85 [PubMedID: 19056394]