ProVE® MHC Class I Pentamer Libraries | ProImmune

ProVE® MHC Class I Pentamer Libraries

ProVE® MHC Class I Pentamer Libraries provide a quick and cost effective way of screening a number of peptides implicated as epitopes in the immune response under investigation.

ProVE® Libraries bridge the gap between the screening of high numbers of T cell epitopes and the in-depth monitoring and characterization of single specificity CD8+ T cell immune responses with our individually manufactured Pro5® MHC Class I Pentamers. A cost reduction of more than 80% is achievable compared to the synthesis of standard MHC reagents.

Application Notes: multiple staining with unlabeled Pentamers

Customer Case Study

REVEAL & ProVE® Rapid Epitope Discovery System

Pro5® MHC Class I Pentamers

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ProVE® Pentamer Libraries Key Features

Rapid high throughput synthesis process

 Fast delivery time (2-3 weeks). A large number of Pentamers can be delivered together.
Flexibility in supplying the peptide Custom peptides can be synthesized by ProImmune or supplied by the customer.
Full QC including conformational ELISA and protein concentration Consistent and reliable performance compared with self-made tetramers or self-loaded dimeric MHC products.

Low price per multimer and no custom set up charges

 Up to 80% cost reduction per reagent when compared to the synthesis of standard Pentamers

Product Specification

A ProVE® Pentamer Library is a set of custom Pentamers. The customer specifies the MHC allele under investigation and provides a small quantity of each of the peptides of interest. Alternatively, a custom synthesis of the chosen peptides may be ordered from ProImmune at competitive rates.

ProImmune manufactures the Pentamers using a rapid, high throughput, parallel synthesis process and supplies them unlabeled and at a minimum quantity of 20 tests. There is a minimum order of 10 Pentamers per ProVE® Library per allele.

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 CD8+ T cells very rapidly and attain conclusive validation of new T cell epitopes.

Quality Control

Each ProVE® Pentamer is QC tested using ELISA to determine that the Pentamer has the correct conformation and therefore the peptide is in place. The protein concentration is determined and translated into quantity for each product. The customer pays for the complete synthesis run and receives those ProVE® Pentamers that pass quality control. The average QC pass rate for HLA-A*02:01 ProVE® Pentamers is 85% for peptides with scores >21 in the SYFPEITHI epitope prediction algorithm (www.syfpeithi.com).

Before proceeding with an order, ProImmune will review the sequences of the peptides submitted for inclusion in the ProVE® Pentamers and will notify the customer of any problems anticipated.

Feature Comparison

 

ProVE® Pentamer Libraries

Custom Pro5® Pentamers
Target Applications Epitope screening
Epitope validation, e.g. following ELISPOT analysis
Mutagenesis studies		 Quantitative epitope confirmation
Immune monitoring of confirmed epitopes
Available Alleles HLA-A*01:01, 02:01, 03:01, 11:01, 24:02, 29:02
HLA-B*07:02, 08:01, 14:02, 15:01, 27:05, 35:01, 40:01
H-2 Kb, Db, Kd, Dd, Ld
Mamu A*01, A*02		 HLA-A*01:01, 02:01, 03:01, 11:01, 11:03, 24:02, 29:02, 68:01
HLA-B*07:02, 08:01, 14:02, 15:01, 27:05, 35:01, 35:08, 40:01, 51:01, 54:01
H-2 Db, Dd, Kb, Kd, Ld
Chimeric A02:01/Kb
Mamu A*01, A*02
Minimum Order 10 ProVE® Pentamers 1 Pro5® Pentamer
Delivery Time 2-3 weeks from receipt of peptides Catalog items 1-2 weeks
Custom items 4-6 weeks
Custom Set Up Charge No Yes
Pack Size 20-50 tests 50, 150 or 500 tests
Synthesis Process Rapid high throughput synthesis Extended synthesis for maximum purity and quality
Quality Control Full QC process incl. pass/fail QC provided for each reagent Full QC process
Fluorescent Labels Pro5® Biotag, R-PE, APC Pro5® Biotag, R-PE, APC
Labeling Method Supplied with separate Pro5® Biotag or Fluorotag (R-PE or APC), for two layer staining Pre-conjugated with Biotin, R-PE or APC
Or supplied with separate Pro5® Biotag or Fluorotag (R-PE or APC), for two layer staining
Who Provides the Peptide? Provided by customer to save cost
(0.5 mg, >70% purity, supplied in an Eppendorf® tube with accurate actual weight provided)
Or synthesized by ProImmune where required		 Synthesized by ProImmune for full quality control
What Do I Receive? All ProVE® Pentamers that pass quality control The custom Pentamer
Guarantee Period 3 months, stored at 4°C Biotin, R-PE or APC labeled: 6 months, stored at 4°C
Unlabeled: 12 months, stored at -80°C

Comparative Staining of PBMC with ProVE® MHC Pentamers and Pro5® MHC Pentamers

Peripheral blood lymphocytes were obtained from a healthy donor previously shown to respond to the A2-restricted CMV epitope, at approx. 0.15% of total PBMC. The left plot shows cells stained with the ProVE® MHC Pentamer, while the right plot shows cells stained with Pro5® MHC Pentamer made by the conventional process. The antigen-specific population is circled in red on both plots.

(Experimental procedure: cells were incubated with 1ug recombinant A2/CMV complex, followed by 1 test of PE-labeled Pro5® Fluorotag & 1 test of FITC-labeled anti-CD8 antibody (Clone LT8). Approximately 30,000 events are shown in each plot).