Flow cytometry Testing with Pro5^® MHC Class I Pentamers

ProImmune offers a flexible flow cytometry testing service for measuring antigen-specific CD8+ T cell responses using MHC Class I Pentamers.

Pro5® and ProVE® MHC Class I Pentamers bind directly to T cell receptors of a particular specificity, determined by the MHC allele and peptide combination, and can detect antigen-specific T cell populations as rare as 0.02% of lymphocytes.

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Pro5® Class I Pentamers

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Staining cells with MHC Pentamers provides a quantitative analysis of lymphocytes that express a specific T cell receptor and in conjunction with other functional assays, such as ELISPOT, gives complementary information about an immune response.

Even for researchers experienced in flow cytometry techniques, cell preparation, staining and analysis all require careful planning and execution. By outsourcing flow cytometry testing to ProImmune, you immediately benefit from many years experience from the team that has developed MHC Pentamer technology. Protocols are carried out to optimized procedures to give reliable and consistent results.

Our affordable, streamlined service is based on simple standardized formats, which follow this step-by-step process:

(1) Customer cryopreserves cells in accordance with optimized protocol recommended by ProImmune or optimized customer protocol.

(2) ProImmune collects cells from customer site using validated shipper (customer may also use their own shipper)

- ProImmune carries out HLA tissue typing as an option if required

(3) ProImmune thaws cells with optimized protocol and carries out Pro5® or ProVE® Pentamer staining and flow cytometry analysis in accordance with one or more of the following assay formats:

a. Immune monitoring: Staining with Pro5® Pentamer vs. anti-CD8 and anti-CD19 (for negative gating of B cells) on frozen, unmodified PBMC.

b. Epitope validation: Staining with individual ProVE® Pentamers or pools of Pentamers vs. anti-CD8 and anti-CD19 (for negative gating of B cells) on frozen ,unmodified PBMC.

(4) Customer receives full assay report and raw data.

By request these processes can be easily adapted to any additional or alternative requirement.

Request the Cellular Analysis Services Survey Form to obtain a quotation for your precise requirements.

Epitope Validation Using Pentamer Staining

The REVEAL & ProVE® Rapid Epitope Discovery System determines candidate T cell epitopes from a starting set of peptides such as overlapping peptides from a protein, or peptides that have been pre-scored or pre-validated in any other way.

Candidate sequences identified with the REVEAL™ MHC-peptide binding assay can be validated either by ELISPOT analysis or with MHC Class I Pentamers in flow cytometry. Our cellular analysis services can be integrated with this process to provide seamless epitope discovery and validation. The customer only needs to provide the cell samples to be used in the validation step.

For example, in the discovery of new CD8+ T cell epitopes, libraries of epitope-specific ProVE® Pentamers can be used to identify and quantify the antigen-specific CD8+ T cells, and thus validate the T cell epitopes. Detection of positive staining with Pentamer against an appropriate negative control means that expanded T cells are present in the donor sample that are specific to the peptide and MHC allele under investigation. This is the most definitive confirmation achievable for a T cell epitope, and confirms its minimal binding sequence and allelic restriction.

It is possible to stain a single cell sample with a pool of unlabeled Pentamers as a rapid screen for positive responses. Pooling MHC Pentamers together for screening purposes reduces the total number of cells required, and subsequently, the time spent staining and analyzing samples.

Case Study: Multiple staining with a pool of unlabeled Pentamers

Overview of Cellular Analysis Services

Cellular Analysis Services (PDF)