PepPool: CEF (CD8), human

Human PBMC stimulated with the CEF peptide pool (product code 3616-1) in human IFN-γ ELISpot. Analysis was done with Mabtech IRIS.

Human PBMC stimulated with the CEF peptide pool (product code 3616-1) and anti-human CD28 antibody in human IFN-γ/Granzyme B FluoroSpot. Analysis was done with Mabtech IRIS.

CEF peptide pool used in Flow cytometry to stimulate IFN-γ production by human CD8+ T cells

PepPool: CEF (CD8), human

3616-1

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Product specifications

Intended use

This peptide pool is intended for use as a positive control in immunoassays such as ELISpot, FluoroSpot, and flow cytometry. It induces cytokine secretion from antigen-specific human CD8 T cells. For research use only. Not for use in diagnostic procedures.

Serum/Plasma samples

Description
The CEF peptide pool induces secretion of e.g., IFN-γ, IL-2, and granzyme B from human CD8 T cells. It consists of 23 MHC class I-restricted viral peptides from human CMV, EBV, and influenza virus. It has been shown to induce IFN-γ responses in almost 90% of all Caucasians. The synthetic peptides are >95% pure.

Published HLA types covered by the CEF peptide pool:
HLA-A1, HLA-A2, HLA-A3, HLA-A11, HLA-A24, HLA-A68, HLA-B7, HLA-B8, HLA-B27, HLA-B35, HLA-B44

Peptides included in the CEF peptide pool are listed in the datasheet.

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Product details

Product	PepPool: CEF (CD8), human
Application	T cell stimulation, ELISpot, FluoroSpot, Flow cytometry
Reactivity	Human
Supplied in	Lyophilized
Contents	Peptide pool with 23 peptides, 25 ug of each peptide. Supplied in one vial (lyophilized).

Shipping and Storage

Shipping	Shipped at ambient temperature.
Storage	Store at -20 °C or below upon receipt.

Performance

Human PBMC stimulated with the CEF peptide pool (product code 3616-1) in human IFN-γ ELISpot. Analysis was done with Mabtech IRIS.

Human PBMC stimulated with CEF peptide pool (2 ug/ml) and anti-CD28 antibody (0.1 ug/ml) for 16 hours (both available from Mabtech) in the presence of Brefeldin A. CD8+ cells start expressing IFN-γ following CEF peptide pool stimulation, whereas CD4+ cells remain quiet. Cells were stained with CD4-PE and CD8-FITC prior to fixation and permeabilization using 4% paraformaldehyde and saponin. IFN-γ was detected using 1D1K-PF647P (Mabtech). The dot plots derive from gated events of typical lymphocyte characteristics in forward and side light scatter. Flow cytometry was performed on a BD FACSVerse system.

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Datasheet/Protocol SDS

Analyte combinations in FluoroSpot

Find out which analyte combinations we have evaluated in T cell FluoroSpot and which combinations are affected by capture effects or not.

What is a capture effect?

When capture antibodies with different specificities are coated together, the capture of one cytokine may affect the secretion of other cytokines. This is usually more pronounced when studying T cell responses with polyclonal stimuli compared to antigen-specific responses. Capture effects are seldom a problem and can often be counteracted by the addition of an anti-CD28 antibody.

Depiction of capture effects observed in FluoroSpot

(1) IL-2 secreted by the activated T cell is captured by coated anti-IL-2 capture antibodies. (2) As a result, IL-2-stimulation of the T cell itself (autocrine stimulation) as well as nearby T cells (paracrine stimulation) is impaired, ultimately leading to (3) decreased IFN-γ secretion.

Co-stimulation with anti-CD28

Anti-CD28 mAb provides a co-stimulatory signal to antigen-specific responses by binding to CD28 on T cells. The addition of an anti-CD28 mAb to the cell culture enhances antigen-specific responses and can counteract capture effects. For example, the presence of IL-2 capture antibodies may result in reduced activation of T cells, as capturing of IL-2 decreases the amount of available IL-2 and thereby dampens the IL-2-mediated stimulation of T cells. The addition of anti-CD28 mAb restores IFN-γ responses (depicted in the below images). Further optimization may be necessary, depending on the cells and stimuli used. Too high concentrations of the anti-CD28 mAb may result in an elevation of non-specific cytokine secretion.

Overcoming capture effects using anti-CD28 anitbodies in FluoroSpot

(1) An anti-CD28 antibody can be added to provide a co-stimulatory signal that can restore (2) e.g. IFN-γ responses.

How to investigate capture effects

Our FluoroSpot Plus kits are evaluated for capture effects, and in studies of T cell responses we recommend co-stimulation with anti-CD28. With FluoroSpo t Flex, it is possible to combine and build your own kit. For guidance look at our analyte combination table (above). Capture effects can be investigated by quantifying spot numbers in wells coated with single capture antibodies and compared to wells coated with a mixture of the capture antibodies. The compensatory effect of the anti-CD28 mAb may be assessed by comparing cells cultured with and without the anti-CD28 mAb.

Tutorials

Tutorial
Published: December 28, 2023 | 2 minute read

Incubation strategies for success in ELISpot and FluoroSpot

We've made a handy table summarizing incubation times for different analytes to make your research planning for the ELISpot and FluoroSpot assay a little easier.

Publications (42)

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