PepPool: CEF extended (CD8), human

PepPool: CEF extended (CD8), human

3618-1

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Validated for our assays.

Content

Peptide pool with 32 peptides, 25 ug of each peptide. Supplied in one vial (lyophilized).

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Delivery 4-9 business days

Shipping $0

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 extended peptide pool induces secretion of e.g., IFN-γ, IL-2, and granzyme B from human CD8 T cells. It consists of 32 MHC class I-restricted viral peptides from human CMV, EBV and influenza virus. The synthetic peptides are >95% pure.

Published HLA types covered by the CEF extended peptide pool:
HLA-A1, HLA-A2, HLA-A3, HLA-A11, HLA-A24, HLA-A68, HLA-B7, HLA-B8, HLA-B27, HLA-B35, HLA-B44, HLA-B18

Recommendation

Product details

ProductPepPool: CEF extended (CD8), human
ApplicationT cell stimulation, ELISpot, FluoroSpot, Flow cytometry
ReactivityHuman
Supplied in

Lyophilized

Contents

Peptide pool with 32 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. 

Human PBMC stimulated with the CEF extended peptide pool (product code 3618-1) in human IFN-γ ELISpot. Analysis was done with Mabtech IRIS.

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 FluoroSpot 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.

Tutorial, Published December 28, 2023

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.
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