Anti-human IL-3 mAb (IL3A), unconjugated

Anti‑human IL‑3 mAb (IL3A), unconjugated

3435-3-250

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Content

Monoclonal antibody IL3A, unconjugated. Supplied at 0.5 mg/ml in PBS with 0.02% sodium azide.

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Product specifications

Intended use

This monoclonal antibody enables specific detection of human IL-3 in immunoassays such as ELISpot, FluoroSpot, and ELISA.

Serum/Plasma samples

Recommendation

IL3A is recommended as capture mAb in ELISpot, FluoroSpot, and ELISA in combination with detection mAb IL3B (product code 3435-6).

Product details

ProductAnti-human IL-3 mAb (IL3A), unconjugated
ApplicationELISpot, FluoroSpot, ELISA
AnalyteIL-3
AntibodyIL3A
ConjugateUnconjugated
ClonalityMonoclonal
ImmunogenRecombinant human IL-3
HostRat
IsotypeIgG1
ReactivityHuman
SpecificityNative and recombinant human IL-3
PurificationPurified from in vitro cultures by protein G affinity chromatography.
Concentration0.5 mg/ml
Supplied inPBS with 0.02% sodium azide. Sterile-filtered (0.2 µm).
ContentsMonoclonal antibody IL3A, unconjugated. Supplied at 0.5 mg/ml in PBS with 0.02% sodium azide.

Shipping and Storage

ShippingShipped at ambient temperature.
StorageStore product at 4-8°C or frozen at -20°C or below. Avoid repeated freezing/thawing.
Shelf lifeAt least 18 months from date of receipt.

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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IL-3

Analyte description

The cytokine interleukin 3 (IL-3) is mainly produced by activated T cells. IL-3 is also called multi-lineage-colony stimulating factor since it induces the growth and differentiation of many cells. IL-3 also activates mast cells, eosinophils, basophils, and macrophages.

Alternative namesInterleukin 3, IL-3, IL3, colony-stimulating factor, multi-CSF, mast cell growth factor, MULTI-CSF, MCGF
Cell typeT cell, Th2
Gene ID3562
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