Anti-human EBI3 mAb (MT23A14), unconjugated

Product specifications

Intended use

This monoclonal antibody enables specific detection of the EBI3 subunit of human IL-27 and IL-35 in immunoassays such as ELISpot, FluoroSpot, and ELISA.

Serum/Plasma samples

Recommendation

MT23A14 is recommended as capture mAb in ELISpot, FluoroSpot, and ELISA in combination with detection mAb MT140 (product code 3458-6).

Product details

Product	Anti-human EBI3 mAb (MT23A14), unconjugated
Application	ELISpot, FluoroSpot, ELISA
Analyte	EBI3
Antibody	MT23A14
Conjugate	Unconjugated
Clonality	Monoclonal
Immunogen	Recombinant human IL-27
Host	Mouse
Isotype	IgG1
Reactivity	Human
Specificity	Native and recombinant human EBI3. MT23A14 binds to the EBI3 subunit of IL-27 and IL-35.
Purification	Purified from in vitro cultures by protein G affinity chromatography.
Concentration	0.5 mg/ml
Supplied in	PBS with 0.02% sodium azide. Sterile-filtered (0.2 µm).
Contents	Monoclonal antibody MT23A14, unconjugated. Supplied at 0.5 mg/ml in PBS with 0.02% sodium azide.

Shipping and Storage

Shipping	Shipped at ambient temperature.
Storage	Store product at 4-8°C or frozen at -20°C or below. Avoid repeated freezing/thawing.
Shelf life	At least 18 months from date of receipt.

Documents

Datasheet/Protocol
SDS

Human IL-12 family mAb guide

Learn how to combine Mabtech’s antibodies for the detection of human IL-12, IL-12/-23 (p40), IL-23, IL-27, and EBI3 in ELISA and ELISpot.

The IL-12 cytokine family

The IL-12 cytokine family consists of IL-12, IL-23, IL-27, IL-35, and IL-39. The existence of IL-39 in human remains, however, to be confirmed. The proteins are heterodimers of different subunits which are either unique or shared by several family members. IL-12 is a heterodimer, (p70), consisting of two covalently linked subunits, p35 and p40. The p40 subunit is shared with IL-23 which is a heterodimer of p40 and a unique p19 subunit. The p40 subunit also exists as a monomer and homodimer. The EBI3 subunit is shared with IL-27, IL-35, and IL-39.

Mabtech supplies monoclonal antibodies with defined subunit specificities to p40, p35, p19, p28, and EBI3. Since both IL-12 and IL-23 heterodimers contain the p40 subunit, capture immunoassays based on antibodies to p40 will detect both cytokines. Such assays can also detect p40 monomers and dimers. The EBI3 assays will detect IL-27, IL-35, and IL-39.

In our IL-12 and IL-23 assays, the same capture antibodies to p40 (MT86/221) are used. Different detection antibodies enable the detection of IL-12 (p70), IL-12/-23 (p40), and IL-23.

Application guide: mAbs to human IL-12, IL-23, IL-27 and EBI3

IL-12 (p70)

Capture mAbs: MT86/221 (p40 specific)

Detection mAb: MT704 (p70 specific)

IL-23

Capture mAbs: MT86/221 (p40 specific)

Detection mAb: MT155 (p19 specific)

IL-27

Capture mAbs: MT2 7/361 (IL-27 and p28 specific)

Detection mAb: MT140 (EBI3 specific)

IL-12/-23 (p40)

Capture mAbs: MT86/221 (p40 specific)

Detection mAb: MT6 18 (p40 specific)

EBI3 (IL-27 and IL-35)

Capture mAbs: M T23A14 (EBI3 specific)

Detection mAb: MT140 (EBI3 specific)

Get inspired!

Make sure to check out how researchers have used Mabtech's IL-23 ELISA, ELISpot, and FluoroSpot kits in everything from infectious disease research to novel immunotherapies. You can find it all in our Publication database.

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

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 (0)

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Analyte information

EBI3

Analyte description	EBI3 is a protein subunit that forms heterodimers with other proteins in the IL-12 family. IL-27 is a heterodimer composed of the subunits EBI3 and p28. IL-35 is a heterodimer composed of the subunits EBI3 and p35. IL-39 is a heterodimer consisting of the subunits EBI3 and p19. Native IL-39 has not yet been detected in humans. The EBI3 has ca. 30% sequence similarity to IL-12 p40.
Alternative names	Epstein–Barr virus-induced gene3, EBI3, Interleukin-27 subunit beta, IL-27B, IL27B, IL35B
Cell type	Monocyte/MΦ, mDC
Gene ID	10148