Human Granzyme B ELISpot Set

The purpose of ELISPOT analysis is to count cytokine producing cells in a single cell suspension. The advantage of this method is that it requires minimal in vitro manipulations, making the analysis of cytokine production as close to in vivo conditions as possible, and in a highly specific manner.

The Elispot test is an ideal tool for TH1/TH2 response, vaccine development, viral infection monitoring and treatment, oncology, infectious diseases, autoimmune diseases and transplantation. Elispot detection is based on sandwich immunoenzyme technology. Cytokines or soluble molecules secreted by cells are captured by coating antibodies to avoid diffusion in the supernatant, protease degradation or binding to soluble membrane receptors. After the cells are removed, the captured cytokines are displayed by the tracer antibody and the appropriate conjugate.

ELISpot (ES)

Small volumes of GZMB ELISpot set vial(s) may occasionally become entrapped in the seal of the product vial during shipment and storage. If necessary, briefly centrifuge the vial on a tabletop centrifuge to dislodge any liquid in the container's cap. Certain products may require to ship with dry ice and additional dry ice fee may apply.

After cell stimulation, locally produced cytokines are captured by IFNg and IL-4 specific monoclonal antibodies. After cell lysis, trapped cytokine molecules are revealed by a secondary anti-IFNg FITC conjugated antibody and a biotinylated anti-IL-4 antibody. Those are in turn recognised by anti-FITC HRP and streptavidin-AP conjugates. PVDF-bottomed-well plates are then incubated first with AEC substrate buffer, washed and subsequently incubated with BCIP/NBT. Coloured red/brownish spots indicate IFNg production while IL-4 is revealed by blue/purple spots.

Granzyme B

Human

Sandwich

Sandwich

cell suspension

Recognizes natural human Granzyme B

1x96 Wells

1.Capture antibody (0.50 mL). Supplied sterile.
2.Biotinylated detection antibody (lyophilised, resuspend in 0.55mL).
3.Streptavidin - Alkaline Phosphatase conjugated (50 µL).
4.Bovine Serum albumin.
5.Ready-to-use substrate buffer (50mL).
6.96 PVDF-bottomed-well plates (5 if ordered).

1.96 PVDF-bottomed-well plates.
2.Cell culture media.
3.CO2 incubator.
4.70% ethanol.
5.Tween 20.
6.Phosphate buffered saline.

100 µL

1.5h

Plate-not-included

1.Detection antibody:Reconstitute the lyophilised antibody with 0.55mL of distilled water. Gently mix the solution and wait until all the lyophilised material is back into solution.
2.Streptavidin alkaline phosphatase:Dilute 1/1000 in PBS 1% BSA.
3.Phosphate buffered saline (10X Concentrate solution):For 1 liter weight : 80g NaCl ; 2g KH2PO4 ; 14.4g Na2HPO4 2H2O. Add distilled water to 1 liter. Check that pH is comprised between 7.4 +/- 0.1. Dilute the solution to 1X before use.
4.1% BSA in PBS:For one plate dissolve 0.2 g of BSA in 20 mL of 1X diluted PBS.
5.0.05% Tween in PBS:For one plate dissolve 50µl of Tween 20 in 100 ml of 1X diluted PBS.
6.35% ethanol in water:For one plate mix 3.5 ml of ethanol with 6.5 ml of distilled water.

1.Incubate PVDF-bottomed-well plates with 25µl / well of 35% ethanol for 30 sec at room temperature.
2.Empty wells and wash three times with 100µl / well of PBS.
3.Pipette 100µl of capture antibody in 10 mL of PBS. Mix and dispense 100 µl into each well, cover the plate and incubate overnight at +4°C.
4.Empty wells and wash once with 100 µl of PBS.
5.Dispense 100 µl of RPMI 10% FCS into wells, cover and incubate for 2 hours at room temperature.
6.Empty wells by flicking the plate over a sink and tapping it on absorbent paper.
7.Wash plate once with PBS.
8.Dispense into wells 100 µl of cell suspension containing the appropriate number of cells and appropriate concentration of stimulator. Cells may have been previously in-vitro stimulated (Indirect ELISPOT). Cover the plate with a standard 96-well plate plastic lid and incubate cells at 37°C in a CO2 incubator for an appropriate length of time (10-15 hours).
9.Empty wells by flicking the plate over a sink and gently tapping it on absorbent paper.
10.Distribute 100µl of PBS-0.0.5% tween 20 in wells and let sit for 10 min at +4°C.
11.Wash wells three times with PBS-0.05% tween 20.
12.For 1 plate dilute 100µl of reconstituted detection antibody into 10 mL of PBS containing 1% BSA. Distribute 100µl in wells, cover the plate and incubate 1 hour 30 min at room temperature.
13.Empty wells and wash three times with PBS-0.05% tween 20.
14.For 1 plate dilute 10µl of streptavidin-Alkaline phosphatase conjugate into 10 mL of PBS-1% BSA. Distribute 100µl of the dilution in wells. Seal the plate and incubate for 1 hour at room temperature.
15.Empty wells and wash three times with PBS-0.05% tween 20.
16.Peel off the plate bottom and wash three times both sides of the membrane under running distilled water. Remove all residual buffer by repeated tapping on absorbent paper.
17.Distribute 100µl of ready-to-use BCIP/NBT buffer in wells.
18.Let the reaction go for about 5-20 min at room temperature. Monitor spot formation visually.
19.Rinse three times both side of the membrane under running distilled water.
20.Dry wells. Read spots. Note that spots may become sharper after one night at +4°C.

Human

NP_004122.2

Liquid

4°C/-20°C

If not used within a short period of time, reconstituted detection antibody should be aliquoted and stored at -20°C.

at least one year at -20°C

Select online data sheet information is drawn from bioinformatics databases, occasionally resulting in ambiguous or non-relevant product information. It is the responsibility of the customer to review, verify, and evaluate the information to make sure it matches their requirements before purchasing the kit. Our ELISA Kit assays are dynamic research tools and sometimes they may be updated and improved.

For Research Use Only. Not for use in diagnostic procedures.

EC 3.4.21.79; CTLA-1; CSP-B; Granzyme B; CTLA1; CCPI; CGL-1; CGL1; Cytotoxic T-lymphocyte proteinase 2; T-cell serine protease 1-3E; HLP; CTSGL1; in-2; Cathepsin G-like 1; SECT; Granzyme-2; Human lymphocyte protein; C11; CSPB; Lymphocyte protease

HLP; CCPI; CGL1; CSPB; SECT; CGL-1; CSP-B; CTLA1; CTSGL1

Granzyme B is a member of the granzyme subfamily of proteins, part of the peptidase S1 family of serine proteases. The encoded preproprotein is secreted by natural killer (NK) cells and cytotoxic T lymphocytes (CTLs) and proteolytically processed to generate the active protease, which induces target cell apoptosis. This protein also processes cytokines and degrades extracellular matrix proteins, and these roles are implicated in chronic inflammation and wound healing. Expression of this gene may be elevated in human patients with cardiac fibrosis.

3002

P10144

Among its related pathways are Allograft rejection and Transcriptional misregulation in cancer.

1.96-PVDF bottomed-well plates are first treated with 70% ethanol and then coated with capture antibody.
2.Incubation of cells in the coated microwell.
3.Cell removal by washing. Incubation with biotinylated antibody.
4.Incubation with streptavidin - alkaline phosphatase conjugated.
5.Addition of substrate BCIP/NBT and monitoring of spot formation.