Human Granzyme B Assay Kit

Sandwich immunoassay kit designed for the rapid detection of Granzyme B

Granzyme B (also called Cathepsin G-like 1; Cytotoxic T-lymphocyte proteinase 2; Fragmentin-2; Granzyme-2; Human lymphocyte protein) is found in cytotoxic lymphocytes, NK cells (natural killer) and cytotoxic T cells. Secreted with perforin, which creates a pore in the cell membrane, it enters the target cell’s cytoplasm and triggers apoptosis through caspase activation.
Based on the TR-FRET homogeneous robust HTRF® technology, this kit is designed for the rapid detection of human Granzyme B in cell supernatant and whole cells. The assay enables high throughput without any washing steps, thereby saving time compared to ELISA.

Assay principle

HTRF® - the homogeneous sandwich immunoassay

Human Granzyme B is measured using a sandwich immunoassay involving two specific anti-human Granzyme B antibodies, respectively labeled with HTRF donor and acceptor dyes. The intensity of signal is directly proportional to the concentration of Granzyme B present in the sample. The assay protocol, using a 384-well small volume white plate or a Cisbio low volume 96 well plate (20 µL final), is described on the right. 16µl of cell supernatant, sample or standard are dispensed directly into the detection plate for the detection by HTRF® reagents. The antibodies labeled with HTRF donor and acceptor can be pre-mixed and added in a single dispensing step, to further streamline the assay procedure. The assay can be run in 96- to 384-well plates by simply resizing each addition volume proportionally.

                                                                    

                                                                          

Product performance

Detection of Granzyme B on cell supernatant (One-plate protocol)

 

Different cell densities (4K and 2K) of TALL-104 cells (Effector cells) were cocultured with or without K-562 cells (Target cells) in suspension under 16µL in 384-well sv microplate. Cells were incubated for 16 hours at 37 °C - 5% CO2. After coculture, 4 µL of the HTRF Granzyme B detection reagents were added. The HTRF signal was recorded after an overnight incubation at room temperature.

One Plate Granzyme B Assay

Detection of Granzyme B on cell supernatant (Two-plate protocol)

 

Different cell densities (50K and 25K) of TALL-104 cells (Effector cells) were plated in suspension under 50µL in 96-well plate. TALL-104 cells were co-cultured with or without K-562 cells (Target cells) for 16 hours at 37 °C - 5% CO2. After co-culture, 16µL of cell supernatants cells were transferred into a 384-well sv white microplate and 4 µL of the HTRF Granzyme B detection reagents were added. The HTRF signal was recorded after an overnight incubation at room temperature.

Two Plate Granzyme B Assay

Analytical performance

Sample size

16 µL

Final assay volume

20 µL

Kit components

Frozen standard, frozen detection antibodies, buffers & protocol

LOD & LOQ (in diluent)

2.3 ng/mL & 3.1 ng/mL

Range

3.1 – 800 ng/mL

Time to result

ON at RT

 

Precision

intra assay (n=24)

Sample

Mean [GranzymeB] (pg/mL)

CV

1

8

24%

2

58

14%

3

578

12%

 

Mean CV

16%

Each of the 3 samples was measured 24 times, and % CV was calculated for each sample.

Inter assay (n=4)

Sample

[GranzymeB]
(pg/mL)

Mean
(deltaRatio)

CV

1

7

292

22%

2

47

1 877

14%

3

339

9 716

8%

 

 

Mean CV

14%

Each of the samples was measured in 4 different experiments, and % CV was calculated for each sample.

Part#, inserts & MSDS

Ordering Info

DescriptionCat. noProduct insertMSDS
Human Granzyme B kit - 500 tests63ADK095PEG
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Human Granzyme B kit - 10,000 tests63ADK095PEH
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Companion products

DescriptionCat. noProduct insertMSDS
Human Granzyme B kit standard63ADK095CDA
-
-

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