Assay principle

Human Granzyme B is a sandwich immunoassay involving two specific anti-human Granzyme B antibodies, respectively labelled with Europium Cryptate (donor) and d2 (acceptor). The intensity of the signal is proportional to the concentration of Granzyme B present in the sample.

Assay Protocol

The assay protocol, using a 384-well small volume white plate or a Revvity low volume 96-well plate (20 µL final), is described on the right. 16 µL of cell supernatant, sample, or standard are dispensed directly into the plate for detection by HTRF reagents. The antibodies labelled 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.

Granzyme B standard curve

Recombinant Granzyme B provided in the kit (standard) was serially diluted in diluent #5, following the procedure given in the kit’s package insert. The HTRF signal, expressed as a delta ratio, was plotted as a function of the Granzyme B concentrations.

intra assay (n=24)

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

Inter assay (n=4)

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

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

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

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 plates. 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 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 RT.