Rat Granzyme B (GZMB) ELISA Kit

Principle of the Assay

The microtiter plate provided in this kit has been pre-coated with an antibody specific to GZMB. Standards or samples are then added to the appropriate microtiter plate wells with a biotin-conjugated antibody preparation specific to GZMB. Next, Avidin conjugated to Horseradish Peroxidase (HRP) is added to each microplate well and incubated. After the TMB substrate solution is added, only those wells that contain GZMB, biotin-conjugated antibody, and enzyme-conjugated Avidin will exhibit a change in color. The enzyme-substrate reaction is terminated by the addition of sulphuric acid solution, and the color change is measured spectrophotometrically at a wavelength of 450nm ± 10nm. The concentration of GZMB in the samples is then determined by comparing the O.D. of the samples to the standard curve.

For Use with serum, plasma, and cell culture supernatants. For Research Use Only. Not for use in diagnostic procedures.

Target Information

Abundant protease in the cytosolic granules of cytotoxic T-cells and NK-cells which activates caspase-independent pyroptosis when delivered into the target cell through the immunological synapse. It cleaves after Asp. Once delivered into the target cell, acts by catalyzing cleavage of gasdermin-E (GSDME), releasing the pore-forming moiety of GSDME, thereby triggering pyroptosis and target cell death. Seems to be linked to an activation cascade of caspases (aspartate-specific cysteine proteases) responsible for apoptosis execution. Cleaves caspase-3, -7, -9 and 10 to give rise to active enzymes mediating apoptosis.

GENE ID	171528
SWISS PROT	P18291
SYNONYMS	GZM-B; HLP; CTLA1; CCPI; CGL1; CSP-B; CSPB; CTSGL1; SECT; Granzyme 2; Cytotoxic T-Lymphocyte-Associated Serine Esterase 1; Fragmentin 2; Cytotoxic Serine Protease B

Materials Supplied

Kit Components	96 Wells Quantity/Size
Pre-coated, ready-to-use 96-well strip plate	1 plate
Plate sealer for 96 wells	2
Standard	2 tubes
Diluent buffer	1 bottle
Detection Reagent A	1 bottle
Detection Reagent B	1 bottle
TMB Substrate	1 tube
Stop Solution	1 tube
Wash Buffer (30 ℅ concentrate)	1 tube
Product data sheet	1 copy

Storage

The TMB Substrate, Wash Buffer (30X concentrate), and the Stop Solution should be stored at 4°C upon receipt, while the other items should be stored at -20°C.

Performance Characteristics

REPEATABILITY	Intra-assay Precision (Precision within an assay): 3 samples with low, middle, and high-level GZMB were tested 20 times on one plate, respectively. Inter-assay Precision (Precision between assays): 3 samples with low, middle, and high-level GZMB were tested on 3 different plates, with 8 replicates in each plate. CV(%) = SD/meanX100 Intra-Assay: CV<10% Inter-Assay: CV<12%
SENSITIVITY	The minimum detectable dose was 6.4pg/mL.
ASSAY RANGE	15.6-1000pg/mL
SPECIFICITY	This assay has high sensitivity and excellent specificity for the detection of GZMB. No significant cross-reactivity or interference between GZMB and analogs was observed. Note: Limited by current skills and knowledge, it is impossible to perform all possible cross-reactivity detection tests between GZMB and all analogs, therefore, cross reactivity may still exist.