Human Granzyme B Quantikine ELISA Kit Summary

Product Summary

The Quantikine® Human Granzyme B Immunoassay is a 3.5 hour solid-phase ELISA designed to measure human Granzyme B in culture supernates. It contains NS0-expressed recombinant human Granzyme B and antibodies raised against the recombinant protein. Results obtained using natural human Granzyme B showed linear curves that were parallel to the standard curves obtained using the Quantikine® kit standards. These results indicate that this kit can be used to determine relative mass values for natural human Granzyme B.

Preparation and Storage

Background: Granzyme B

Granzyme B is a member of the granzyme family of serine proteases found specifically in granules of cytotoxic T lymphocytes (CTL) and natural killer (NK) cells (1, 2). Granzyme B plays an essential role in granule-mediated apoptosis utilizing the substrates in this pathway, such as Caspase 3, Caspase 8 and Bid (3, 4). Recent research indicates expanded Granzyme B functionality to include extracellular roles along with its classical pro-apoptotic function. It has been found that Granzyme B is an important mediator of skin injury, repair and inflammation (4) through extracellular substrates including Laminin, VE-Cadherin, Fibronectin and the proteoglycans Aggrecan (3) and Decorin (4). 

As one of the five Granzymes (A, B, H, K and M) identified in the human genome, Granzyme B (32kDa) (5) is the most widely researched in terms of its biological function and its utility in health and disease (4). It is synthesized as a precursor (247 residues) with a signal peptide (residues 1-18), a pro-peptide (residues 19-20), and a mature chain (residues 21-247) (6-8). Once inside granules, Granzyme B is fully processed into the mature chain and becomes an active protease when the pro-peptide, Gly-Glu is removed from the N-terminus by cleavage with Cathepsin C (9). The protease activity of Granzyme B is tightly controlled by Serpin B9/ Protease Inhibitor 9 (9). The amino acid sequence of human Granzyme B is 71%, 69%, and 68% identical to its canine, rat, and mouse counterparts, respectively. 

Granzymes have been shown to modulate inflammation, and Granzyme B plasma levels have been found higher with atopic dermatitis and psoriasis when compared to healthy controls. This is in contrast to Granzyme A plasma levels which remain unchanged (10). Serum from patients with Crohn's disease have significantly higher Granzyme B levels than controls (11).

Assay Procedure

Refer to the product datasheet for the complete assay procedure.

Bring all reagents and samples to room temperature before use. It is recommended that all samples, standards, and controls be assayed in duplicate.

1.     Prepare all reagents, standard dilutions, and samples as directed in the product insert.

2.     Remove excess microplate strips from the plate frame, return them to the foil pouch containing the desiccant pack, and reseal.

3.     Add 50 μL of Assay Diluent to each well.

4.     Add 50 μL of Standard, control, or sample to each well. Cover with a plate sealer, and incubate at room temperature for 2 hours.

5.     Aspirate each well and wash, repeating the process 4 times for a total of 5 washes.

6.     Add 100 μL of Conjugate to each well. Cover with a new plate sealer, and incubate at room temperature for 2 hours.

7.     Aspirate and wash 5 times.

8.     Add 100 μL Substrate Solution to each well.

9.     Add 100 μL of Stop Solution to each well. Read at 450 nm within 30 minutes. Set wavelength correction to 540 nm or 570 nm.