Human Granzyme B DuoSet ELISA

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Human Granzyme B ELISA Standard Curve
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Product Details
Citations (17)
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Human Granzyme B DuoSet ELISA Summary

Assay Type
Solid Phase Sandwich ELISA
96-well strip plate
Assay Range
39.1 - 2,500 pg/mL
Sufficient Materials
For five 96-well plates*
Please see the product datasheet

* Provided that the recommended microplates, buffers, diluents, substrates and solutions are used, and the assay is run as summarized in the Assay Procedure provided.

This DuoSet ELISA Development kit contains the basic components required for the development of sandwich ELISAs to measure natural and recombinant human Granzyme B. The suggested diluent is suitable for the analysis of most cell culture supernate samples. Diluents for complex matrices, such as serum and plasma, should be evaluated prior to use in this DuoSet.

Product Features

  • Optimized capture and detection antibody pairings with recommended concentrations save lengthy development time
  • Development protocols are provided to guide further assay optimization
  • Assay can be customized to your specific needs
  • Economical alternative to complete kits

Kit Content

  • Capture Antibody
  • Detection Antibody
  • Recombinant Standard
  • Streptavidin conjugated to horseradish-peroxidase (Streptavidin-HRP)

Other Reagents Required

DuoSet Ancillary Reagent Kit 2 (5 plates): (Catalog # DY008) containing 96 well microplates, plate sealers, substrate solution, stop solution, plate coating buffer (PBS), wash buffer, and Reagent Diluent Concentrate 2.

The components listed above may be purchased separately:

PBS: (Catalog # DY006), or 137 mM NaCl, 2.7 mM KCl, 8.1 mM Na2HPO4, 1.5 mM KH2PO4, pH 7.2 - 7.4, 0.2 µm filtered

Wash Buffer: (Catalog # WA126), or 0.05% Tween® 20 in PBS, pH 7.2-7.4

Reagent Diluent: (Catalog # DY995), or 1% BSA in PBS, pH 7.2-7.4, 0.2 µm filtered

Substrate Solution: 1:1 mixture of Color Reagent A (H2O2) and Color Reagent B (Tetramethylbenzidine) (Catalog # DY999)

Stop Solution: 2 N H2SO4 (Catalog # DY994)

Microplates: R&D Systems (Catalog # DY990)

Plate Sealers: ELISA Plate Sealers (Catalog # DY992)

Scientific Data

Human Granzyme B ELISA Standard Curve

Product Datasheets

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Preparation and Storage

The product is shipped at ambient temperature. Upon receipt, store it immediately at the temperature recommended below.
Stability & Storage
Store the unopened product at 2 - 8 °C. Do not use past expiration date.

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). 

Entrez Gene IDs:
3002 (Human); 14939 (Mouse)
Alternate Names:
C11; CCPI; CGL1; CGL-1; CSPB; CSP-B; CTLA1; CTLA-1; CTSGL1; Fragmentin-2; Granzyme B; Granzyme-2; GranzymeB; GRB; GrzB; GZMB; HLP; SECT

Assay Procedure


Plate Preparation

  1. Dilute the Capture Antibody to the working concentration in PBS without carrier protein. Immediately coat a 96-well microplate with 100 μL per well of the diluted Capture Antibody. Seal the plate and incubate overnight at room temperature.
  2. Aspirate each well and wash with Wash Buffer, repeating the process two times for a total of three washes. Wash by filling each well with Wash Buffer (400 μL) using a squirt bottle, manifold dispenser, or autowasher. Complete removal of liquid at each step is essential for good performance. After the last wash, remove any remaining Wash Buffer by aspirating or by inverting the plate and blotting it against clean paper towels.
  3. Block plates by adding 300 μL Reagent Diluent to each well. Incubate at room temperature for a minimum of 1 hour.
  4. Repeat the aspiration/wash as in step 2. The plates are now ready for sample addition.

Assay Procedure

  1. Add 100 μL of sample or standards in Reagent Diluent, or an appropriate diluent, per well. Cover with an adhesive strip and incubate 2 hours at room temperature.
  2. Repeat the aspiration/wash as in step 2 of Plate Preparation.
  3. Add 100 μL of the Detection Antibody, diluted in Reagent Diluent, to each well. Cover with a new adhesive strip and incubate 2 hours at room temperature.
  4. Repeat the aspiration/wash as in step 2 of Plate Preparation.
  5. Add 100 μL of the working dilution of Streptavidin-HRP to each well. Cover the plate and incubate for 20 minutes at room temperature. Avoid placing the plate in direct light.
  6. Repeat the aspiration/wash as in step 2.
  7. Add 100 μL of Substrate Solution to each well. Incubate for 20 minutes at room temperature. Avoid placing the plate in direct light.
  8. Add 50 μL of Stop Solution to each well. Gently tap the plate to ensure thorough mixing.
  9. Determine the optical density of each well immediately, using a microplate reader set to 450 nm. If wavelength correction is available, set to 540 nm or 570 nm. If wavelength correction is not available, subtract readings at 540 nm or 570 nm from the readings at 450 nm. This subtraction will correct for optical imperfections in the plate. Readings made directly at 450 nm without correction may be higher and less accurate.

Citations for Human Granzyme B DuoSet ELISA

R&D Systems personnel manually curate a database that contains references using R&D Systems products. The data collected includes not only links to publications in PubMed, but also provides information about sample types, species, and experimental conditions.

17 Citations: Showing 1 - 10
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  1. SCD1 inhibition enhances the effector functions of CD8+ T cells via ACAT1-dependent reduction of esterified cholesterol
    Authors: Sugi, T;Katoh, Y;Ikeda, T;Seta, D;Iwata, T;Nishio, H;Sugawara, M;Kato, D;Katoh, K;Kawana, K;Yaguchi, T;Kawakami, Y;Hirai, S;
    Cancer science
    Species: Human
    Sample Types: Cell Culture Supernates
  2. Characterizing the regulatory Fas (CD95) epitope critical for agonist antibody targeting and CAR-T bystander function in ovarian cancer
    Authors: Mondal, T;Gaur, H;Wamba, BEN;Michalak, AG;Stout, C;Watson, MR;Aleixo, SL;Singh, A;Condello, S;Faller, R;Leiserowitz, GS;Bhatnagar, S;Tushir-Singh, J;
    Cell death and differentiation
    Species: Human
    Sample Types: Cell Culture Supernates
  3. T Cells Expressing a Modified FcgammaRI Exert Antibody-Dependent Cytotoxicity and Overcome the Limitations of CAR T-cell Therapy against Solid Tumors
    Authors: D Rasoulouni, N Santana-Ma, A Gutwillig, L Farhat-You, L Tal, S Amar, M Milyavsky, SSNA Muddineni, N Solomon, H Shpilt, S Dotan, N Pilpel, C Waskow, M Feinmesser, P Rider, Y Carmi
    Cancer Immunology Research, 2023-06-02;0(0):OF1-OF18.
    Species: Human
    Sample Types: Cell Culture Supernates
  4. NK cell-derived extracellular granzyme B drives epithelial ulceration during HSV-2 genital infection
    Authors: YS Lim, AG Lee, X Jiang, JM Scott, A Cofie, S Kumar, D Kennedy, DJ Granville, H Shin
    Cell Reports, 2023-04-17;42(4):112410.
    Species: Human
    Sample Types: Vaginal Lavage Fluid
  5. Genome-wide gain-of-function screening characterized lncRNA regulators for tumor immune response
    Authors: Y Wang, Y Zhao, W Guo, GS Yadav, C Bhaskarla, Z Wang, X Wang, S Li, Y Wang, Y Chen, D Pattarayan, W Xie, S Li, B Lu, US Kammula, M Zhang, D Yang
    Science Advances, 2022-12-07;8(49):eadd0005.
    Species: Human
    Sample Types: Cell Culture Supernates
  6. Large libraries of single-chain trimer peptide-MHCs enable rapid antigen-specific CD8+ T cell discovery and analysis
    Authors: J Heath, W Chour, J Choi, J Xie, M Chaffee, T Schmitt, K Finton, D Delucia, A Xu, Y Su, D Chen, R Zhang, D Yuan, S Hong, A Ng, J Butler, R Edmark, L Jones, K Murray, S Peng, G Li, R Strong, J Lee, J Goldman, P Greenberg
    Research square, 2022-11-18;0(0):.
    Species: Human
    Sample Types: Cell Culture Supernates
  7. Combined IgE neutralization and Bifidobacterium longum supplementation reduces the allergic response in models of food allergy
    Authors: SB An, BG Yang, G Jang, DY Kim, J Kim, SM Oh, N Oh, S Lee, JY Moon, JA Kim, JH Kim, YJ Song, HW Hyun, J Kim, K Lee, D Lee, MJ Kwak, BK Kim, YK Park, CP Hong, JH Kim, HS Lim, MS Ryu, HT Jin, SW Lee, YS Chang, HS Park, YC Sung, MH Jang
    Nature Communications, 2022-09-27;13(1):5669.
    Species: Human
    Sample Types: Cell Culture Supernates
  8. Novel Combinations of Human Immunomodulatory mAbs Lacking Cardiotoxic Effects for Therapy of TNBC
    Authors: C Vetrei, M Passariell, G Froechlich, R Rapuano Le, E Sasso, N Zambrano, C De Lorenzo
    Cancers, 2021-12-27;14(1):.
    Species: Human
    Sample Types: Cell Culture Supernates
  9. CRISPR/Cas9-mediated TGFbetaRII disruption enhances anti-tumor efficacy of human chimeric antigen receptor T cells in vitro
    Authors: K Alishah, M Birtel, E Masoumi, L Jafarzadeh, HR Mirzaee, J Hadjati, RH Voss, M Diken, S Asad
    Journal of Translational Medicine, 2021-11-27;19(1):482.
    Species: Human
    Sample Types: Cell Culture Supernates
  10. Monocyte-dependent co-stimulation of cytokine induction in human gammadelta T cells by TLR8 RNA ligands
    Authors: R Serrano, C Coch, C Peters, G Hartmann, D Wesch, D Kabelitz
    Scientific Reports, 2021-07-27;11(1):15231.
    Species: Human
    Sample Types: Cell Culture Supernates
  11. The Search for an Interesting Partner to Combine with PD-L1 Blockade in Mesothelioma: Focus on TIM-3 and LAG-3
    Authors: E Marcq, JRM Van Audena, J De Waele, C Merlin, P Pauwels, JP van Meerbe, SA Fisher, ELJ Smits
    Cancers, 2021-01-14;13(2):.
    Species: Human
    Sample Types: Cell Culture Supernates
  12. Influence of Indoleamine-2,3-Dioxygenase and Its Metabolite Kynurenine on gammadelta T Cell Cytotoxicity against Ductal Pancreatic Adenocarcinoma Cells
    Authors: H Jonescheit, HH Oberg, D Gonnermann, M Hermes, V Sulaj, C Peters, D Kabelitz, D Wesch
    Cells, 2020-05-06;9(5):.
    Species: Human
    Sample Types: Cell Culture Supernates
  13. Targetable mechanisms driving immunoevasion of persistent senescent cells link chemotherapy-resistant cancer to aging
    Authors: DP Muñoz, SM Yannone, A Daemen, Y Sun, F Vakar-Lope, M Kawahara, AM Freund, F Rodier, JD Wu, PY Desprez, DH Raulet, PS Nelson, LJ van 't Vee, J Campisi, JP Coppé
    JCI Insight, 2019-06-11;5(0):.
    Species: Human
    Sample Types: Cell Culture Supernates
  14. Soluble markers of neutrophil, T-cell and monocyte activation are associated with disease severity and parasitemia in falciparum malaria
    Authors: K Otterdal, A Berg, AE Michelsen, S Patel, MG Tellevik, CG Haanshuus, B Fevang, P Aukrust, N Langeland, T Ueland
    BMC Infect. Dis., 2018-12-18;18(1):670.
    Species: Human
    Sample Types: Plasma
  15. Folate receptor 1 (FOLR1) targeted chimeric antigen receptor (CAR) T cells for the treatment of gastric cancer
    Authors: M Kim, S Pyo, CH Kang, CO Lee, HK Lee, SU Choi, CH Park
    PLoS ONE, 2018-06-06;13(6):e0198347.
    Species: Human
    Sample Types: Cell Culture Supernates
  16. Preclinical assessment of galunisertib (LY2157299 monohydrate), a first-in-class transforming growth factor-? receptor type I inhibitor
    Authors: JM Yingling, WT McMillen, L Yan, H Huang, JS Sawyer, J Graff, DK Clawson, KS Britt, BD Anderson, DW Beight, D Desaiah, MM Lahn, KA Benhadji, MJ Lallena, RB Holmgaard, X Xu, F Zhang, JR Manro, PW Iversen, CV Iyer, RA Brekken, MD Kalos, KE Driscoll
    Oncotarget, 2017-12-31;9(6):6659-6677.
    Species: Human
    Sample Types: Cell Culture Supernates
  17. JAK2 inhibitor combined with DC-activated AFP-specific T-cells enhances antitumor function in a Fas/FasL signal-independent pathway
    Onco Targets Ther, 2016-07-20;9(0):4425-33.
    Species: Human
    Sample Types: Cell Culture Supernates


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Reviews for Human Granzyme B DuoSet ELISA

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Human Granzyme B DuoSet ELISA
By Supriya Sen on 10/31/2023

Reason for Rating: Worked great. Sample Tested: T Cell culture supernatant

Worked great. Granzyme B was detected in T Cell culture supernatant stimulated with target cell. Minimal background in T cell only sample.

Human Granzyme B DuoSet ELISA
By Robert Berahovich on 09/29/2020
Sample Tested: Tissue Culture Media

Detection of granzyme B produced by human BCMA-specific CAR-T cells in response to BCMA-negative and BCMA-positive cell lines.

Human Granzyme B DuoSet ELISA
By Anonymous on 04/02/2020
Sample Tested: Serum and Plasma

We used this kit for the quantification of Granzyme B in human serum and plasma. Works very well and well-described protocol.