Granzyme B ELISA Kit (Gzm-B)

INTENDED USE

Human granzyme B ELISA kit is a protocol that is intended to measure in vitro quantitative levels of Gzm-b (granzyme B, GzmB) in human human serum, other biological fluids, plasma and tissue homogenates. This assay has a minimum sensitivity limit of 9.375 pg/ml.

BACKGROUND

Granzyme B (Gzm-b) is found in granules of cytotoxic lymphocytes, cytotoxic T cells and natural killer cells. These are all serine protease which are secreted along with pore forming proteins (called perforin) in order to mediate the process of apoptosis in target cells. It is through the transmembrane pores the granzymes are able to enter the cytosol of the target cell. Upon entering they are able to activate the intracellular cascade of many caspases which ultimately leads to the eventual killing of the target cells.

Granzyme B are involved in a number of other secondary functions which include inducing inflammation and extracellular matrix remodelling. There are recent evidence that it can be produced by a number of non-cytotoxic cells for example mast cells, smooth muscle cells and basophils cells. Elevated levels have been implicated in many autoimmune diseases, type 1 diabetes and in several skin diseases.

It has the ability to cleave Mcl-1 that can be found within the outer mitochondrial membrane and this can lead to relieving its inhibition of Bim. Bim has been identified in stimulating apoptosis, mitochondrial membrane permeability and BAX/BAK oligomerisation. It can also cleave Hs-1 associated protein X-1 (HAX1) in order to facilitate mitochondria polarisation.

CONTENT

All reagents supplied need to be stored at 2 °C – 8 °C, unopened reagents will retain reactivity until expiration date. Do not use reagents beyond this date.

• One 96-Well Plate: Pre-coated with anti Gzm-B antibody.
• Standards: Concentrations 0, 15.625, 31.25, 62.5, 125, 250, 500, 1000 pg/ml, made up from lyophilized recombinant.
• Sample/Standard Dilution Buffer.
• Biotinylated-Labelled Antibody.
• Antibody Dilution Buffer.
• HRP-Streptavidin Conjugate (SABC).
• SABC Dilution Buffer.
• TMB Substrate.
• Wash Buffer (25x).
• Plate Sealer.
• Product Instructions.

SENSITIVITY

Minimum detection limits of a  Gzm-b (GzmB, granzyme B) using this Gzm-B ELISA kit was 9.375 pg/ml. The dynamic range for this assay is 15.625 – 1000.0 pg/ml.

ASSAY CHARACTERISTICS

– Specificity: Highly specific for GzmB, no cross reactivity or interference between GzmB and analogues was detected.
– Recovery: Serum (86 – 105%), EDTA Plasma (90 – 102%), Heparin Plasma (85 – 104%).
– Linearity: Serum (91 – 99%), EDTA Plasma (82 – 96%), Heparin Plasma (82 – 100%).
– Precison Intra-Assay: CV < 8%.
– Precison Inter-Assay: CV < 10%.
– Stability: Less than 10%.

REFERENCES

1. Granzyme B-induced apoptosis in cancer cells and its regulation (review). Int J Oncol. (2010) 37 (6): 1361-78. Review. Rousalova I. and Krepela E.
2. Perforin and granzymes: function, dysfunction and human pathology. Nat Rev Immunol. (2015) 15 (6): 388-400. Review. Voskoboinik I., et al.
3. Delivery and therapeutic potential of human granzyme B. Immunol Rev. (2010) 235 (1): 159-71. Review. Kurschus F.C. and Jenne D.E.
4. Non-cytotoxic antiviral activities of granzymes in the context of the immune antiviral state. Immunol Rev. (2010) 235 (1): 128-46. Review. Andrade F.

ADDITIONAL INFORMATION

• Full Name: Granzyme B ELISA Kit (Gzm-B)
• Reactivity: Human
• Sample Type: Serum, Tissue Homogenates, Plasma, Other Biological Fluids
• Sensitivity: 9.375 pg/ml

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