The enzyme site (ELISPOT) technology is a novel assay developed in recent years for antiviral immunity and anti-tumor immunity.
The granzyme-B enzyme detection kit from Sanquin uses the latest technology and is highly sensitive. The presence of granzyme-B can be detected in only one positive cell. Strong specificity and good repeatability. Provides a new and powerful testing tool for antiviral and anti-tumor research.
Comparison of enzyme point technology and enzyme labeling technology
1. The obvious feature of enzyme point technology is the increase in sensitivity.
2, the enzyme point technology only needs 1 positive cell / hole, the sensitivity is up to 1:1,000,000 positive and accurate analysis
3. The enzyme labeling technique requires at least 400 cells/well to achieve meaningful detection levels.
4. Ability to analyze cytokine-producing T cells at the single cell level
Introduction of Granzyme-B
Granzymes are exo-serine proteases derived from cytosolic granules released from cytotoxic T lymphocytes (CTLs) and natural killer cells (NK). These particles contain granzyme zymogen and other proproteinogens, including perforin. Since the CTL cells bind to the target cells (by binding the CTL receptor to the antigen peptide-MHCI-like molecular complex on the surface of the target cell), the granzyme enters the target cell through the exocytosis process of the particle, and the perforated protein aggregates in the cell membrane and enters the target. The cells form transmembrane pores that perforate the target cell membrane. Perforin releases granzyme by perforating the granzyme-endosomal membrane. Granzyme B activates the intracellular caspases reaction and ultimately causes death of target cells. Granzyme A also induces apoptosis in target cells.
In many diseases, the percentage of granzyme B-positive CTLs can be determined by flow cytometry and immune cells. Some granzymes can also "leak" into peripheral blood and other body fluids. Granzymes can also be measured in healthy individuals. These soluble granzymes can be assayed by ELISA.
The PeliSPOT Human Granzyme B Kit is used for repetitive specific quantification of the frequency of granzyme B released by human CTL cells and NK cells.
method
The cells were incubated overnight in pre-coated microplates with pre-coated high affinity anti-granzyme-B monoclonal antibodies. The secreted granzyme-B binds to the pre-coated anti-granzyme-B antibody. The unbound components were washed to remove the biotinylated anti-granzyme-B antibody. Streptavidin-HRP polymer and substrate were added separately, and blue/purple spots were observed in the secreted cytokines.
Granzyme-B clinical significance
Viral infections EBV, HIV, CMV, hepatitis and dengue virus infections can enhance CTL responses in patients with increased levels of soluble granzymes in the body.
Lymphoma and sarcoma Hemorrhage patients such as He Jiejin disease have increased granzyme B concentration.
Rheumatoid arthritis Soluble granzyme B is significantly elevated in synovial fluid of patients with rheumatoid arthritis than primary systemic osteoarthrosis.
Transplantation Granzymes involve acute rejection following kidney transplantation. Infiltrating lymphocytes strongly express plasma soluble granzymes, and elevated levels of plasma soluble granzymes in kidney transplant patients suggest systemic viral infections, such as CMV infection.
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