Human Granzyme B ELISA Kit is a sandwich Enzyme-Linked Immunosorbent Assay (sELISA) designed for the in vitro quantitative determination of human Granzyme B in serum, plasma, and cell culture supernatant.
Assay Type
Sandwich (quantitative)
Principle of Assay
Human Granzyme B ELISA Kit (A334814) employs the sandwich enzyme immunoassay technique for the quantitative measurement of human Granzyme B in serum, plasma, and cell culture supernatant. An antibody specific for Granzyme B has been pre-coated onto a 96-well microtiter plate. The standards and test samples are added into the wells and the Granzyme B present in each sample is bound to the wells by the immobilized antibody. Following incubation, the wells are washed and then incubated with Biotinylated Anti-Granzyme B Antibody, which binds the captured Granzyme B present in each well. Following incubation, unbound biotinylated detection antibody is removed by washing, and an HRP-Streptavidin conjugate is added to the wells and the microtiter plate is incubated. Following incubation and washing, TMB substrate solution is then used to visualize the HRP enzymatic reaction by catalysis to produce a blue-coloured product that changes to yellow after addition of acidic stop solution. The density of yellow is proportional to the amount of Granzyme B captured in each well. The concentration of Granzyme B can then be calculated by reading the O.D. absorbance at 450nm in a microplate reader and referring to the standard curve.
Components
This ELISA Kit contains 10X 600 µL Capture Antibodies, 10X 600 µL Detector Antibodies, 2 Lyophilized Recombinant Protein Vials, 50 mL Assay Diluent #10, 10X 20 mL Wash Buffer, 12 mL TMB Solution, 12 mL Stop Solution, Pre-Coated 96-Well H-Microplate (12 x 8 well strips), and 2 Plate Seals.
A standard curve was generated by plotting optical density (O.D.) at 450 nm versus the concentration of human Granzyme B (pg/mL) in Assay Diluent #10. The curve demonstrates a strong linear relationship over the tested concentration range, indicating good assay sensitivity and dynamic range.
Human Granzyme B levels were quantified in undiluted and serially diluted PBMC stimulated supernatants. The concentration remained consistent across dilutions, indicating good assay linearity and minimal matrix interference. Error bars represent the standard deviation of replicate measurements.
Human Granzyme B concentrations were determined in undiluted and serially diluted PBMC unstimulated supernatants. The measured levels were low and consistent across dilutions, indicating minimal endogenous Granzyme B production and good assay performance. Error bars represent the standard deviation of replicate measurements.
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