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 a one-step sandwich enzyme-linked immunosorbent assay (ELISA) for the quantitative determination of human Granzyme B in serum, plasma, and cell culture supernatant. A 96-well microtiter plate is provided pre-coated with an antibody specific for Granzyme B. Standards and test samples are added to the wells, allowing Granzyme B to bind to the immobilized capture antibody. A prepared antibody mixture is added and incubated to form antibody–antigen complexes. After incubation, unbound material is removed by washing, and TMB substrate is added to develop colour through an HRP-catalysed reaction. The reaction is stopped with acidic stop solution, producing a yellow endpoint signal. The intensity of the colour is proportional to the amount of Granzyme B captured in each well. The concentration of Granzyme B is determined by measuring absorbance at 450 nm and interpolating from 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.
Schematic overview of the 60 Minute ELISA workflow illustrating antigen capture and colorimetric signal development. The target antigen is captured on the solid phase by an immobilized capture antibody while an HRP conjugated detection antibody binds simultaneously, forming a ternary immune complex during a 45 minute incubation. After washing to remove unbound components, tetramethylbenzidine (TMB) substrate is added. The horseradish peroxidase enzyme catalyzes oxidation of TMB, generating a blue reaction product that reflects the amount of bound antigen. Addition of stop solution terminates the reaction and converts the signal to a stable yellow color, which is measured spectrophotometrically after a 10 minute incubation. The entire assay is completed within approximately 60 minutes.
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