The GZMK Kit ELISA (Granzyme K Enzyme-Linked Immunosorbent Assay) is a laboratory kit designed to measure the concentration of Granzyme K in biological samples, such as serum, plasma, or cell culture supernatants. Granzyme K is a serine protease enzyme produced by cytotoxic T cells and natural killer (NK) cells, playing a key role in immune response by inducing apoptosis in target cells.

Overview

• Purpose: To quantify Granzyme K levels in various biological samples.
• Applications: Useful in immunology research, particularly in studies related to cytotoxic T cells and NK cell activity, cancer research, and autoimmune diseases.
• Sensitivity and Specificity: High sensitivity and specificity for Granzyme K.

Kit Components

• Microplate: Pre-coated with an antibody specific to Granzyme K.
• Standards: Known concentrations of Granzyme K to create a standard curve.
• Detection Antibody: Biotin-labeled antibody specific to Granzyme K.
• Enzyme Conjugate: Streptavidin-HRP (Horseradish Peroxidase) that binds to the biotin-labeled detection antibody.
• Substrate Solution: TMB (3,3',5,5'-tetramethylbenzidine), which produces a color change when acted upon by HRP.
• Stop Solution: Usually an acidic solution to stop the enzymatic reaction.
• Wash Buffer: To remove unbound substances.
• Dilution Buffer: For sample and reagent preparation.

Protocol Summary

• Sample Preparation: Collect and prepare biological samples (serum, plasma, etc.). Dilute if necessary.
• Standard Preparation: Prepare standard dilutions from the provided Granzyme K stock.
• Plate Loading: Add standards and samples to the pre-coated microplate wells.
• Incubation: Allow the plate to incubate, enabling Granzyme K in the samples/standards to bind to the coated antibody.
• Detection: Add the biotin-labeled detection antibody to each well and incubate.
• Enzyme Conjugation: Add the Streptavidin-HRP conjugate and incubate.
• Substrate Reaction: Add the TMB substrate solution. The enzyme-substrate reaction produces a blue color.
• Stop Reaction: Add the stop solution to halt the reaction, turning the blue color to yellow.
• Measurement: Measure the absorbance at 450 nm using a microplate reader. The intensity of the color is proportional to the concentration of Granzyme K in the samples.

Key Points

• Ensure all reagents are at room temperature before use.
• Follow precise pipetting techniques to ensure accuracy and reproducibility.
• Avoid contamination of samples and reagents.
• Thorough washing of wells is critical to reduce background noise.