Interferon-gamma (IFN-γ, also known as Type II interferon) is a crucial cytokine with immunomodulatory functions. IFN-γ signaling is via IFN-γ RI (the alpha subunit) and IFN-γ RII. IFN-γ plays a key role in host defense through its antiviral, antiproliferative, and immunomodulatory functions. IFN-γ can induce the production of cytokines and upregulate the expression of different membrane proteins, including MHC-I/II (major histocompatibility complex), Fc receptors, leukocyte adhesion molecules, and B7 family antigens in various types of cells. IFN-γ effectively activates macrophages, guides B cell immunoglobulin synthesis, class switching, and secretion. It also influences the development of T helper cell subtypes by inhibiting Th2 differentiation and stimulating Th1 growth.

This assay kit employs the double antibody sandwich enzyme-linked immunosorbent assay (ELISA) technique. Specific anti-human IFN-γ antibodies with high affinity are pre-coated onto the enzymelinked plate. In the wells of the enzyme-linked plate, standards, test samples, and biotinylated detection antibodies are added. After incubation, IFN-γ present in the samples binds to the solid-phase antibody and the detection antibody, forming an immune complex. After removing unbound substances, streptavidin-horseradish peroxidase labeled with horseradish peroxidase (HRP) is added. After wash step to remove any unbound substances, 3,3′,5,5′-Tetramethylbenzidine (TMB)substrate is added and color develops in proportion to the amount of IFN-γ bound initially. The assay is stopped, and the optical density of the wells is determined using a micro-plate reader at 450nm wavelength (with a reference wavelength of 540nm or 570nm) for quantification.

Recovery
Human IFN-γ of different levels was spiked into cell culture medium samples to determine its recovery rate. The recovery rate ranged from 90-116%, with an average recovery rate of 103%.
Sensitivity
The minimum detectable dose (MDD) of Human IFN-γ is typically less than 7.8 pg/mL.

The MDD was determined by adding two standard deviations to the mean O.D. value of twenty zero standard replicates and calculating the corresponding concentration.
Linearity
To assess the linearity of the assay, four samples spiked with high concentrations of Human IFN-γ were diluted with 1×Reagent Diluent to bring them within the detection range.

Specificity
This assay recognizes natural and recombinant Human IFN-γ.
The factors listed below were prepared at 50 ng/mL in 1×Reagent Diluent and assayed for cross-reactivity.
Preparations of the following factors prepared at 50 ng/mL in a mid-range Human IFN-γ control were assayed for interference. No significant cross-reactivity or interference was observed.