Assay Principle

This kit employs double antibody sandwich ELISA technology: Capture Antibody is coated on the microplate to capture IFN-γ from samples and standards. After washing, biotin-labeled Detection Antibody is added and incubated, followed by washing to form a "Capture Antibody-Antigen-Detection Antibody" immune-complex. Subsequently, Streptavidin-Horseradish Peroxidase (SA-HRP) is added and incubated. After incubation and washing, TMB Substrate is added for color development. If the target analyte is present in the sample, a blue color develops. Stop Solution is then added to terminate the reaction. During the assay, unbound components are washed away. The Optical Density (OD) is measured at 450 nm using a microplate reader. The intensity of the color is proportional to the IFN-γ concentration in the sample, and the concentration is calculated by plotting a standard curve.

 

Precision

Intra-assay and inter-assay Coefficients of Variation (CV) are both <10%.

· Intra-assay Precision: Three known concentration samples were assayed 20 times on one plate. The CV of the concentrations was calculated.

· Inter-assay Precision: Three known concentration samples were assayed in 20 replicates across three different plates. The CV of the concentrations was calculated.

	Intra-assay Precision			Inter-assay Precision
Sample	1	2	3	1	2	3
n	20	20	20	20	20	20
Mean (pg/mL)	12.48	24.97	50.02	12.48	24.97	50.02
Standard Deviation	0.191	0.619	1.576	0.308	0.859	1.606
CV (%)	1.53	2.48	3.15	2.47	3.44	3.21

Recovery

Recovery was tested by spiking known concentrations of Bovine IFN-γ into different sample matrices. The recovery range and average recovery are shown below.

 

Sample Type	Range (%)	Average Recovery (%)
Serum (n=8)	88-101	96
Plasma (n=8)	93-104	98
Cultured Cells (n=8)	99-115	106

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