Components
Human IFN-gamma Microplate: A 96-well polystyrene microplate (12 strips of 8 wells) coated with a polyclonal antibody against human IFN- gamma. Sealing Tapes: Each kit contains 3 precut, pressure sensitive sealing tapes that can be cut to fit the format of the individual assay. Human IFN-gamma Standard: Human IFN-gamma in a buffered protein base (2 ng, lyophilized). Biotinylated Human IFN-gamma Antibody (50x): A 50-fold concentrated biotinylated polyclonal antibody against human IFN-gamma (120 μl). EIA Diluent Concentrate (10x): A 10-fold concentrated buffered protein base (30 ml). Wash Buffer Concentrate (20x): A 20-fold concentrated buffered surfactant (30 ml, 2 bottles). SP Conjugate (100x): A 100-fold concentrate (80 l). Chromogen Substrate (1x): A stabilized peroxidase chromogen substrate tetramethylbenzidine (8 ml). Stop Solution (1x): A 0.5 N hydrochloric acid solution to stop the chromogen substrate reaction (12 ml).
Material not included
Microplate reader capable of measuring absorbance at 405 nm. Pipettes (1-20 µL, 20-200 µL, and multiple channel). Deionized or distilled reagent grade water. Incubator (37 °C).
Reagent Preparation
Freshly dilute all reagents and bring all reagents to room temperature before use. EIA Diluent Concentrate (10x): If crystals have formed in the concentrate, mix gently until the crystals have completely dissolved. Dilute the EIA Diluent Concentrate 10-fold with reagent grade water to produce a 1x solution. Store for up to 30 days at 2-8 °C.
Assay Procedure
Add 50ul of Human IFN-gamma Standard or sample to each well. Cover wells with a sealing tape and incubate for 2 hours. Wash five times with 200ul of Wash Buffer. Add 50ul of Biotinylated Human IFN-gamma Antibody to each well. Cover wells with a sealing tape and incubate for 2 hours. Wash the microplate as described above. Add 50ul of SP Conjugate to each well. Cover wells with a sealing tape and incubate for 30 minutes. Wash the microplate as described above. Add 50ul of Chromogen Substrate to each well. Incubate for 25 minutes or until the optimal blue color density develops. Add 50ul of Stop Solution to each well. The color will change from blue to yellow. Read the absorbance on a microplate reader at a wavelength of 450 nm immediately.
Calculation of Results
Calculate the mean absorbance for each set of duplicate standards, controls and samples, and subtract the average zero standard optical density. Plot the standard curve on log-log graph paper or using Sigma plot software, with standard concentration on the x-axis and absorbance on the y-axis. Draw the best-fit straight line through the standard points.
Assay Precision
Intra-assay and inter-assay coefficients of variation were 4.4 % and 7.3 % respectively.
Handling Advice
This product is for Research Use Only and is not intended for use in diagnostic procedures. Prepare all reagents as instructed, prior to running the assay. Prepare all samples prior to running the assay. The dilution factors for the samples are suggested in this insert. However, the user should determine the optimal dilution factor.
Storage Comment
Store SP Conjugate and Biotinylated Antibody at -20°C. Store Microplate, Diluent Concentrate (10x), Wash Buffer, Stop Solution, and Chromogen Substrate at 2-8°C. Unused microplate wells may be returned to the foil pouch with the desiccant packs and resealed. May be stored for up to 30 days in a vacuum desiccator.
Note
Store SP Conjugate and Biotinylated Antibody at -20°C. Store Microplate, Diluent Concentrate (10x), Wash Buffer, Stop Solution, and Chromogen Substrate at 2-8°C. Unused microplate wells may be returned to the foil pouch with the desiccant packs and resealed. May be stored for up to 30 days in a vacuum desiccator.
Restrictions
For Research Use only
