This ELISA kit is designed for the analysis of cell supernatants. I successfully used this kit to quantify natural INF-gamma in human PBMC’s conditioned medium. Each ELISA kit contains sufficient materials to run approximately 18 full 96-well plates. It contains all of the components that are required for measurement of human INF-gamma. These materials include:
: mouse anti-human IFN-gamma
It should be reconstituted in 1 ml of sterile PBS. After reconstituion, the antibody's concentration will be 720 μg/ml. Its working concentration is 4 μg/ml; thus, it should be diluted 1:180 in PBS without carrier protein.
: biotinylated mouse anti-human IFN-gamma
It should be reconstituted in 1 ml of Reagent Diluent, which consists of 1% BSA in sterile PBS. After reconstitution, it’s concentration will be 9 μg/ml. The working concentration is 50 ng/ml, which means that this antibody should be diluted 1:180 to achieve this concentration.
: The standards are human recombinant IFN-gamma protein. Addition of 0.5 ml of PBS will bring the concentration to 120 ng/ml. Allow the standards to completely dissolve by gently agitating for 15 minutes at room temperature. Make a standard curve of at least seven points by using a 2-fold dilution series, starting from a 1000 pg/ml maximum concentration. In order to achieve this concentration, dilute the reconstituted standards 1:8.3.
: Streptavidin is provided conjugated to horseradish peroxidase. It should be stored in the dark at 2-8ºC.
A few additional solutions should be prepared in advance and according the manual. These are:
Wash buffer: 0.05% Tween-20 in PBS. Prepare at least 2 L of this solution.
Reagent Diluent: 1% BSA in PBS, prepare about 100 ml per one 96-well plate.
Stop solution: 2N H2S04. I used ready-made stop solution from R&D (catalog number DY994). All reconstituted materials must be stored at –20ºC or –80ºC. All the components are stable even after 6 months if stored at this temperature.
Start the ELISA by coating your plate with 100 μl of capture antibody per well. Dilute the appropriate amount of the antibody from the stock. Calculate this amount according to the number of the plates you will be using. From my experience, about 12 ml of diluted capture antibody is needed to coat one plate. Cover the plate and leave it overnight at room temperature. The next day, aspirate the capture antibody solution and wash the plate at least 3 times with 400 μl/well of Wash Buffer. After the last wash, invert and vigorously blot the plate against blotting paper. If you proceed with several plates, note that the plates should not be left to dry, this will increase background dramatically.
Block the plate with 300 μl/well of Reagent Diluent for 1 hour at room temperature. If you need to stop, wash the plate and leave it in the last wash at 2-8ºC overnight or even over the weekend. If you want to proceed, add 100 μl/well of the assay solution and incubate for 2 hours at room temperature. If you need to stop the reaction, you may stop at this point also. Just wash the plate and leave it with Wash Buffer at 2-8ºC overnight.
After incubation with assay solution, add 100 μl of the detection antibody, diluted in
Reagent Diluent, to each well. Cover it with a new adhesive strip and incubate 2 hours at room temperature. Repeat washing step and then add 100 μl/well working dilution of
Streptavidin-HRP to each well. Cover the plate with aluminum foil to protect it from the light and incubate the plate at room temperature. Note that color may develop more quickly than the 20 minutes stated in the manual. In my system, I add the 50 μl of stop solution about 1-2 minutes after substrate addition. The moment to add stop solution should be estimated empirically for each plate. Note when color starts to develop in the 2-3 most concentrated wells in the standard curve, which takes about 1-2 minutes in my system, and then wait for about 1 minute and then add stop solution. If you wait too long, color will start to develop even in empty or blank wells, thus increasing background and making it impossible to detect small differences between samples. It is very important to keep the same conditions for the all plates in the all experiments in order to be able to compare results across all experiments. Therefore, I always wait the same time before addition of the stop solution.