Human NT-4 DuoSet ELISA Development Kit From R&D Systems

This ELISA Kit contains the components that are required for human BDNF measurement. It is designed for the analysis of cell supernatants. I sucessfully used this kit to measure the amount of natural BDNF in the conditioned media of human PBMCs. Each ELISA kit contains sufficient materials to run aproximately 15 full 96-well plates. These materials include:
Capture antibody: mouse anti-human BDNF
It should be reconstituted in 1 ml of sterile PBS. After reconstituion, the antibody's concentration will be 360 μg/ml. Its working concentration is 2 μg/ml; thus, it should be diluted 1:180 in PBS without carrier protein.
Detection antibody: biotinilated mouse anti-human BDNF
It should be reconstituted in 1 ml of Reagent Diluent, which consists of 1% BSA in sterile PBS. After reconstitution, its consentration will be 4.5 μg/ml. The working concentration is 25 ng/ml, meaning that this antibody should be diluted 1:180 to acheive this concentration.
Standards: The standards are human recombinant BDNF protein. Addition of 0.5 ml of PBS will bring the concentration to 170 ng/ml. Allow the standard 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 1500 pg/ml maximum concentration. In order to achive this concentration, dilute the reconstituted standards 1:133.3.
Streptavidin: 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 2L 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.

You 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 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 of Wash Buffer, after the last wash invert and virogously 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 of Reagent Diluent for 1 hour at room temperature. If you need to stop, wash the plate and you may leave it in the last wash at 2-8ºC for overnight or even for over the weekend. If you want to proceed, add 100 μl 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 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 5-7 minutes after substrate addition. It is difficult to predict when you have to add stop solution in each case because the concentration of BDNF in each assay varies. Usually, I note when the color starts to develop in the 2-3 most concentrated wells of the standard curve, which takes about 3 minutes in my system and then I wait for another 2-5 minutes and then I add the stop solution. If you wait too long, the color will start to develop even in empty or blank wells, thus increasing background and making impossible for you to detect small differences between samples.