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

This ELISA kit is designed for the analysis of cell supernatants. I successfully used this kit to quantify secreted BMP-4 in the conditioned medium of a human neuroblastoma cell line. 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 BMP-4. These materials include:

Capture Antibody (Part 840880, 1 vial) is lyophilized mouse anti-human BMP-4. The lyophilized antibody should be reconstitution with 1 mL of PBS and stored at 2 – 8°C for up to 60 days or aliquoted and stored at -20°C to -70°C in a manual defrost freezer for up to 6 months or even longer. The concentration of the stored stock is 360 μg/mL; the achieve the working concentration of 2 μg/ml, it should be diluted 1:180 in PBS without carrier protein.
Detection Antibody (Part 840881, 1 vial) is lyophilized biotinylated mouse anti-human BMP-4. After reconstitution with 1 mL of Reagent Diluent, which consists of 1% BSA in sterile PBS, its concentration will be 180 μg/mL. To achieve this concentration, the detection antibody should be diluted 1:180. After reconstitution, you may store at 2 - 8°C for up to 60 days or aliquot and store at -20°C to -70°C in a manual defrost freezer for up to 6 months.
Standard (Part 840882, 3 vials) consists of lyophilized recombinant human BMP-4. Addition of 0.5 ml of Reagent Diluent brings the concentration to 30 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 1:30. Aliquot and store reconstituted standard at -70° C for up to 2 months.
Streptavidin-HRP (Part 890803, 1 vial) is 1 mL of streptavidin conjugated to horseradish-peroxidase. Store at 2 - 8° C for up to 6 months after initial use. DO NOT FREEZE. Dilute to the working concentration specified on the vial label using Reagent Diluent.

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 a ready-made stop solution from R&D. 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 have to proceed with many plates and to save Reagent Diluent, you may use only 100-150 μl/well. If you need to stop, wash the plate and leave it in the last wash at 2-8ºC overnight or even over the weekend. To continue, 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 the 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 15 minutes after substrate addition. The moment to add stop solution should be estimated empirically for each plate. 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. Also, I would like to mention that it would be good idea to run just standard curve once prior to experiment if you try a new kit of human BMP4. It happened that I lost whole plate with this kit because standards were not good.