The 17-beta estradiol ELISA Kit from IBL Hamburg contains all that is needed to perform the estimates of 17-beta estradiol in plasma or serum samples. It is equipped with a 96-well plate that comes in 12 strips of 8 wells each which simplifies setting up the measurements according to the number of samples used. However, it is always an advantage to use all wells in one run in order to avoid wasting wells; in addition to wasting the unused wells, each additional run requires the use of additional controls and standards.
The plate wells are coated with an antibody against estradiol. The estimate of the estradiol levels in the samples is based on the competition of sample estradiol for binding sites with estradiol conjugated to horseradish peroxidase (HRP). The amount of bound peroxidase conjugate is inversely proportional to the concentration of estradiol in the sample.
The experiment is run by first bringing the reagents to room temperature. The kit contains 7 ready-to-use standards, ranging from 0 to 2000 pg/ml of estradiol. Standard 0 is utilized for dilution of samples if necessary. The kit also contains ready-to-use HRP conjugate, substrate solution, stop solution and 40x concentrate of wash solution that needs to be prepared by diluting with deionized water.
The assay is run by adding 25 µl of each standard, sample and control to the wells of the microplate. 200 µl of HRP conjugate is then added to all used wells and incubated for120 min at room temperature. After that time, wells are emptied by flipping the plate and washing 3 times with washing solution. It is good to use an 8-channel manual washer that quickly dispenses the washing solution contained in large enough container to accommodate desired volume for all washes. After the last wash, the plate should be tapped against a paper towel to discard any residual liquid. Next, 100 µl of substrate solution is added to the each well and the plate is incubated for 15 minutes at room temperature in the darkness. After that time, 50 ml of stop solution is added to each well. Results of the reaction should be read within 10 min on an absorbance reader at 450+10 nm. Results of the concentrations of estradiol in samples are read against the curve plot from the readings obtained for standards. We use Bio-Rad’s Model 550 microplate reader and the values were estimated automatically by software supplied with the reader.
In addition to being used for human samples, this assay can be utilized for materials from different animals, including mice as is my experience. This kit allows for this because the antibody cross-reacts with estradiol from different animals as is indicated by information sheet of the producer.