Androstenedione is synthesized in the adrenal gland. It is the primary precursor of testosterone for women. Therefore, in women, androstenedione contributes significantly to the level of androgenic activity, while in men this contribution is nil. Quantitation of androstenedione may indicate the androgenic activity in women.
Principle of the Assay:This is an ELISA for the quantitative analysis of Androstenedione levels in biological fluid. This test kit operates on the basis of competition between the enzyme conjugate and the Androstenedione in the sample for a limited number of binding sites on the antibody coated plate.
The sample or standard solution is first added to the microplate. Next, the diluted enzymeconjugate is added and the mixture is shaken and incubated at room temperature for onehour. During the incubation, competition for binding sites is taking place. The plate is thenwashed removing all the unbound material. The bound enzyme conjugate is detected by the addition of substrate, which generates an optimal color after 30 minutes.
Quantitative test results may be obtained by measuring and comparing the absorbance reading of the wells of the samples against the standards with a microplate reader at 650nm. The extent of color development is inversely proportional to the amount of Androstenedione in the sample or standard. For example, the absence of Androstenedione in the sample will result in a bright blue color, whereas the presence of Androstenedione will result in decreased or no color development.
Kit Components:A2292-20A: ELISA Buffer, 1x30ml
To be used to dilute enzyme conjugate and Androstenedione standards
A2292-20B: Wash Buffer (10x), 1x20ml
To be diluted 10x with deionized water. This is used to wash all unbound enzyme conjugate, samples and standards from the plate after the one hour incubation
A2292-20C: TMB Substrate, 1x20ml
Stabilized 3,3', 5,5' Tetramethylbenzidine (TMB) plus hydrogen peroxide (H2O2) in a single bottle. It is used to develop the color in the wells after they have been washed. Keep substrate refrigerated. It is light-sensitive
A2292-20D: Extraction Buffer (5x), 1x30ml
To be diluted 5x with deionized water. This is used for diluting extracted and non-extracted samples
A2292-20E: Androstenedione (HRP), 1x150ul
A2292-20F: Androstenedione Standard, 1x100ul. Androstenedione standard with a concentration of 1mg/ml
A2292-20G: Microtiter Strips, 1x96wellsA 96-well microtiter plate coated with rabbit anti- Androstenedione in each well. The plate is ready for use as is. Do not wash.
Storage and Stability:Store components at 4°C. Stable for 6 months. For maximum recovery of product, centrifuge the original vial after thawing and prior to removing the cap.
