13,14-dihydro-15-keto-Prostaglandin F2a (13,14-d-15-k-PGF2a) is a major metabolite of PGF2a in tissues. It is derived from PGF2a through the consecutive actions of 15-hydroxy prostaglandin dehydrogenase and prostaglandin Delta13-reductase. Because of the rapid metabolism of PGF2a, quantitation of 13, 14-d-15-k-PGF2a in circulation can be a good index for PGF2a synthesis in tissue.
Principle of the Assay:
This is an ELISA (Enzyme-Linked Immunosorbent Assay) for the quantitative analysis of
13,14-dihydro-15-keto-Prostaglandin F2a levels in biological fluid. This test kit operates on the basis of competition between the enzyme conjugate and the 13,14-d-15-k-PGF2a 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 enzyme conjugate is added and the mixture is shaken and incubated at room temperature for one hour. During the incubation, competition for binding sites is taking place. The plate is then washed 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 13,14-d-15-k-PGF 2a in the sample or standard. For example, the absence of 13,14-d-15-k-PGF 2a in the sample will result in a bright blue color, whereas the presence of 13,14-d-15-k-PGF2a will result in decreased or no color development.
1. EIA Buffer, 1x30ml. To be used to dilute enzyme conjugate and 13,14-d-15-k-PGF2a standards.
2. 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.
3. 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.
4. Extraction Buffer (5X), 1x30ml. To be diluted 5x with deionized water. This is used for diluting extracted and non-extracted samples.
5. 13,14 Dihydro-15-Keto-Prostaglandin F2 Enzyme Conjugate, 1x150ul. 13,14-d-15-k-PGF 2a HRP concentrate. Blue capped vial.
6. 13,14 Dihydro-15-Keto-Prostaglandin F2 Standard, 1x100ul. 13,14-d-15-k-PGF 2a standard at the concentration of 1ug/ml. Green capped vial.
7. 13,14 Dihydro-15-Keto-Prostaglandin F2 Antibody Coated Plate, 1x 96-well microplate with anti-13,14-d-15-k-PGF 2a rabbit antibody precoated on each well. The plate is ready for use as is. Do not wash.
Storage and Stability:
Store components at -20 degrees C. Stable for 6 months. For maximum recovery of product, centrifuge the original vial after thawing and prior to removing the cap.