11b-Prostaglandin F2a (11b-PGF2a) is derived from PGD2 by the action of 11-ketoreductase. It is a potent broncho-constrictor agent. It also causes hypertension in animals. Plasma levels of 11b-PGF2a are highly elevated in patients with systemic mastocytosis with an overproduction of PGD2.
Principle of the Assay:
This is an ELISA (Enzyme-Linked Immunosorbent Assay) for the quantitative analysis of 11b-Prostaglandin F2a levels in biological fluid. This test kit operates on the basis of competition between the enzyme conjugate and the 11b-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 11b-PGF2a in the sample or standard. For example, the absence of 11b-PGF2a in the sample will result in a bright blue color, whereas the presence of 11b-PGF2a will result in decreased or no color development.
1. EIA Buffer, 1x30ml. To be used to dilute enzyme conjugate and 11b-PGF2a standards.
2. Wash Buffer (10X), 120ml. 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. 11-Prostaglandin F Enzyme Conjugate, 1x150ul. 11b-PGF2a HRP concentrate. Blue capped vial.
6. 11-Prostaglandin F Standard, 1x100ul. 11b-PGF2a standard at the concentration of 1ug/ml. Green capped vial.
7. 11-Prostaglandin F2 Antibody Coated Plate, 1xA 96-well microplate with anti-11b-PGF2a 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.