Progesterone is a C-21 steroid secreted by the corpus luteum and the placenta. It is an important intermediate in steroid biosynthesis in all tissues that secrete steroid hormones. Progesterone is responsible for the progestational changes in the endometrium and cyclic changes in the cervix and vagina. The plasma progesterone level is useful in investigating the luteal function and monitoring ovulation.
Principle of the Assay:
This is an ELISA for the quantitative analysis of Progesterone levels in biological fluid. This test kit operates on the basis of competition between the enzyme conjugate and the Progesterone 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 Progesterone in the sample or standard. For example, the absence of Progesterone in the sample will result in a bright blue color, whereas the presence of Progesterone will result in decreased or no color development.
Usually, urine and tissue culture supernatant can be assayed directly by diluting them with the diluted extraction buffer. Plasma and most other mediums will need to be extracted.
P9006-08A: ELISA Buffer 1x30ml. To be used to dilute enzyme conjugate and Progesterone standards.
P9006-08B: 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.
P9006-08C: 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.
P9006-08D: Extraction Buffer (5x) 1x30ml. To be diluted 5x with deionized water. This is used for diluting extracted and non-extracted samples.
P9006-08E: Progesterone (HRP) 1x150ul. Progesterone HRP concentrate.
P9006-08F: Progesterone Standard 1x100ul. Progesterone standard at the concentration of 1ug/ml. Green capped vial.
P9006-08G: Microplate 1x96 well microplate with anti-Progesterone rabbit antibody precoated on each well. The plate is ready for use as is. Do not wash.
Storage and Stability:
Store at 4 degrees C. Do Not Freeze. Stable for 6 months. For maximum recovery of product, centrifuge the original vial after thawing and prior to removing the cap.