Enzyme Immunoassay for the Quantitative Determination of Progesterone Concentration in Human Serum or Plasma

for in vitro diagnostic use

Product Description
Progesterone is a C21 steroid which is synthesized from both tissue and circulating cholesterol. Cholesterol is transformed to pregnenolone which is then converted via a combined dehydrogenase and isomerase to progesterone. The principle production sites are the adrenals and ovaries and the placenta during pregnancy. The majority of this steroid is metabolized in the liver to pregnanediol and conjugated as a glucuronide prior to excretion by the kidneys.

Progesterone exhibits a wide variety of end organ effects. The primary role of progesterone is exhibited by the reproductive organs. In males, progesterone is a necessary intermediate for the production of corticosteroids and androgens. In females, progesterone remains relatively constant throughout the follicular phase of the menstrual cycle. The concentration then increases rapidly following ovulation and remains elevated for 4-6 days and decreases to the initial level 24 hours before the onset of menstruation. In pregnancy, placental progesterone production rises steadily to levels of 10 to 20 times those of the luteal phase peak. Progesterone measurements are thus performed to determine ovulation as well as to characterize luteal phase defects. Monitoring of progesterone therapy and early stage pregnancy evaluations comprise the remaining uses of progesterone assays.

The Progesterone EIA kits are designed for the measurement of total progesterone in human serum or plasma.

Principle
The progesterone EIA is based on the principle of competitive binding between progesterone in the test specimen and progesterone-HRP conjugate for a constant amount of rabbit anti-progesterone. In the incubation, goat anti-rabbit IgG-coated wells are incubated with 25µl progesterone standards, controls, patient samples, 100µl progesterone-HRP Conjugate Reagent and 50µl rabbit anti-progesterone reagent at room temperature (18-25°C) for 90 minutes. During the incubation, a fixed amount of HRP-labeled progesterone competes with the endogenous progesterone in the standard, sample, or quality control serum for a fixed number of binding sites of the specific progesterone antibody. Thus, the amount of progesterone peroxidase conjugate immunologically bound to the well progressively decreases as the concentration of progesterone in the specimen increases.

Unbound progesterone peroxidase conjugate is then removed and the wells washed. Next, a solution of TMB Reagent is then added and incubated at room temperature for 20 minutes, resulting in the development of blue color.
The color development is stopped with the addition of Stop Solution, and the absorbance is measured spectrophotometrically at 450nm. The intensity of the color formed is proportional to the amount of enzyme present and is inversely related to the amount of unlabeled progesterone in the sample.
A standard curve is obtained by plotting the concentration of the standard versus the absorbance. The progesterone concentration of the specimens and controls run concurrently with the standards can be calculated from the standard curve.

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