Calbiotech 17-OH Progesterone Elisa Kit

PRINCIPLE OF THE TEST

The Calbiotech 17α-OH Progesterone (17-OHP) is a based on the principle of competitive binding between 17- OHP in the test specimen and 17-OHP-HRP for a constant amount of Rabbit anti-17-OHP antibody. In the assay, goat anti-rabbit IgG-coated wells are incubated with 25µl 17-OHP standards, 50µl 17-OHP-HRP conjugate, and 50µl rabbit anti-17-OHP. HRP-labeled-17-OHP competes with 17-OHP in the standards and sample for a fixed number of binding sites of the specific anti-17-OHP antibody. Thus, the amount of 17-OHP peroxidase conjugate immunologically bound to the well progressively decreases as the concentration of 17- OHP in the specimen increases. Unbound 17-OHP is then removed by the washing steps. TMB Substrate is added, resulting in the development of blue color. The color development is stopped with the addition of stop solution, and the absorbance is spectrophotometrically measured at 450nm. A standard curve is prepared relating color intensity to the concentration of 17-OHP.

STORAGE AND STABILITY

1. Store the kit at 2-8°C.
2. Keep microwells sealed in a dry bag with desiccants.
3. The reagents are stable until expiration of the kit.
4. Do not expose test reagents to heat, sun or strong light.

SPECIMEN COLLECTION AND HANDLING
1. Collect blood specimens and separate the serum immediately.
2. Specimens may be stored refrigerated at (2-8°C) for 5 days. If storage time exceeds 5    days, store frozen at (-20°C) for up to one month.
3. Avoid multiple freeze-thaw cycles.
4. Prior to assay, frozen sera should be completely thawed and mixed well.
5. Do not use grossly lipemic specimens.

REAGENT PREPARATION
1. Working Enzyme Conjugate: Prepare 1X working dilution at 1:20 with assay diluents as needed, e.g. 0.1ml of the stock conjugate in 1.9ml of assay diluent for 40 wells. The diluted conjugate has to be used on the same day.
2. Wash Buffer: Prepare 1X Wash Buffer by adding the contents of the bottle (25ml, 20X) to 475ml of distilled water. Store at room temperature (20-25°C).

CALCULATION OF RESULTS
A standard curve is constructed as follows:
1. Calculate the average absorbance values for each set of standards and patient samples
2. To construct the standard curve, plot the mean absorbance of each 17-OHP standards (vertical axis) against its concentration in ng/ml (horizontal axis)
3. Draw the best-fit curve through the plotted points.
4. Read the absorbance for each unknown sample from the curve to determine the corresponding concentration of 17-OHP.