Reagent Provided

Reagents are stable until the expiry date of the kit, if stored at 2-8°C.

• Progesterone -HRP enzyme conjugate (7 mL), ready to use.
• Secondary-Ab coated –96 wells (8 well strip) ready to use.
• Progesterone-Ab (2.5mL), ready to use.
• Eight standards (each vial with 0.5 mL of standards), ready to use. The concentration of progesterone ranges from 0 to 40ng/mL with 0.03% Proclin 300. The exact concentration is marked on each vial.
• TMB/H₂O₂ solution (12 mL), ready to use.
• Stop solution (12.0 mL 0.5 M H₂SO₄)

Materials Required But Not Provided

In addition to standard laboratory equipment the following items are required

• Precision micropipette (100 µL – 1000 µL)
• Precision micropipette (10 µL – 100 µL)
• ELISA – Reader (Microtiter Plate Reader)

Precautions

For satisfactory results:

• Do not mix reagents from different lots
• Let reagents come to room temperature before use

All blood components and biological materials should be handled as potentially hazardous. Decontaminate and dispose specimens and all potentially contaminated materials as they could contain infectious agents.

Sample Collection

Collect the blood in plain tube, separate the serum from cells by centrifugation. Store at 2-8oC, if the assay is to be performed within 24 hours. For longer storage keep frozen at – 20o C or – 8Oo C after aliquoting so as to avoid repeated freezing thawing.

Assay Procedure

Let reagents come to room temperature.

• Secure the desired number of coated wells in the holder.
• Dispense 20µL of progesterone -Ab into each well.
• Dispense 50µL of standard followed by serum samples or tissue homogenate into appropriate wells in duplicate.
• Dispense 50µL of progesterone-HRP enzyme conjugate into each well.
• Keep at room temperature for 60 minutes.
• After 60 minutes remove the mixture by flicking the plate contents into a waste container.
• Fill and flick the microtiter wells 7-10 times under running tap water.
• Tap the plate 15 to 20 times on a cloth towel and ensure that water has been removed from wells.
• Dispense 100 µL of substrate solution in each well.
• Keep at room temperature for 15 minutes.
• Stop the reaction by adding 100µL of stop solution.
• Read OD at 450 nm with a microtiter well reader immediately.

Summary Of Assay Procedure



Results

Progesterone concentration in samples are obtained from the standard curve.

• Standards curve (sigmoid in nature). Plot standard curve on semi- logarithmic paper. Optical density or % A/Ao on the vertical axis. The progesterone standard concentrations
  (ng/mL) on the horizontal axis.
• ii) Obtaining of progesterone concentration in samples: For each sample, locate the OD or %A/Ao on the vertical axis and read of the corresponding progesterone concentration on the horizontal axis. It will directly show the concentration in terms of ng/mL in serum.

  Ao = optical density at zero dose
  A = optical density at varying concentrations of standard dose or unknown sample
  % A/Ao = A/Ao X 100

Example Of Progesterone Standard Curve



Specfic Performance Characteristics

• a) Sensitivity: The minimum concentration of progesterone, which can be detected from 0 standard, is 0.25 ng/mL (B0-2SD)
• b) Intra-assay and inter-assay variation: The precision profile of the assay was determined from 4 quality control serum samples. The results were –
  
• c) Accuracy: For recovery study, different concentrations of progesterone were added to serum sample wherein its basal concentration was already estimated. The recovery performance of the assay is given below.
  
• d) Specificity: The cross-reactivity of this antibody used in this kit is less than 0.025% with naturally occurring C27, C21, C19, and C18 steroids except 6.8% cross-reaction with pregnenolone and 6.1% with 17- α-OH-P.