Reagent Provided

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

• Cortisol-HRP enzyme conjugate (6 mL), ready to use.
• Secondary-Ab coated –96 wells (8 well strip) ready to use.
• D-Ab (2.5mL), ready to use.
• Eight standards (each vial with 0.5 mL of standards), ready to use. The concentration of nandrolone 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)
• 250 mL PBS
• 60 mL homogenizing buffer

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)
• Centrifuge, Balance, Glass Homogenizer, Ultrasonicator or deep fridge

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 milk sample in a plain tube and store at 2-8oC, if the assay is to be performed within 24 hours. For longer storage keep frozen at – 20o C or – 8OoC after aliquoting so as to avoid repeated freezing thawing.

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 – 20oC or – 8OoC after aliquoting so as to avoid repeated freezing thawing.

Tissue homogenates – Rinse the tissue in PBS (0.02mol/L, pH 7.0-7.2) to remove excess blood thoroughly and weighed 500mg before homogenization. Minced the tissues to small pieces and homogenized them in 500L of homogenizing buffer with a glass homogenizer. The resulting suspension are subjected to either ultrasonication or to two freeze-thaw cycles to further break the cell membranes.

Assay Procedure

Let reagents come to room temperature.

• Secure the desired number of coated wells in the holder.
• Dispense 50µL of standard or unknown serum samples, into appropriate wells in duplicate.
• Dispense 50µL of dexamethasone -HRP enzyme conjugate into each well.
• Dispense 20µL of t dexamethasone -antibody into each well.
• Incubate for 60 minutes at room temperature.
• After incubation 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.
• Incubate 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

Dexamethasone 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/A0 on the vertical axis. The dexamethasone standard concentrations (ng/mL) on the horizontal axis.
• Obtaining of dexamethasone concentration in samples: For each sample, locate the OD or %A/Ao on the vertical axis and read of the corresponding dexamethasone 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 Dexamethasone Standard Curve



Specfic Performance Characteristics

• a) Sensitivity: The minimum concentration of dexamethasone, 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 5 quality control serum samples. The results were –
  
• c) Accuracy: For recovery study, different concentrations of testosterone 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 is less than 0.1% with naturally occurring C27, C21, C19, and C18, except testosterone 2.46%, androstenediol 0.89%, and 5 α –dihydrotesto sterone 5.66%.