Medroxyprogestrone Acetate ELISA Kit

Intended Use

For in-vitro research use. The progesterone ELISA KIT is to be used for quantitative determination of Medroxyprogesterone acetate (MPA) levels in serum and tissues.

Introduction

MPA is a synthetic substitute for human progesterone, produced by acetylating medroxyprogesterone. MPA is primarily used as a contraceptive to prevent pregnancy for three months. Unfortunately, it has also been misused as a fattening agent and to improve the texture of meat, making it more appealing to consumers. This misuse poses significant health risks such as venous thromboembolism, reduced bone mineral density, and obesity.

To prevent its misuse, monitoring MPA levels in food and animal feed is crucial. The kit is capable of measuring MPA in various biological samples and will facilitate effective monitoring of its presence. This monitoring is essential to ensure public health and safety.

Principle Of The Assay

The ELISA of MPA is a competitive solid-phase assay with sequential addition of reagents.  Standards followed by serum samples along with MPA-antibody (MPA-Ab) are incubated for 15 minutes in second antibody coated wells thereafter MPA-horseradish peroxidase (MPA-HRP) enzyme conjugate is added and further incubated for 45 minutes. After incubation, the liquid contents of the wells are decanted, and the wells are washed in running tap water for removing the unbound enzyme conjugate. The bound enzyme activity is measured by developing colored product from colorless substrate after incubation. The quantity of color developed is directly related to the bound enzyme conjugate and is inversely related to the concentration of analyte. Unknown values are determined by interpolation from the standard curve

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Reagent Provided

Reagents are stable until the expiry date (see COA) of the kit, if stored at 2-80 C.

  • MPA -HRP enzyme conjugate (7 mL), ready to use.
  • Secondary-Ab coated –96 wells (8 well strip) ready to use.
  • MPA -Ab (5.0 mL), ready to use.
  • Eight standards (each vial with 1.0 mL of standards), ready to use. The concentration of MPA ranges from 0 to 40ng/mL in stripped serum 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 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 blood in plain tube, separate the serum from cells by centrifugation. Store at 2-8o C, if the assay is to be performed within 24 hours. For longer storage keep frozen at – 20o C or – 😯o C after aliquoting so as to avoid repeated freezing thawing.

Tissue homogenates – Rinse the tissue in PBS (0.02 mol/L, pH 7.0-7.2) to remove excess blood thoroughly and weighed 500 mg before homogenization. Minced the tissues to small pieces and homogenized them in 500 mL 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. After that, centrifuge the homogenates for 15 minutes at 1500×g (or 5000 rpm). Take out the supernatant and assay within 24 hours or aliquot and store it at -20°C or -80°C.

Assay Procedure

Let reagents come to room temperature.

      • Secure the desired number of coated wells in the holder.
      • Dispense 40µL of MPA-Ab into each well.
      • Dispense 100µL of standard followed by serum samples or tissue homogenate into appropriate wells in duplicate.
      • Dispense 50µL of of MPA-HRP enzyme conjugate into each well.
      • Keep at room temperature for 45 minutes.
      • After 45 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

MPA 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 MPA standard concentrations (ng/mL) on the horizontal axis.
      • Obtaining of MPA 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 or tissue homogenate.
        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 17 Α 20 Β Dioh-P Standard Curve

Specfic Performance Characteristics

    • a)Sensitivity: The minimum concentration of MPA, 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 MPA 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 fifty one C27, C21, C19, and C18
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