17 α 20 β Dihydroxyprogesterone ELISA Kit

Intended Use

For in-vitro research use. The 17 α 20 β Dihydroxyprogesterone ELISA KIT is to be used for quantitative determination of 17 α 20 β Dihydroxyprogesterone (17α, 20β-diOH-P) levels in fish serum.

Introduction

In lower vertebrates like fishes, maturation of oocyte is elicited by 17α, 20β-diOH-P which is universally known as Maturation Inducing Steroid (MIS). 17α, 20β-diOH-P is a key mediator hormone in fishes (spawning and non-spawning fishes) to detect oocyte maturation/ovulation in aquaculture sector and thus the estimation of this steroid would help the ichthyologists to improve further the quality and quantity of aquaculture to fulfil the demand of the food market in current scenario.

Principle Of The Assay

The ELISA of 17 α 20 β DiOH-P is a competitive solid-phase assay. Standards followed by serum samples along with 17 α 20 β DiOH-P-horseradish peroxidase (17 α 20 β DiOH-P-HRP) enzyme conjugate and 17 α 20 β Di-OHP-antibody (17 α 20 β DiOH-P-Ab) are incubated in second antibody coated wells. 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 of the kit, if stored at 2-8°C.

  • 17 α 20 β DiOH-P -HRP enzyme conjugate (3.0 mL), ready to use.
  • Secondary-Ab coated –96 wells (8 well strip) ready to use.
  • 17 α 20 β DiOH-P -Ab (2.5mL), ready to use.
  • Eight standards (each vial with 0.5 mL of standards), ready to use. The concentration of 17 α 20 β DiOHP 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 2-8°C, if the assay is to be performed within 24 hours. For longer storage keep frozen at – 20°C or – 8O°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 17 α 20 β DiOH-P-Ab into each well.
    • Dispense 50µL of standard followed by serum samples or tissue homogenate into appropriate wells in duplicate.
    • Dispense 25µL of 17 α 20 β DiOH-P-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

    17 α 20 β DiOH-P 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 17 α 20 β DiOH-P standard concentrations (ng/mL) on the horizontal axis.
    • Obtaining of 17 α 20 β DiOH-P concentration in samples: For each sample, locate the OD or %A/Ao on the vertical axis and read of the corresponding 17 α 20 β DiOH-P 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 17 Α 20 Β Dioh-P Standard Curve

    Specfic Performance Characteristics

    • a)The minimum concentration of 17 α 20 β DiOH-P, which can be detected from 0 standard, is 0.25 ng/mL (B0-2SD)
    • b) 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 17 α 20 β DiOH-P 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.
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