Principle of The Assay

The ELISA of 11-KT is a competitive solid-phase assay. Serum samples or standards along with 11-KT-horseradish peroxidase enzyme conjugate (11-KT-HRP) and 11-KT- antibody are incubated in second antibody coated wells. After incubation, the liquid contents of the wells are decanted, and the wells are washed under 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.

Reagent Provided

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

• 11-KT-horseradish peroxidase enzyme conjugate (3.0 mL), ready to use.
• Secondary-Ab coated –96 wells (8 well strip) ready to use.
• 11-KT- antibody (2.5mL), ready to use.
• Eight standards (each vial with 1.0 mL of standards), ready to use. The concentration of 11-KT 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 2-8o C, 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 50µL of standard or unknown serum samples, into appropriate wells in duplicate.
• Dispense 50µL of 11-KT-HRP enzyme conjugate into each well.
• Dispense 20µL of 11 KT-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

11 KT concentration in samples are obtained from the standard curve.

• i) Standards curve (sigmoid in nature). Plot standard curve on semi- logarithmic paper. Optical density or % A/A0 on the vertical axis. The 11 KT standard concentrations (ng/mL) on the horizontal axis.
• ii) Obtaining of 11 KT concentration in samples: For each sample, locate the OD or %A/Ao on the vertical axis and read of the corresponding 11 KT 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
  % Aµ Ao = Aµ Ao X 100

Example Of 11 Ketotestosterone Standard Curve



Specfic Performance Characteristics

• a) Sensitivity: The minimum concentration of 11 KT, which can be detected from 0 standard, is 0.1 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 11 KT 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 0.1% with naturally occurring C27, C21, C19, and C18, except 5 α-DHT (8.5%), Testosterone (3.5%) and Androstendione (5.6%).