Mouse/Rat Estradiol ELISA Protocol (Calbiotech)

Contributed by Calbiotech, Edited by Youssef Farhat on 5/14/13, Last updated 5/6/14

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This Mouse/Rat Estradiol ELISA Kit is designed for the measurement of Estradiol in Mouse/Rat serum and urine.  It can also be adapted for other fluids.  Store the kit at 2 – 8°C.


  • Mouse/Rat Estradiol ELISA Kit (Calbiotech, ES180S-100).  This kit includes the following components:
    • ELISA Plate Pre-Coated with Anti-Estradiol Capture Antibodies
    • Estradiol Standards (ready to use)
    • Assay Diluent (ready to use)
    • TMB Substrate Reagent (ready to use)
    • Stop Solution (ready to use)
    • 20X Estradiol Enzyme Conjugate Concentrate
    • 20X Wash Buffer
  • Distilled or deionized water
  • Micropipettes
  • Pipette tips
  • Paper towels
  • Plate reader capable of reading absorbance at 450 nm

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  1. Bring reagents to room temperature (18-26 °C) before use.
  2. Prepare working solutions:
  3. Pipette 25 μL of standards, controls and specimens into selected wells in duplicate.
  4. Add 100 μL of 1X Enzyme Conjugate to each well. Shake the plate 10-30 seconds to ensure proper mixing.
  5. Incubate for 2 hours at room temperature, preferably in the dark (cover with aluminum foil, for example).
  6. Wash the wells 3 times with 300 μL of 1X wash buffer using either a suitable plate washer or wash bottle taking care not to cross contaminate wells.
  7. Invert wells and vigorously slap dry on absorbent paper to ensure all residual moisture is removed. This step is critical to ensure that residual enzyme conjugate, does not skew results. If using an automated system, ensure that the final aspiration on the wash cycle aspirates from either side of the well.
  8. Add 100 μL of TMB Substrate Reagent to each well.  Gently shake the plate for 10 seconds to mix.
  9. Incubate for 30 minutes at room temperature, preferably in the dark.
  10. Add 50 μL of Stop Solution to each well. Shake the plate gently to mix the solution for 30 seconds to ensure that the blue color completely changes to yellow.
  11. Read absorbance on a plate reader set to 450 nm within 15 minutes.
    1. 1X Wash Buffer: Combine 25 mL of the kit’s 20X Wash Buffer with 475 mL of distilled or deionized water into a clean bottle or similar container
    2. 1X Enzyme Conjugate: Combine 0.1 mL of 20X Estradiol Enzyme Conjugate Concentrate with 1.9 mL of Assay Diluent


Citing this Protocol

Calbiotech & Farhat, Y. “Mouse/Rat Estradiol ELISA Protocol (Calbiotech).” Protocol Place. May 2013. Accessed on mm/dd/yyyy. <>

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