Cotinine ELISA Protocol (Calbiotech)

Contributed by Calbiotech, Edited by Youssef Farhat on 4/19/13, Last updated 5/6/14

Download the PDF or Microsoft Word versions of this protocol


This Cotinine ELISA Kit is designed for the detection Cotinine in serum and urine. It can also be adapted for other fluids.  Store the kit at 2 – 8°C.


  • Cotinine ELISA Kit (Calbiotech, #CO096D) This kit includes the following components:
    • ELISA Plate Pre-Coated with Anti-Cotinine Capture Antibodies
    • Cotinine Standards (ready to use)
    • Cotinine HRP Enzyme Conjugate Concentrate (ready to use)
    • TMB Substrate Reagent (ready to use)
    • Stop Solution (ready to use)
  • Distilled or deionized water
  • Micropipettes
  • Pipette tips
  • Paper towels
  • Plate reader capable of reading absorbance at 450nm

Click to see Calbiotech's ELISA Kits


  1. Bring reagents to room temperature (18-26 °C) before use.
  2. Pipette 10 μl of standards, controls and specimens into selected wells in duplicate.
  3. Add 100 μl of the Enzyme Conjugate to each well. Shake the plate 10-30 seconds to ensure proper mixing.
  4. Incubate for 60 minutes at room temperature, preferably in the dark (cover with aluminum foil, for example).
  5. Wash the wells 6 times with 300 μL of distilled water using either a suitable plate washer or wash bottle taking care not to cross contaminate wells.
  6. 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.
  7. Add 100 μl of Substrate reagent to each well.
  8. Incubate for 30 minutes at room temperature, preferably in the dark.
  9. Add 100 μl of Stop Solution to each well. Shake the plate gently to mix the solution.
  10. Read absorbance on a plate reader set to 450nm within 15 minutes.


Citing this Protocol

Calbiotech & Farhat, Y. “Cotinine ELISA Protocol (Calbiotech).” Protocol Place. Apr 2013. Accessed on mm/dd/yyyy. <>

Leave a Reply

Your email address will not be published. Required fields are marked *

You may use these HTML tags and attributes: <a href="" title=""> <abbr title=""> <acronym title=""> <b> <blockquote cite=""> <cite> <code> <del datetime=""> <em> <i> <q cite=""> <strike> <strong>