ELISA Leptin Bench Protocol: R&D Quantikine Leptin

Revision #: 2.0

Prepared by HS, Rev. Jan. 31, 2005, RVS

Kit used: R&D Systems Quantikine mouse Leptin Kit, Catalog No. MOB00

Equipment Used: Molecular Devices SPECTRAmax 190 and the Skan Washer 300, Model 12010

  1. Bring all Reagents, Plates, and Samples to room temperature before use.
  2. Make Template Sheet
  3. Dilute Plasma Samples 20 fold:
  4. We use 8.5ul of sample to 161.5ul of Calibrator Diluent RD5-3
    Suggested dilution: 20ul of sample to 380ul of Calibrator Diluent RD5-3
    *Using this method we cannot run plasma that was taken from a pregnant mouse. Serum, not plasma, needs to be obtained and then activated using the methods stated in the procedure booklet provided with each kit. 
  5. Prep Mouse Leptin Standard:
     Reconstitute Leptin Standard with 2.0 ml of Calibrator Diluent RD5-3
     (This produces the stock solution  =  4000 pg/ml) Allow to sit for a minimum of 5 minutes with gentle mix prior to use.
  6. Prep Mouse Leptin Kit Control:
    Reconstitute control with 1.0 ml of deionized or distilled water. (Assay control un-diluted.)
  7. Prep Mouse Leptin Conjugate:
    1 plate: add 500ul (0.5ml) of Conjugate Concentrate to 11ml Conjugate Diluent. Use sterile container and PROTECT FROM LIGHT.
  8. Prepare Standard Dilutions:
    Pipette 200ul of Calibrator Diluent RD5-3 into each of seven tubes. Use stock solution as the starting high standard. Calibrator Dilutent RD5-3 serves as the zero standard. Dilute as follows:
    *MIX THOROUGHLY BEFORE NEXT TRANSFER


  9. Take enough micro-plate strips out for the test and place remaining strips into sealed pouch.
  10. Add 50ul of Assay Diluent RD1W to each well.
  11. Add 50ul of standard, control, and sample per well according to template sheet.
  12. Mix gently by tapping for 1 minute.
  13. Cover and incubate for 1 hour at room temp.
  14. Prep Wash Buffer:
    For 1 plate add 25 ml of wash buffer concentrate to 600 ml of distilled or deionized water  to prepare 625ml of wash buffer.
  15. After incubation wash 5 times with wash buffer with ~400ul per well each time. Make sure all liquid is removed when finished.
  16. Add 100ul of diluted Mouse Leptin Conjugate to each well, cover and incubate 2 hours at room temp.
  17. ~ 3 minutes before incubation is done, prep Substrate Solution. 
    Add Color reagent A to Color reagent B in equal volumes.
    PROTECT FROM LIGHT (5ml of each is good for one plate)
  18. After incubation wash plate 5 times as above.
  19. Add 100ul of Substrate Solution that was made prior to washing the plate, to each well.
  20. Incubate for 30 minutes at room temp in dark drawer, protected from light
  21. Add 100ul of stopping solution to each well. Gently tap plate to ensure mixing.
  22. Determine optical density of plate within 30 minutes. Read at 450nm with subtracting wavelength 540nm or 570nm.