Leptin protocol

Protocol for: ELISA leptin protocol
Prepared by: Rong Yuan
Reviewed by: David Harrison
Last revision date: 05.01.2006

Kit used: Crystal Chem (CrystalChem, Inc., USA; tel. 630-889-9003) Mouse Leptin ELISA Kit, Cat. No: 90030

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

Samples: The “sample” used in this application refers to mouse plasma. Whole blood is obtained via retro-orbital sinus through heparinized capillary tubes from non-fasted mice. Blood is collected into 1.5ml Eppendorf tubes containing sodium heparin, and tubes are spun in a microcentrifuge at 14,000 rpm for 5 minutes. Plasma is removed into a clean Eppendorf tube and remaining material is discarded. Samples may be frozen for later analysis or used directly.

**Bring microplate to room temperature**

  • Step 1: Prepare template sheet: Label a sheet that looks like the plate with the corresponding sample numbers, and standards that will be run.
  • Step 2: Prepare mouse leptin standard by reconstituting with 100ul SampleDiluent and dissolve completely. This stock is 25,600 pg/ml.
  • Step 3: Prepare working mouse standards: Label eight microtubes 0, 200, 400, 800, 1,600, 3,200, 6,400 and 12,800. Pipette 50ul of sample diluent into each tube. Start by pipetting 50ul of mouse leptin stock into the tube labeled 12,800 and mix well. Then take 50ul of 12,800 standard and pipette into 6,400 and mix well. Pipette 50ul of 6,400 standard into 3,200 and mix well. Repeat dilution procedure on remaining tubes, but stop at 0. The 0 standard should only contain 50ul of sample diluent. See below:

Mouse leptin concentration (pg/ml)

12,800

6,400

3,200

1,600

800

400

200

0

MLSS*

50ul

SD**

50ul

50ul

50ul

50ul

50ul

50ul

50ul

50ul

50ul

50ul

50ul

50ul

50ul

50ul

***

***

***

***

***

***

Total

100ul

100ul

100ul

100ul

100ul

100ul

100ul

50ul

* MLSS: Mouse Leptin Stock Solution
** SD: Sample Diluent
*** Place 50ul of previous dilution into new dilution to create the desired dilution.
Discard leptin standards after use.

First reaction:

  • Step 4: After modules are placed in the micoplate, wash each well with 300ul (which is about the whole well) of washing buffer, discard liquid and wash once more; discard liquid again.
    Washing buffer solution preparation: With distilled water, take washing buffer stock solution and fill to 1000ml and mix well.
  • Step 5: Pipette 45ul of Sample diluent and 50ul of Guinea Pig anti-mouse leptin insulin to each well.
  • Step 6: Pipette 5ul of each standard into the wells that correspond to the template.
  • Step 7: Pipette 5ul of each sample into the wells that correspond to the template.
  • Step 8: Cover plate with plastic microplate cover and let stand overnight (16-20 hours) at 4°C.

Second reaction:

  • Step 9: Discard well content and wash 5 times with 300ul of washing buffer.
  • Step 10: Pipette 100ul of Anti-guinea pig IgG enzyme conjugate to each well. Preparation of Anti-Guinea Pig IgG enzyme conjugate: Dilute one bottle of Anti-guinea pig IgG enzyme conjugate stock solution by adding one bottle of Enzyme conjugate diluent and mix until clear.
  • Step 11: Cover plate and incubate for 3 hours at 4°C.
  • Step 12: Discard contents of wells and wash 7 times with 300ul of washing buffer each time.
  • Step 13: IMMEDIATELY AFTER washing, pipette 100ul of Enzyme SubstrateSolution, and react for 30 minutes. **Protect plate from light**
  • Step 14: Stop reaction by adding 100ul of enzyme reaction stopping solution to each well. **Wear gloves (stop solution is sulfuric acid)**
  • Step 15: Measure absorbance within 30 minutes, measuring wave length 450nm, subtracting wavelength 630nm.