HUMAN LEPTIN
This kit is used for the non-radioactive quantification of human leptin in serum, plasma and other biological media. One kit is sufficient to measure 37 unknown samples in duplicate. This kit is for research purposes only.
This assay is a direct Sandwich ELISA based, sequentially, on: 1) capture of human leptin by a polyclonal rabbit anti-human leptin antibody immobilized on a 96-well microtiter plate, 2) wash away unbound materials, 3) binding of a biotinylated monoclonal antibody to the captured human leptin, 4) wash away unbound materials, 5) binding of streptavidin-horseradish peroxidase to the immobilized biotinylated antibodies, 6) wash away free enzyme conjugates, and 7) quantification of bound streptavidin-horseradish peroxidase with the substrate 3,3’,5,5’-tetramethylbenzidine. The enzyme activity is measured spectrophotometrically by the increased absorbency at 450 nm - 590nm after acidification of formed products. Since the increase in absorbency is directly proportional to the amount of captured human leptin in the unknown sample, the latter can be derived by interpolation from a reference curve generated in the same assay with reference standards of known concentrations of human leptin.
Each kit is sufficient to run one 96-well microtiter plate and contains the following reagents:
A. Human Leptin ELISA Plate
Coated with Rabbit anti-Human Leptin Antibody
Quantity: 1 plate
Preparation: Ready to use
B. Adhesive Plate Sealer
Quantity: 2 Sheets
Preparation: Ready to use
C. 10X HRP Wash Buffer Concentrate
10X concentrate of 50 mM Tris Buffered Saline containing Tween 20
Quantity: Two bottles containing 50 ml each
Preparation: Dilute 1:10 with deionized water
D. Human Leptin ELISA Standards
Human Leptin in Assay Buffer: 0.5, 1, 2, 5, 10, 20, 50 and 100 ng/ml
Quantity: 1 ml/vial
Preparation: Ready to use
E. Quality Controls 1 and 2
Purified Recombinant Human Leptin in QC Buffer
Quantity: 0.5 ml/vial
Preparation: Ready to use
F. Assay Buffer
0.05M PBS, pH 7.4, containing 0.025M EDTA, 0.08% Sodium Azide, 1% BSA and 0.05% Triton X-100
Quantity: 10 ml/vial
Preparation: Ready to use
G. Human Leptin Detection Antibody
Biotinylated Mouse anti-Human Leptin Antibody
Quantity: 11 ml/vial
Preparation: Ready to use
H. Enzyme Solution
Streptavidin-Horseradish Peroxidase Conjugate in Buffer
Quantity: 12 ml/vial
Preparation: Ready to use
I. Substrate
3,3’5,5’-tetramethylbenzidine
Quantity: 12ml
Preparation: Ready to use
J. Stop Solution
3M HCL
Quantity: 12 ml/vial
Preparation: Ready to use (Caution: Corrosive Material)
Upon receipt, all components of the kit should be stored at 2-8° C. For prolonged storage (>2 weeks), store the Wash Buffer, Assay Buffer, Standards, and Controls at £ -20° C and store the Detection Antibody, Enzyme Solution, Substrate, and Plate at 2-8° C. Refer to expiration dates on all reagents prior to use. Do not mix reagents from different kits unless they have the same lot numbers.
A. Hydrochloric Acid
Hydrochloric Acid is corrosive and can cause eye and skin burns. It is harmful if swallowed and can cause respiratory and digestive tract burns. Avoid contact with skin and eyes. Do not swallow or ingest.
B. Sodium Azide
Sodium Azide has been added to some reagents as a preservative at a concentration of 0.08%. Although it is at a minimum concentration, Sodium Azide may react with lead and copper plumbing to form highly explosive metal azides. On disposal, flush with large volume of water to prevent azide build up.
VI. MATERIALS REQUIRED BUT NOT PROVIDED
1. Pipette with Tips, 10m l-200m l
2. Multi-channel Pipette, 50m l-300m l
3. Buffer and Reagent Reservoirs
4. Vortex Mixer
5. Absorbent Paper or Cloth
6. Deionized Water
7. Microtiter Plate Reader capable of reading absorbency at 450 nm
8. Orbital Microtiter Plate Shaker
VII. SAMPLE COLLECTION AND STORAGE
1. To prepare serum, whole blood is directly drawn into a Vacutainer® serum tube that contains no anti-coagulant. Let blood clot at room temperature for 30 minutes.
2. Promptly centrifuge the clotted blood at 2000 to 3000 x.g. for 15 minutes at 4 ± 2ºC.
3. Transfer and store serum samples in separate tubes. Date and identify each sample.
4. Use freshly prepared serum or store samples at £ -20° C for later use. Avoid multiple (>5) freeze/thaw cycles.
5. To prepare plasma samples, whole blood should be collected into Vacutainer® EDTA-plasma tubes and centrifuged immediately after collection. Observe same precautions in the preparation of serum samples.
6. If heparin is to be used as an anti-coagulant, the effect on the assay outcome at the dose of heparin used should be pre-determined.
7. Avoid using samples with gross hemolysis or lipemia.
(Sensitivity: 0.5 ng/ml – 100 ng/ml)
Pre-warm all reagents to room temperature immediately before setting up the assay.
1. Dilute the concentrated Wash Buffer 10 fold by adding the entire contents of both bottles of buffer to 900 ml de-ionized or glass distilled water.
2. Remove the microtiter assay plate from the foil pouch and add 300 µl of diluted Wash Buffer to each well. Incubate at room temperature for 5 minutes. Decant wash buffer and remove the residual amount from all wells by inverting the plate and tapping it smartly onto absorbent towels several times. Do not let wells dry before proceeding to the next step. If an automated machine is used for the assay, follow the manufacturer’s instructions for all washing steps described in this protocol.
3. Add 75 µl Assay Buffer into all wells.
4. Add in duplicate 25 µl Assay Buffer to blank wells. (Refer to Section IX for suggested well Orientations.)
5. Add in duplicate 25 µl Human Leptin Standards in order of ascending concentration to the appropriate wells. Add in duplicate 25 µl QC1 and 25 µl QC2 to the appropriate wells. Add sequentially 25 µl of samples in duplicate to the remaining wells. For best results all additions should be completed within one hour.
6. Cover the plate with plate sealer and incubate at room temperature for 2 hours on an orbital microtiter plate shaker set to rotate at moderate speed, about 400 to 500 rpm.
7. Remove plate sealer and decant solutions from the plate. Tap as before to remove residual solutions in the wells.
8. Wash wells 3 times with diluted Wash Buffer, 300 µl per well per wash. Decant and tap after each wash to remove residual buffer.
9. Add 100 µl Detection Antibody to each well. Cover the plate with sealer and incubate at room temperature for 30 minutes on the microtiter plate shaker.
10. Remove sealer and decant solution from the plate. Tap as before to remove residual solutions in the wells.
11. Add 100 µl Enzyme Solution to each well. Cover plate with sealer and incubate with moderate shaking at room temperature for 30 minutes on the microtiter plate shaker.
12. Remove sealer, decant solution from the plate, and tap plate to remove the residual fluid.
13. Wash wells 5 times with diluted Wash Buffer, 300 µl per well per wash. Decant and tap firmly after each wash to remove residual buffer.
14. Add 100 µl of Substrate Solution to each well, cover plate with sealer and shake on the plate shaker for ~5 minutes. Blue color should be formed in wells of Leptin standards with intensity proportional to increasing concentrations of Leptin.
15. Remove sealer and add 100 µl of Stop Solution (Caution: Corrosive solution) and shake plate by hand to ensure complete mixing of solution in all wells. The blue color should turn to yellow after acidification. Read absorbance at 450nm and 590nm in a plate reader within 5 minutes and ensure that there are no air bubbles in any well. Record the difference in absorbance units.