HUMAN ADIPONECTIN
This Human Adiponectin (ACRP30) ELISA kit is used for the non-radioactive quantification of Human Adiponectin in serum, plasma, and adipocyte extracts or cell culture media samples. This kit specifically measures native Human Adiponectin and has no cross reactivity to Mouse Adiponectin. One kit is sufficient to measure 38 unknown samples in duplicate. This kit is for research purpose only.
This assay is a Sandwich ELISA based, sequentially, on: 1) concurrent capture of Human Adiponectin molecules from samples to the wells of a microtiter plate coated with a monoclonal anti-human adiponectin antibodies, and binding of a second biotinylated monoclonal anti-human antibody to the captured molecules , 2) washing of unbound materials from samples, 3) binding of streptavidin-horseradish peroxidase conjugate to the immobilized biotinylated antibodies, 4) washing of excess of free enzyme conjugates, and 5) quantification of immobilized antibody-enzyme conjugates by monitoring horseradish peroxidase activities in the presence of the substrate 3,3’,5,5’-tetramethylbenzidine. The enzyme activity is measured spectrophotometrically by the increased absorbance at 450 nm – 590nm after acidification of formed products. Since the increase in absorbance is directly proportional to the amount of captured Human Adiponectin 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 Adiponectin.
Each kit is sufficient to run one 96-well plate and contains the following reagents:
A. Human Adiponectin ELISA Plate
Coated with Mouse anti-Human Adiponectin Antibodies
Quantity: 1 plate
Preparation: Ready to Use
Note: Unused strips should be resealed in the foil pouch with the desiccant provided.
B. Adhesive Plate Sealer
Quantity: 1 sheet
Preparation: Ready to Use
C. 10X HRP Wash Buffer Concentrate
10X concentrate of 50 mM Tris Buffered Saline containing Tween-20
Quantity: 2 bottles containing 50 ml each
Preparation: Dilute 1:10 with distilled or deionized water
D. Human Adiponectin Standard
Purified Recombinant Human Adiponectin, lyophilized.
Quantity: 100 ng/0.5 ml (200 ng/ml) upon hydration.
Preparation: Contents Lyophilized. Reconstitute with 0.5 ml distilled or deionized water to obtain 200 ng/ml.
E. Human Adiponectin Quality Controls 1 and 2
One vial each, lyophilized, containing diluted human serum at two different levels of Adiponectin.
Quantity: 0.5ml/bottle upon hydration
Preparation: Contents Lyophilized. Reconstitute each vial with 0.5ml distilled or deionized water
F. 10X Assay Buffer (Sample Diluent)
Quantity: 50 ml
Preparation: Dilute 1:10 with distilled or deionized water to make 1X Assay Buffer
(0.05M Phosphosaline containing 0.025M EDTA, 0.08% Sodium Azide, 1% BSA)
Note: Use 1X Assay Buffer to dilute samples (Section VIII, SAMPLE PREPARATION) and Standard Curve (Section IX, STANDARD AND QUALITY CONTROLS PREPARATION)
G. Assay Buffer A (Assay Running Buffer)
0.05M Phosphosaline containing 0.025M EDTA, 0.08% Sodium Azide, 1% BSA
Quantity: 7 ml
Preparation: Ready to Use
H. Human Adiponectin Detection Antibody
Pre-titered Biotinylated Mouse anti-Human Adiponectin Antibody
Quantity: 3 ml
Preparation: Ready to Use
I. Enzyme Solution
Pre-titered Streptavidin-Horseradish Peroxidase Conjugate in Buffer
Quantity: 12 ml
Preparation: Ready to Use
J. Substrate (Light sensitive, avoid unnecessary exposure to light)
3, 3’, 5, 5’-tetramethylbenzidine in buffer
Quantity: 12 ml
Preparation: Ready to Use.
K. Stop Solution (Caution: Corrosive Solution)
0.3 M HCl
Quantity: 12 ml
Preparation: Ready to Use
Prior to use, all components in the kit can be stored up to 2 weeks at 2-8oC. For longer storage (> 2 weeks), freeze diluted Wash Buffer, Assay Buffer, and reconstituted Standards and Controls at £ –20oC. Minimize repeated freeze and thaw of the Adiponectin Standards and Quality Controls. Refer to expiration dates on all reagents prior to use. Do not mix reagents from different kits unless they have the same lot numbers. Unused microtiter strips should be resealed in the foil pouch with the desiccant provided.
A. Sodium Azide
Sodium azide has been added to certain reagents as a preservative. Although the concentrations are low, sodium azide may react with lead and copper plumbing to form highly explosive metal azides. On disposal, flush with a large volume of water to prevent azide build up.
B. 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.
VI. MATERIALS REQUIRED BUT NOT PROVIDED
1. Pipettes and Pipette Tips: 10m l - 20 m l or 20m l - 100 m l
2. Multi-Channel Pipettes and Pipette Tips: 5 ~ 50 µl and 50 ~ 300 m l
3. Buffer and Reagent Reservoirs
4. Vortex Mixer
5. Deionized Water
6. Microtiter Plate Reader capable of reading absorbency at 450 nm
7. Orbital Microtiter Plate Shaker
8. Absorbent Paper or Cloth
VII. SAMPLE COLLECTION AND STORAGE
1. To prepare serum samples, whole blood is directly drawn into a centrifuge tube that contains no anti-coagulant. Let blood clot at room temperature for 30 min.
2. Promptly centrifuge the clotted blood at 2,000 to 3,000 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 aliquot and store samples at £ –20oC for later use. For long-term storage, keep at -70 oC. Avoid freeze/thaw cycles.
5. To prepare plasma samples, whole blood should be collected into centrifuge tubes containing enough K3EDTA to achieve a final concentration of 1.735 mg/ml and centrifuged immediately after collection. Observe the same precautions in the preparation of serum samples.
6. If heparin is to be used as an anticoagulant, 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.
1. Allow all the reagents to come to room temperature.
2. Dilute serum or plasma samples 1:500 in 1X Assay Buffer (Sample Diluent). Cellular extract and culture media dilutions will vary.
3. Make Dilution A with 10µl sample to 990µl of 1X Assay Buffer (Sample Diluent) and mix well.
4. Make Dilution B by adding 100µl of Dilution A to 400µl of 1X Assay Buffer (Sample Diluent) and mixing well. Use Dilution B (1:500) for the assay procedure.
IX. STANDARD AND QUALITY CONTROLS PREPARATION
Human Adiponectin Standard Preparation
1. Use care in opening the lyophilized Standard vial. Using an Eppendorf pipette, reconstitute the Human Adiponectin Standard with 0.5 ml distilled or deionized water into the glass vial to give a 200 ng/mL concentration of Standard. Invert and mix gently, let sit for 5 minutes then vortex gently.
2. Label seven tubes 100, 50, 25, 12.5, 6.25, 3.125, 1.56 ng/ml. Add 0.2 ml Assay Buffer (Sample Diluent) to each of the seven tubes. Prepare serial dilutions by adding 0.2 ml of the 200 ng/ml reconstituted standard to the 100 ng/ml tube, mix well and transfer 0.2 ml of the 100 ng/ml reconstituted standard to the 50 ng/ml tube, mix well and transfer 0.2 ml of the 50 ng/ml Standard to the 25 ng/ml tube, mix well and transfer 0.2 ml of the 25 ng/ml Standard to the 12.5 ng/ml tube, mix well and transfer 0.2 ml of the 12.5 ng/ml Standard to the 6.25 ng/ml tube, mix well and transfer 0.2 ml of the 6.25 ng/ml Standard to the 3.125 ng/ml tube, mix well and transfer 0.2 ml of the 3.125 ng/ml Standard to the 1.56 ng/ml tube and mix well.
Note: Do not use a Repeater pipette. Change tip for every dilution. Wet tip with Standard before dispensing. Unused portions of standard should be stored at £ -20°C. Avoid multiple freeze/thaw cycles.