MOUSE ADIPONECTIN
This Mouse Adiponectin (ACRP30) ELISA kit is used for the non-radioactive quantification of Mouse Adiponectin in serum, plasma, and adipocyte extracts or cell culture media samples. This kit has 100% cross reactivity to Mouse Adiponectin. There is no binding to Mouse Adiponectin globular domain, or to Rat 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 Mouse Adiponectin molecules from samples to the wells of a microtiter plate coated by a pre-titered amount of anti- mouse adiponectin monoclonal antibodies, and binding of a second biotinylated anti- mouse polyclonal antibody to the captured molecules, 2) wash away of unbound materials from samples, 3) conjugation of horseradish peroxidase to the immobilized biotinylated antibodies, 4) wash away 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 absorbency at 450 nm, corrected from the absorbency at 590nm, after acidification of formed products. Since the increase in absorbency is directly proportional to the amount of captured Mouse 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 Mouse Adiponectin.
Each kit is sufficient to run one 96-well plate and contains the following reagents:
A. Mouse Adiponectin ELISA Plate
Coated with Rat Monoclonal anti-Mouse Adiponectin Antibodies
Quantity: 1 plate
Preparation: Ready to Use
B. Adhesive Plate Sealer
Quantity: 1 sheet
Preparation: Ready to Use
C. 10X HRP Wash Buffer Concentrate
10X concentrate of 50mM Tris Buffered Saline containing Tween-20.
Quantity: Two bottles containing 50 ml each
Preparation: Dilute 1:10 with distilled or deionized water.
D. Mouse Adiponectin Standard
Purified Recombinant Mouse Adiponectin, lyophilized.
Quantity: 1ml upon hydration
Preparation: Contents Lyophilized. Reconstitute with 1 ml distilled or deionized water. The actual concentration of Mouse Adiponectin present in the vial will be lot dependent. Please refer to the analysis sheet for exact concentration present in a specific lot.
E. Quality Controls 1 and 2
Purified Recombinant Mouse Adiponectin in Assay Buffer, lyophilized.
Quantity: 1ml/vial upon hydration
Preparation: Contents Lyophilized. Reconstitute with 1 ml distilled or deionized water.
F. 10X Assay Buffer
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 CONTROL PREPARATION) and in Assay Procedure (SECTION X, ASSAY PROCEDURE).
G. Mouse Adiponectin Detection Antibody
Pre-titered Biotinylated Goat anti-Mouse Adiponectin Polyclonal Antibody
Quantity: 3.0 ml
Preparation: Ready to Use
H. Enzyme Solution
Pre-titered Streptavidin-Horseradish Peroxidase Conjugate in Buffer
Quantity: 12 ml
Preparation: Ready to Use
I. Substrate (Light sensitive, avoid unnecessary exposure to light)
3, 3’, 5, 5’-tetramethylbenzidine in buffer
Quantity: 12 ml
Preparation: Ready to Use.
J. 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, Adiponectin Standards, Controls 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.
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.
Promptly centrifuge the clotted blood at 2,000 to 3,000 x g for 15 minutes at 4 ± 2oC.
Transfer and store serum samples in separate tubes. Date and identify each sample.
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.
2. 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.
3. 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.
4. Avoid using samples with gross hemolysis or lipemia.
1. Allow all the reagents to come to room temperature.
2. Dilute serum or plasma samples 1:1000 in 1X Assay Buffer (See Section III, F). Cellular extract and culture media dilutions will vary.
a. Make Dilution A by adding 10 µl sample to 990 µl Assay Buffer and vortex.
b. Make Dilution B by adding 100 µl of Dilution A to 900 µl Assay Buffer and vortexing. Use Dilution B (1:1000) for the assay procedure.
IX. STANDARD AND QUALITY CONTROLS PREPARATION
A. Mouse Adiponectin Standard Preparation
1. Use care in opening the lyophilized Standard vial. Using an Eppendorf pipette, reconstitute the Mouse Adiponectin Standard with 1.0 ml distilled or deionized water into the glass vial to give a concentration prescribed in the analysis sheet. Invert and mix gently, let sit for 5 minutes then mix well.
2. Label seven tubes 1, 2, 3, 4, 5, 6, and 7. Add 0.5 ml Assay Buffer to each of the seven tubes. Prepare serial dilutions by adding 0.5 ml of the reconstituted standard to tube 1, mix well and transfer 0.5 ml of tube 1 to tube 2, mix well and transfer 0.5 ml of tube 2 to tube 3, mix well and transfer 0.5 ml of tube 3 to tube 4, mix well and transfer 0.5 ml of tube 4 to tube 5, mix well and transfer 0.5 ml of tube 5 to tube 6, mix well and transfer 0.5 ml of tube 6 to tube 7 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.