HUMAN RESISTIN
This Human Resistin ELISA kit is used for the non-radioactive quantification of human resistin in serum, plasma and other biological media. One kit is sufficient to measure 38 unknown samples in duplicate. This kit is for research purposes only.
This assay is based, sequentially, on: 1) capture of human resistin from sample by a monoclonal antibody, immobilized in the wells of a microwell plate, 2) washing off unbound materials including free materials from samples, 3) binding of the biotinylated monoclonal human resistin antibody to the other side of captured human resistin molecules, 4 ) conjugation of SA-HRP (Poly-HRP-labeled streptavidin) enzyme to the biotinylated antibodies, and 5) quantification of bound detection conjugate by monitoring SA-HRP enzyme activity in the presence of TMB (tetramethylbenzidine) substrates. The enzyme activity is measured spectrophotometrically by the absorbency at 450 nm due to production of the photometric product. Since the amount of photometric product is directly proportional to the concentration of human resistin 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 resistin.
Each kit is sufficient to run one 96-well microtiter plate and contains the following reagents:
A. Human Resistin ELISA Plate
Coated with anti- Human Resistin Monoclonal 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 TBS Buffer containing 0.05%Tween 20
Quantity: 2 bottles containing 50 ml each
Preparation: Dilute 1:10 with distilled or deionized water
D. Human Resistin Standards
Human Resistin, lyophilized.
Quantity: 2.5 ng/0.25 ml (10 ng/ml) upon hydration.
Preparation: Contents Lyophilized. Reconstitute with 250 μL distilled or deionized water to obtain 10.0 ng/ml.
E. Human Resistin Quality Controls 1 and 2
One vial each, lypholized, containing Human Resistin in Assay Buffer.
Quantity: 0.25 ml/vial upon hydration.
Preparation: Contents Lyophilized. Reconstitute each vial with 250 μL distilled or deionized water.
F. Assay Buffer
0.05M PBS, pH 7.4, containing 0.025 M EDTA, 0.08% Sodium Azide and 1% BSA.
Quantity: 12 ml
Preparation: Ready to use
G. Human Resistin Detection Antibody
Biotinylated anti-human resistin monoclonal antibody
Quantity: 9 ml
Preparation: Ready to use
H. Enzyme Solution
Pre-titered Streptavidin-Horseradish Peroxidase Conjugate (SA-HRP)
Quantity: 12 ml
Preparation: Ready to use
I. Substrate (Light sensitive, avoid unnecessary exposure to light)
3, 3’, 5, 5’-tetramethylbenzidine (TMB)
Quantity: 12 ml
Preparation: Ready to use
J. Stop Solution (Caution: Corrosive Solution)
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, Standards, Controls and reconstituted Standards and Controls at £ –20oC. Minimize repeated freeze and thaw of the 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.
Sodium Azide
Sodium Azide has been added to 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.
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.
VI. MATERIALS REQUIRED BUT NOT PROVIDED
1. Pipet with tips, 10m l-200m l
2. Multi-channel pipette, 50m l-300m l
3. Reagent reservoirs
4. Vortex mixer
5. Refrigerator
6. Deionized water
7. Microtiter plate reader capable of reading absorbency at 450 nm
8. Microtiter plate shaker
9. 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 minutes. 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 1:10 in Assay Buffer. Cellular extract and culture media dilutions will vary.
IX. STANDARD AND QUALITY CONTROLS PREPARATION
Human Resistin Standard Preparation
1. Use care in opening the lyophilized Standard vial. Using a pipette, reconstitute the Human Resistin Standard with 250 μL distilled or deionized water into the glass vial to give a 10 ng/mL concentration of Standard. Invert and mix gently, let sit for 5 minutes then vortex gently.
2. Label six tubes 5, 2.5, 1.25, 0.625, 0.312, and 0.16 ng/ml. Add 100 μL Assay Buffer to each of the six tubes. Prepare serial dilutions by adding 100 μL of the 10 ng/ml reconstituted standard to the 5 ng/ml tube, mix well and transfer 100 μL of the 5 ng/ml reconstituted standard to the 2.5 ng/ml tube, mix well and transfer 100 μL of the 2.5 ng/ml Standard to the 1.25 ng/ml tube, mix well and transfer 100 μL of the 1.25 ng/ml Standard to the 0.625 ng/ml tube, mix well and transfer 100 μL of the 0.625 ng/ml Standard to the 0.312 ng/ml tube, mix well and transfer 100 μL of the 0.312 ng/ml Standard to the 0.16 ng/ml tube and mix well.