RAT/MOUSE INSULIN
This Rat / Mouse Insulin ELISA kit is used for the non-radioactive quantification of insulin in mouse and rat sera. Plasma samples may also be used but application to samples of other biological fluids may need validation by the user. One kit is sufficient to measure 39 unknown samples in duplicate. This kit is for research purpose only.
This assay is a Sandwich ELISA based, sequentially, on: 1) capture of insulin molecules from samples to the wells of a microtiter plate coated by pre-titered amount of a monoclonal mouse anti-rat insulin antibodies and the binding of biotinylated polyclonal antibodies to the captured insulin, 2) wash away of unbound materials from samples, 3) binding 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 insulin 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 rat insulin.
Each kit is sufficient to run one 96-well plate including, in duplicates, background, 6 rat insulin standards, 2 quality controls and 39 unknown samples.
A. Rat/Mouse Insulin ELISA Plate
Coated with mouse monoclonal anti-rat insulin 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 50 mM Tris Buffered Saline containing Tween-20.
Quantity: Two bottles containing 50 ml each
Preparation: Dilute 10 times with de-ionized water.
D. Rat/Mouse Insulin Standards
Rat insulin in Assay Buffer: 0.2, 0.5, 1, 2, 5 and 10 ng/ml.
Quantity: 0.1 ml/vial
Preparation: Ready to use.
E. Rat/Mouse Insulin Quality Controls 1 and 2
Rat insulin in QC buffer.
Quantity: 0.1 ml/vial
Preparation: Ready to use.
F. Matrix Solution
Charcoal stripped pooled mouse serum
Quantity: 0.5 ml
Preparation: Ready to use.
G. Assay Buffer
0.05 M phosphosaline, pH 7.4, containing 0.025 M EDTA, 0.08% sodium azide, and 1% BSA.
Quantity: 20 ml
Preparation: Ready to use.
H. Rat/Mouse Insulin Detection Antibody
Pre-titered biotinylated anti-insulin antibody.
Quantity: 10 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
0.3 M HCl
Quantity: 12 ml
Preparation: Ready to use.
IV. STORAGE AND STABILITY
Prior to use, all components in the kit can be stored up to 2 weeks at 2 - 8°. For longer storage (> 2 weeks), freeze diluted Wash Buffer, Insulin Standards, Quality Controls, and Matrix Solution at < 20oC. Minimize repeated freeze and thaw of the Insulin Standards, Quality Controls and Matrix Solution. 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. 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 eye. Do not swallow or ingest.
VI. MATERIALS REQUIRED BUT NOT PROVIDED
1. Pipette with tips, 10m l - 100 m l.
2. Multi-channel Pipette: 50 ~ 300 m l
3. Reagent Reservoirs
4. Vortex Mixer
5. Refrigerator
6. De-ionized Water
7. Microtiter Plate Reader capable of reading absorbency at 450 nm and 590nm
8. Orbital Microtiter Plate Shaker
9. Absorbent Paper or Cloth
VII. SAMPLE COLLECTION AND STORAGE
A. To prepare serum, whole blood is directly drawn into a centrifuge tube that contains no anti-coagulant. Let blood clot at room temperature for 30 min.
B. Promptly centrifuge the clotted blood at 2,000 to 3,000 x g for 15 minutes at 4 ± 2oC.
C. Transfer and store serum samples in separate tubes. Date and identify each sample.
D. Use freshly prepared serum or aliquot and store samples at –20 ± 5oC for later use. For long-term storage, keep at -70 oC. Avoid freeze/thaw cycles.
E. To prepare plasma sample, 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 same precautions in the preparation of serum samples.
F. If heparin is to be used as anti-coagulant, the effect on the assay outcome at the dose of heparin used should be pre-determined.
G. Avoid using samples with gross hemolysis or lipemia.
Pre-warm all reagents to room temperature prior to setting up assay.
1. Dilute the 10X Wash Buffer concentrate 10 fold by mixing the entire content of each bottle of Wash Buffer with 450ml de-ionized water. (dilute both bottles with 900 ml deionized water)
2. Remove the Microtiter Assay Plate from the foil pouch and wash each well 3 times with 300 m l of diluted Wash Buffer per wash. 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 automated machine is used for assay, follow the manufacturer’s instructions for all washing steps described in this protocol.
3. Add 10 µl Assay Buffer to the NSB wells and to each of the sample wells. Refer to Section IX for suggested well orientations.
4. If samples to be assayed are serum or plasma, add 10 m l Matrix Solution to the NSB, Standard, and Control wells (Option A). If samples are free of significant serum matrix components, add 10 m l Assay Buffer instead (Option B).
5. Add in duplicate 10 m l Rat Insulin Standards in the order of ascending concentration to the appropriate wells.
6. Add 10 m l QC1 and 10 m l QC2 to the appropriate wells.
7. Add sequentially 10 m l samples of the unknown samples in duplicates to the remaining wells.
8. Add 80 µl Detection Antibody to all wells. For best result all additions should be completed within one hour. Cover the plate with plate sealer and incubate at room temperature for 2 hours on a orbital microtiter plate shaker set to rotate at moderate speed, about 400 to 500 rpm.
9. Remove plate sealer and decant solutions from the plate. Tap as before to remove residual solutions in well.
10. Wash wells 3 times with diluted Wash Buffer, 300 µl per well per wash. Decant and tap after each wash to remove residual buffer.
11. Add 100 µl Enzyme Solution to each well. Cover plate with sealer and incubate with moderate shaking at room temperature for 30 min on the microtiter plate shaker.
12. Remove sealer, decant solutions from the plate and tap plate to remove the residual fluid.
13. Wash wells 6 times with diluted Wash Buffer, 300 µl per well per wash. Decant and tap after each wash to remove residual buffer.
14. Add 100 µl of Substrate Solution to each well, cover plate with sealer and shake in the plate shaker for 15 minutes. Blue color should be formed in wells of Insulin Standards with intensity proportional to increasing concentrations of insulin. Remove sealer and add 100 µl Stop Solution [CAUTION: CORROSIVE SOLUTION] and shake plate by hand to ensure complete mixing of solution in all wells. The blue color should turn into yellow after acidification. Read absorbance at 450 nm and 590 nm in a plate reader within 5 minutes and ensure that there is no air bubbles in any well. Record the difference of absorbance units.