INTACT HUMAN PROINSULIN
This Intact Proinsulin ELISA kit is used for the non-radioactive quantification of intact human proinsulin in serum and plasma. This kit does not cross-react with human insulin as high as 10 nM. In addition, presence of insulin (up to 208µU/ml) in serum or plasma does not interfere with the assay result. The kit has no cross-reactivity with the major species of proinsulin metabolites des(31,32) proinsulin while cross-reacts only weakly with the minor intermediate des(64,65) proinsulin. One kit is sufficient to measure 37 unknown samples in duplicate. This kit is for research purpose only.
This assay is a Sandwich ELISA based, sequentially, on: 1) capture of human proinsulin molecules from samples to the wells of a microtiter plate coated by a pre-titered amount of polyclonal guinea pig anti-human insulin antibodies, 2) wash away of unbound materials from samples, 3) binding of a second biotinylated monoclonal antibody specific to the B chain-C peptide junctional sequences of the captured molecules , 4) wash away of unbound materials from samples, 5) conjugation of horseradish peroxidase to the immobilized biotinylated antibodies, 6) wash away of free enzyme, and 7) quantification of immobilized antibody-enzyme conjugates by monitoring horseradish peroxidase activities in the presence of the substrate 3,3’,5,5’-tetra-methylbenzidine. 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 human total proinsulin 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 proinsulin.
Each kit is sufficient to run one 96-well plate and contains the following reagents:
A. Human Proinsulin ELISA Plate
Coated with pretitered guinea pig anti -human insulin antibodies.
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. Intact Proinsulin Standards
Human Proinsulin in Buffer: 0.5, 1, 2, 5, 10, 20, 50, and 100 pM.
Quantity: 0.5ml/bottle
Preparation: Ready to Use
E. Quality Controls 1 and 2
Purified Recombinant Human Proinsulin in Buffer.
Quantity: 0.5ml/bottle
Preparation: Ready to Use
F. Matrix Solution
Charcoal stripped pooled human Serum.
Quantity: 1.5 ml/vial
Preparation: Ready to Use
G. Assay Buffer
0.025 M Phosphate Buffer, pH 6.8, containing 0.025 M EDTA, 0.08% Sodium Azide, 1% BSA.
Quantity: 9 ml/vial
Preparation: Ready to Use
H. Human Intact Proinsulin Detection Antibody
Pre-titered biotinylated mouse monoclonal antibody.
Quantity: 12 ml/vial
Preparation: Ready to Use
I. Enzyme Solution
Pre-titered Streptavidin-Horseradish Peroxidase Conjugate in Buffer.
Quantity: 12 ml/vial
Preparation: Ready to Use
J. Substrate
3, 3’,5,5’-tetramethylbenzidine in Buffer.
Quantity: 12 ml/vial
Preparation: Ready to Use.
Minimize exposure to light.
K. Stop Solution
0.3 M HCl
Quantity: 12 ml/vial
Preparation: Ready to Use
Caution: Corrosive Solution
Prior to use, all components in the kit can be stored up to 2 weeks at 2 - 8°. For longer storage (> 2 weeks), freeze Assay Buffer, Wash Buffer, Matrix Solution, Proinsulin Standards and Quality Controls at £ –20oC. Minimize repeated freeze and thaw of the Proinsulin 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. Pipettes and Pipette Tips: 20 m l ~ 100 m l
2. Multi-Channel Pipettes and Pipette Tips: 50 m l ~ 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 and 590nm
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 Vacutainer® serum tube that contains no anticoagulant. Immediately add enough aprotinin to a final concentration of 500 KIU/ml. Mix well and 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 store samples in aliquots at £ –20oC for later use. Avoid repeated freeze/thaw cycles.
5. To prepare plasma samples, whole blood should be collected into Vacutainer® EDTA-plasma tubes and placed on ice. Immediately add enough aprotinin to a final concentration of 500 KIU/ml, mix well and centrifuge at 2,000 to 3,000 x g for 15 min at 4 ± 2oC. Observe the same precautions in the preparation of serum samples.
6. Other protease inhibitors or cocktails of inhibitors may be used instead of aprotinin, but the optimal concentrations to prevent degradation of intact proinsulin should be pre-determined.
7. 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.
8. Avoid using samples with gross hemolysis or lipemia.
Pre-warm all reagents to room temperature immediately before setting up the assay.
1. Dilute the 10X Wash Buffer concentrate 10 fold by mixing the entire content of each bottle of Wash Buffer with 450 ml deionized water. (dilute both bottles with 900 ml deionized water).
2. Remove the Microtiter Assay Plate from the foil pouch and fill each well with 300 m l of diluted Wash Buffer. Decant Wash Buffer and remove the residual amount from all wells by inverting the plate and tapping it smartly onto absorbent towels several times. Wash assay plate using this procedure 3 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 50 m l Matrix Solutions to Blank, Standards and Quality Control wells (refer to IX for suggested well orientations)..
4. Add 50 m l Assay Buffer to each of the Blank and sample wells.
5. Add in duplicate 50 m l Human Proinsulin Standards in the order of ascending concentration to the appropriate wells.
6. Add in duplicate 50 m l QC1 and 50 m l QC2 to the appropriate wells.
7. Add sequentially 50 m l of the unknown samples in duplicate to the remaining wells. For best result all additions should be completed within one hour. Cover the plate with plate sealer and incubate at room temperature for 1 hour on an orbital microtiter plate shaker set to rotate at moderate speed (approximately 400 to 500 rpm).
8. Remove plate sealer and decant solutions from the plate. Tap as before to remove residual solutions in the wells.
9. Wash wells 3 times with diluted Wash Buffer, 300 µl per well per wash. Decant and tap firmly after each wash to remove residual buffer.
10. Transfer Detection Antibody solution to a reagent reservoir and add 100 µl of this solution to each well with a multi-channel pipette. Cover the plate with plate sealer and incubate at room temperature for 1 hour on an orbital microtiter plate shaker set to rotate at moderate speed (approximately 400 to 500 rpm).
11. Remove plate sealer and decant solutions from the plate. Tap as before to remove residual solutions in the wells
12. Wash wells 3 times with diluted Wash Buffer, 300 µl per well per wash. Decant and tap firmly after each wash to remove residual buffer.
13. Add 100 µl Enzyme Solution to each well. Cover the plate with sealer and incubate with moderate shaking at room temperature for 30 minutes on the microtiter plate shaker.
14. Remove sealer, decant solutions from the plate, and tap plate to remove the residual fluid.
15. Wash wells 6 times with diluted Wash Buffer, 300 µl per well per wash. Decant and tap firmly after each wash to remove residual buffer.
16. Add 100 µl of Substrate Solution to each well, cover plate with sealer and shake in the plate shaker for 15 ~ 25 minutes. Blue color should be formed in wells of reference standards with intensity proportional to increasing concentrations of proinsulin. 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. Alternatively, the increase in blue color can be monitored at 630 nm wavelength and the stop solution to be added when the absorbance of the highest standard wells reached 0.8 to 0.9. The optimal time for substrate incubation may vary among laboratories.