HUMAN TOTAL PROINSULIN
This Human Total Proinsulin ELISA kit is used for the non-radioactive quantification of human total proinsulin in serum and plasma. This kit has 100% cross reactivity to intact human proinsulin and its major processed intermediate, des(31,32) proinsulin, and 81% cross reactivity to its processed intermediate des(64,65) proinsulin in serum and plasma. Human Insulin (up to 200µU/ml) and Human C-Peptide (up to 10ng/ml) do not interfere with the assay result. One kit is sufficient to measure 38 unknown samples in duplicate. This kit has no cross reactivity to human insulin. 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 mouse anti-human antibody to the c-peptide section 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: 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 1:10 with deionized water
D. Human Proinsulin Standards
Human Proinsulin in Buffer: 2, 5, 10, 20, 50, 100 and 200 pM.
Quantity: 0.5ml/bottle
Preparation: Ready to Use
E. ELISA Quality Controls 1 and 2
Purified Recombinant Human Proinsulin in Assay Buffer.
Quantity: 0.5ml/bottle
Preparation: Ready to Use
F. Matrix Solution
Charcoal stripped Proinsulin Depleted Human Serum.
Quantity: 1 ml/vial
Preparation: Ready to Use
G. Assay Buffer
0.025 M Phosphosaline, pH 6.8, containing 0.025 M EDTA, 0.08% Sodium Azide, 1% BSA.
Quantity: 9 ml/vial
Preparation: Ready to Use
H. Human Total Proinsulin Detection Antibody
Pre-titered Biotinylated Monoclonal Mouse anti-Human C-Peptide 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, HRP 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: 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 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. 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 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.
Avoid using samples with gross hemolysis or lipemia.7.
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 HRP 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 60 m l Assay Buffer to the Standard and Quality Control wells.
4. Add 80 m l Assay Buffer to each of the NSB and sample wells.
5. Add 20 m l Matrix Solution to the NSB, Standard, and Quality Control wells (refer to IX for suggested well orientations).
6. Add in duplicate 20 m l Human Proinsulin Standards in the order of ascending concentration to the appropriate wells.
7. Add in duplicate 20 m l QC1 and 20 m l QC2 to the appropriate wells.
8. Add sequentially 20 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 a orbital microtiter plate shaker set to rotate at moderate speed (approximately 400 to 500 rpm).
9. Remove plate sealer and decant solutions from the plate. Tap as before to remove residual solutions in the wells.
10. Wash wells 3 times with diluted HRP Wash Buffer, 300 µl per well per wash. Decant and tap firmly after each wash to remove residual buffer.
11.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).
12. Remove plate sealer and decant solutions from the plate. Tap as before to remove residual solutions in the wells
13.Wash wells 3 times with diluted HRP Wash Buffer, 300 µl per well per wash. Decant and tap firmly after each wash to remove residual buffer.
14.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.
15.Remove sealer, decant solutions from the plate, and tap plate to remove the residual fluid.
16.Wash wells 6 times with diluted HRP Wash Buffer, 300 µl per well per wash. Decant and tap firmly after each wash to remove residual buffer.
17.Add 100µl of substrate solution to each well, cover plate with sealer and shake on the plate shaker for approximately 15-20 minutes.
NOTE: Please be aware that the color may develop more quickly or more slowly than the recommended incubation time depending on the localized room temperature. Please visually monitor the color development to optimize the incubation time.
18.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 to yellow after acidification. Read absorbance at 450 nm in a plate reader within 5 minutes and ensure that there are no air bubbles in any well. If wavelength correction is available, set the instrument to dual wavelength measurement at 450 nm with background wavelength correction set at 590, 600, or 620 nm. If the absorbance readings exceed the limitations of the plate reader, a second reading at 405 nm is needed (reference filter 590, 600 or 620 if available). In this case, proceed to construct a second standard curve as above with the absorbance readings of all Standards at 405 nm. The concentrations of the off-scale samples at 450 nm are than read from the new standard curve. The readings at 405 nm should not replace the on-scale readings at 450 nm.