HUMAN C-PEPTIDE
This kit is for non-radioactive quantification of Human C-Peptide (HCP) 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 C-Peptide from samples by a monoclonal antibody immobilized to the wells of a microtiter plate, 2) binding of the biotinylated monoclonal HCP antibody to capture Human C-Peptide molecules, 3) wash away of unbound materials including free materials from samples and free detection antibody, 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 C-Peptide 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 HCP.
Each kit is sufficient to run one 96-well microtiter plate and contains the following reagents:
A. Human C-Peptide ELISA Plate
Coated with anti-Human C-Peptide Monoclonal Antibody
Quantity: 1 plate
Preparation: Ready to use
Note: Unused strips should be resealed in the foil pouch with the desiccant provided and stored at 2-8°C.
B. Adhesive Plate Sealer
Quantity: 1 Sheet
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 deionized water
D. Human C-Peptide Standards
Human C-Peptide in Assay Buffer: 0.2, 0.5, 1, 2, 5, 10, and 20 ng/ml
Quantity: 0.5 ml/vial
Preparation: Ready to use
E. ELISA Human C-Peptide Quality Controls 1 and 2
Human C-Peptide in QC Buffer
Quantity: 0.5 ml/vial
Preparation: Ready to use
F. Matrix Solution
Quantity: 1 ml
Preparation: Ready to use
G. Assay Buffer
0.05M PBS containing 0.025 M EDTA, 1% BSA, with 0.08% Sodium Azide, with proprietary reagent, pH 7.4
Quantity: 8 ml
Preparation: Ready to use
H. Human C-Peptide Detection Antibody
Biotinylated anti-Human C-Peptide Monoclonal Antibody
Quantity: 3.0 ml
Preparation: Ready to use
I. Enzyme Solution
Pre-titered Streptavidin-Horseradish Peroxidase Conjugate (SA-HRP)
Quantity: 12 ml
Preparation: Ready to use
J. Substrate (TMB)
3, 3’, 5, 5’-tetramethylbenzidine (TMB)
Quantity: 12 mL
Preparation: Ready to use
K. ELISA Stop Solution
0.3M HCl
Quantity: 12 ml
Preparation: Ready to use
All components of the kit should be stored at 2-8° C upon receipt. For prolonged storage (> 2 weeks), store the Wash Buffer, Standards and Matrix Solution at ≤ -20° C. Unused strips should be resealed in the foil pouch with the desiccant provided and stored at 2-8°C.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 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 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.
VI. MATERIALS REQUIRED BUT NOT PROVIDED
1. Pipets with tips, 10m l-200m l
2. Multi-channel pipette, 50m l-300m l
3. Buffer and 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. Human C-Peptide must be protected from proteolysis during assay procedures and sample storage. Trasylol (Aprotinin) at a concentration of 500 KIU per ml of serum or plasma should be added to samples to protect from proteolysis.
2. 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 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.
3. Samples can be stored at 4°C if they will be tested within 3 hours of collection. For longer storage, specimens should be stored at £ -20°C. Avoid multiple (>3) freeze/thaw cycles. Aliquot samples before freezing if necessary.
4. 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.
5. 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.
6. Avoid using samples with gross hemolysis or lipemia.
Pre-warm all reagents to room temperature (20-25° C) immediately before setting up the assay.
1. Dilute the concentrated 10X Wash Buffers 10 fold. Mix the entire contents of both bottles of 10X Wash Buffer with 900 ml distilled or deionized water.
2. Remove the required number of strips from the Microtiter Assay Plate. Unused strips should be resealed in the foil pouch and stored at 2-8°C. Assemble the strips in an empty plate holder and fill each well with 300 µl of 1X Wash Buffer. Incubate at room temperature for 5 minutes. 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.
3. Wash the wells two additional times with 1X Wash Buffer, 300 µl per well per wash. Decant and tap after each wash to remove residual buffer.
4. Add 40 m l of Assay Buffer into each Blank, Standard, and QC well. Refer to Section IX for suggested well orientations.
5. Add 50 m l of the Assay Buffer into each Sample well.
6. Add 10 m l Assay Buffer to assay background well, i.e. the blank wells #A1 and #B1.
7. Add 10 m l of Matrix Solution into each Blank, Standard, and QC well.
8. Add 10 m l of Standards in duplicate into appropriate wells.
9. Add 10 m l of QC1 and QC2 in duplicate into appropriate wells.
10. Add 10 m l of serum or plasma samples in duplicate into appropriate wells.
11. Add 20 m l of the Detection Antibody into each well. For best results all additions should be completed within one hour.
12. Cover the plate with plate sealer.
13. Incubate at room temperature (20-25°C) for 2 hours while shaking on a microtiter plate shaker.
14. Remove plate sealer and decant solutions from the plate. Tap as before to remove residual solutions in the well.
15. Wash the wells 5 times with 1X Wash Buffer, 300 µl per well per wash. Decant and tap after each wash to remove residual buffer.
16. Add 80 m l of the Enzyme Solution into each well.
17. Cover the plate with plate sealer. Incubate 30 minutes at room temperature while shaking on a microtiter plate shaker.
18. Wash the wells 5 times with 1X Wash Buffer, 300 µl per well per wash. Decant and tap after each wash to remove residual buffer.
19. Add 80 µl of the Substrate Solution to each well, cover plate with sealer and shake on the plate shaker for approximately 12 to 19 minutes. Blue color should be formed in wells of Human C-Peptide standards with intensity proportional to increasing concentrations of Human C-Peptide.
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.
20. Remove the plate sealer, and stop the reaction by adding 80 m l of Stop Solution into each well of the plate. Shake the plate by hand to ensure complete mixing of solution in all wells. The blue color should turn to yellow after acidification.
21. Read absorbance at 450 nm in a plate reader within 5 minutes and ensure that there is no air bubbles in any well. The absorbance of highest Human C-Peptide standard should be approximately 2.4 ~ 2.8
The dose-response curve of this assay fits best to a sigmoidal 5-parameter logistic equation. The results of unknown samples can be calculated with any computer program having a 5-parameter logistic function.
Sensitivity
The lowest level of Human C-Peptide that can be detected by this assay is 0.2 ng/mL.
Crossreactivity
The specificity of the Human C-Peptide ELISA is the ability to selectively measure the analytes in the presence of other like components in the sample matrix.
| Human C-Peptide | 100% |
| Intact Human Proinsulin | 63% |
| Human Insulin | n.d.* |
| Porcine C-Peptide | n.d.* |
| Rat C-Peptide | n.d.* |
| Canine C-Peptide | n.d.* |
n.d.: Not detectable at concentrations up to 200 ng/ml.
Precision
Within and Between Assay Variation
|
Sample |
Mean HCP Levels |
Within |
Between |
|
1 |
1 |
4.75 | 8.72 |
|
2 |
3 |
2.95 | 5.00 |
|
3 |
7 |
1.60 | ---- |
The between assay variation of Linco Human C-Peptide ELISA kits were studied using two serum samples with varying concentrations of Human C-Peptide. The mean variation of each sample was calculated using results from eight separate assays with duplicate samples in each assay.
Recovery
Spike & Recovery of Human C-Peptide in Human Serum
|
Sample # |
HCP added ng/mL |
Expected ng/mL |
Observed ng/mL |
% of Recovery |
| 1 |
0 |
2.31 |
2.31 |
100 |
|
0.5 |
2.81 |
2.83 |
104 |
|
|
2.0 |
4.31 |
4.21 |
95 |
|
|
5.0 |
7.31 |
7.70 |
108 |
|
| 2 |
0 |
1.68 |
1.68 |
100 |
|
0.5 |
2.18 |
2.19 |
101 |
|
|
2.0 |
3.68 |
3.57 |
93 |
|
|
5.0 |
6.68 |
6.77 |
102 |
|
| 3 |
0 |
2.05 |
2.05 |
100 |
|
0.5 |
2.55 |
2.50 |
98 |
|
|
2.0 |
4.05 |
3.92 |
91 |
|
|
5.0 |
7.05 |
6.93 |
97 |
|
| 4 |
0 |
3.84 |
3.84 |
100 |
|
0.5 |
4.34 |
4.34 |
100 |
|
|
2.0 |
5.84 |
5.87 |
102 |
|
|
5.0 |
8.84 |
9.34 |
111 |
|
Varying concentrations of Human C-Peptide were added to four human serum samples and the Human C-Peptide content was determined by ELISA. Mean of the observed levels from four duplicate determinations are shown. Percent recovery = observed ÷ expected x 100%.
Linearity
Effect of Serum Dilution
|
Sample |
Sample |
Expected |
Observed |
% of |
|
1 |
0 |
3.38 |
3.38 |
100 |
| 2 |
1.69 |
1.83 |
108 | |
| 4 |
0.85 |
0.88 |
104 | |
| 8 |
0.42 |
0.43 |
102 | |
|
2 |
0 |
7.24 |
7.24 |
100 |
| 2 |
3.62 |
3.17 |
88 |
|
| 4 |
1.81 |
1.65 |
92 |
|
| 8 |
0.91 |
0.88 |
98 |
|
|
3 |
0 |
8.42 |
8.42 |
100 |
| 2 |
4.21 |
3.60 |
86 |
|
| 4 |
2.11 |
2.10 |
100 |
|
| 8 |
1.05 |
1.01 |
96 |
|
Dilutions of human sera containing varying concentrations of Human C-Peptide were analyzed. The mean Human C-Peptide level and percent of expected from four duplicates determinations are shown.