ImmunoDiagnostics Rat C-Peptide ELISA kit

Reagents Supplied

Each kit is sufficient for one 96-well plate and contains the following components:

1. Microtiter Strips (96 wells) – Coated with a monoclonal antibody against C-peptide, sealed.
2. (10×) Wash buffer, 40ml.
3. Assay buffer, 25ml, ready for use.
4. (100×) Detection antibody solution – A biotin labeled monoclonal antibody against C-peptide (0.12ml).
5. C-peptide standard solutions – 0ng/ml (1ml). 0.15ng/ml, 0.3ng/ml, 0.6ng/ml, 1.2ng/ml and 2.5ng/ml (0.15ml each), ready for use.
6. 200×STP-HRP solution, 0.06ml.
7. Substrate solution, 12ml, ready for use.
8. Stop solution, 12ml, ready for use.
9. Plate cover, 2.

Other Materials Required, But Not Provided

1. Pipettes and pipette tips.
2. 96-well plate or manual strip washer.
3. Buffer and reagent reservoirs.
4. Paper towels or absorbent paper.
5. Plate reader capable of reading absorbency at 450nm.
6. Distilled water or deionized water.
7. Horizontal micro-plate shaker capable of 600rpm.

Storage

The kit should be stored at 2-8°C upon receipt. Remove any unused antibody-coated strips from the micro-plate, return them to the foil pouch and reseal. Once opened, the strips may be stored at 2-8°C for up to one month.

Sample Handling

If a sample has a C-peptide level greater than the highest standard, the sample should be diluted with 0ng/ml C-peptide standard solution and the assay should be repeated.

Calculation

1. Subtract the absorbance of the blank from that of standards and samples.
2. Generate a standard curve by plotting the absorbance obtained (y-axis) against C-peptide concentrations (x-axis). The best fit line can be generated with any curve-fitting software by regression analysis. Log-log curve fitting or curve of 4-parameter can be used for calculation.
3. Determine C-peptide concentration of samples from standard curve.

Assay Principle

This assay is a quantitative sandwich ELISA. The micro-plate is pre-coated with a monoclonal antibody against C-peptide. Standards and samples are added into the wells and co-incubated with a biotin labeled monoclonal antibody. After wash step to remove any unbound reagents, streptavidin-HRP conjugate (STPHRP) is added. After the last wash step, TMB substrate is added and color develops in proportion to the amount of C-peptide bound initially. The assay is stopped and the optical density of the wells determined using a micro-plate reader. Since the increases in absorbance are directly proportional to the amount of captured C-peptide, the unknown sample concentration can be interpolated from a reference curve included in each assay.

Assay Procedure

It is recommended that all standards and samples should be run in duplicate.

1. Add 100µl of 1x Detection antibody solution per well.
2. Add 10µl of standard or sample to its respective well.
3. Seal the plate with a plate cover. Incubate at room temperature for 60 minutes, shaking the plate at 600rpm on a horizontal micro-plate shaker.
4. Discard the content and tap the plate on a clean paper towel to remove residual solution in each well. Add 300µl of 1×Wash buffer to each well. Incubate at room temperature for 30 seconds. Discard the 1× Wash buffer and tap the plate on a clean paper towel to remove residual wash buffer. Repeat the wash step for a total 3 washes.
5. Add 100µl of 1×STP-HRP solution to each well, seal the plate with a plate cover. Incubate at room temperature for 30 minutes, shaking the plate at 600rpm on a horizontal micro-plate shaker.
6. Wash each well 4 times as described in step 2.
7. Add 100µl of Substrate solution to each well, incubate at room temperature for 15 minutes. Protect from light.
8. Add 100µl of Stop solution to each well, gently tap the plate frame for a few seconds to ensure thorough mixing.
9. Measure absorbance of each well at 450nm immediately.

Assay Procedure Summary

Add 100μl of 1× Detection antibody solution per well.
↓
Add 10μl of standard or sample to each well.
↓
Incubate at room temperature for 60 minutes (600rpm).
↓
Wash each well 3 times.
↓
Add 100μl of 1×STP-HRP solution to each well.
↓
Incubate at room temperature for 30 minutes (600rpm).
↓
Wash each well 4 times.
↓
Add 100μl of Substrate solution to each well.
↓
Incubate at room temperature for 15 minutes.
↓
Add 100μl of Stop solution to each well.
↓
Measure absorbance of each well at 450nm.
↓
Calculation

The following standard curve is provided for demonstration only. A standard curve should be generated for each set of sample assay.

C-peptide standard curve (4-parameter)

Insulin is a kind of peptide hormone which is critical for glucose homeostasis. The mature Insulin peptide is derived from Proinsulin, which includes the Insulin A and B chains connected by a peptide fragment (C-peptide). Proinsulin is processed within the endoplasmic reticulum of pancreatic beta cells into equimolar ratios of mature Insulin and C-peptide. Mouse C-peptide 1 is a single chain peptide composed of 29 amino acids, while C-peptide 2 is composed of 31 residues. C-peptide is secreted together with insulin.

The role of C-peptide has been considered to keep the best configuration to form three disulfide bonds, and has no biological activity, however, recent studies indicated that C-peptide can bind, probably, a G-protein coupling specific receptor present on the surface of endothelial cells, kidney microtubule cells and fibroblasts, resulting in activation of calcium-dependent intracellular signaling, activation of Na+-K+-ATPase, and enhancement of NO synthesis. Administration of C-peptide to DM1 samples enhances blood circulation in the skeletal muscle and skin, and also minimizes kidney glomerular hyperfiltration, decreasing albumin excretion into urine, and also improves nervous function, indicating that C-peptide should be given together with insulin to DM1 samples. Important region to bind receptor has been reported to be C-terminal penta peptide.

Click here for more information about ImmunoDiagnostics Limited (IMD)

Search this site for “ImmunoDiagnostics”