TECO® Intact Proinsulin ELISA

TECO® Intact Proinsulin ELISA is a biomarker for advanced beta-cell dysfunction and research of insulin resistance.

Proinsulin is produced in the pancreatic ß-cells and is normally further processed to insulin and C-peptide. It is only seen in low concentrations in the plasma of normal samples. An increase in the insulin demand, as provided by insulin resistance in later stages of type 2 diabetes mellitus research, can result in increased expression of proinsulin into the sample. The intact molecule and its degradation products are known to block fibrinolysis because of plasminogen-activator inhibitor (PAI-1) stimulation.

• Diabetes II
• Staging of insulin resistance and b-cell dysfunction
• Identification of high risk subjects for CAD
• Polycystic ovary Syndrome (PCOS)
• Insulinoma

Reagents and materials supplied

Symbol	Description	Format
1	Intact Proinsulin Antibody Coated Microtiter Plate 12 strips of 8 wells (96 breakable wells in total), in a frame, Ready to use	1 plate
2	Blocking Buffer Ready to use	1 x 1.5 ml
3	Antibody-HRP Conjugate Ready to use	1 x 11 ml
4	TMB Substrate Ready to use	2 x 15 ml
5	Wash Solution 10 times concentrated	1 x 40 ml
6	Stop Solution – 0.5 M H2SO4 0.5 M sulfuric acid, ready to use	1 x 15 ml
A	Standard A 0 pmol/L, lyophilized	2 x 3.0 ml
B	Standard B lyophilized, Concentration see data sheet	1 x 1.0 ml
C	Standard C lyophilized, Concentration see data sheet	1 x 1.0 ml
D	Standard D lyophilized, Concentration see data sheet	1 x 1.0 ml
E	Standard E lyophilized, Concentration see data sheet	1 x 1.0 ml
F	Standard F lyophilized, Concentration see data sheet	1 x 1.0 ml
L	Control 1 lyophilized, Range see data sheet	1 x 1.0 ml
H	Control 2 lyophilized, Range see data sheet	1 x 1.0 ml
I	Kit Instruction	1 x

Materials Required and not Supplied

• Pipettes capable of dispensing 50 µl, 100 µl, 150 µl and 300 µl
• Graduated cylinders for reconstituting or diluting reagents
• Manual aspiration system and multi-channel pipette or automatic washer
• Aqua dest
• Vortex mixer
• ELISA plate reader suitable for 96 well formats and capable of measuring at 450 and 405 nm and with 590-650 for reference
• ELISA plate shaker (400 rpm) (orbital shaker)
• Software package for data reduction and analysis

Assay Principle

The TECO® human Proinsulin ELISA Kit is a sensitive two-site sandwich enzyme-linked immunosorbent assay. The microtiter plates are coated with a monoclonal antibody (S2) specific for an epitope at the C-peptide/insulin A chain junction. The antibody is able to bind intact proinsulin, des (31,32)-proinsulin and split (32,33)- proinsulin but not insulin, C-peptide and the other “des” and “split” forms.

First, a blocking buffer is added to the allocated wells. An aliquot of patient sample is then added to the wells. After incubation, the wells are washed to remove unbound antibody and other serum compounds. In a second incubation time, an enzyme labelled monoclonal proinsulin antibody is added. This antibody is specific for the epitopes at insulin β chain/C-peptide junction. S53 is able to bind to intact proinsulin, des (64,65)- proinsulin and split (65,66)- proinsulin but not insulin, C-peptide and other “des” and “split” forms. The combination of these two monoclonal antibodies has the ability to detect only the intact human proinsulin.

After washing, the remaining oder bound enzyme activity is measured by adding a chromogenic substrate. The intensity of colour development is proportional to the concentration of proinsulin in the patient sample.

Assay Procedure

In order to obtain an optimal differentiation in the cut-off range (11 pmol/l) it is recommended to use standards  A  till  E  (0~60 pmol/l) and to measure the absorption at 450 nm with a reference filter of 590–650 nm. A second measurement of standards  A  till  F  (0~100 pmol/l) can be done at 405 nm with a reference filter of 590–650 nm.

Allow all reagents to stand at room temperature (20–25 °C) for at least 30 minutes.

1. Prepare the frame and the required number of coated strips  1 . Allocate the wells of the microtiter plate for standards, controls and samples.
2. Pipette 50 µl of blocking buffer working solution  2  directly into the bottom of the wells.
3. Pipette 50 µl of each standards  A  till  F , controls 1 and 2 ( L  and  H ) and samples into the corresponding wells.
4. Cover the strips and incubate for 60 minutes at room temperature (20–25 °C) on an orbital shaker (400 rpm).
5. After incubation, aspirate the wells by using a plate washer or manually decant by inverting the plate. Wash the wells 3 x with 300 ml diluted washing buffer. After the last wash cycle tap the inverted wells gently on a dry absorbent surface to remove excess wash solution.
6. Add 100 µl of HRP conjugate  3  into the wells.
7. Cover the strips and incubate for 60 minutes at room temperature (20–25 °C) on an orbital shaker (400 rpm).
8. Repeat wash step 5.
9. Pipette 150 µl of TMB substrate  4  into the wells and incubate for 15–25 minutes at room temperature on an orbital shaker (400 rpm).
10. Add 100 µl of stop solution  6  into the wells, shake for 5 seconds on a plate shaker and read the absorbance within 15 minutes.
11. Read the absorbance of the wells (450, 405 nm). Reference filter at 590–650 nm.
12. If dilution of samples is required, dilution should be done with zero standard (recommended dilution 1:4).

Protocols for the different automatic ELISA systems are available.

Range, Sensitivity & Specificity

Range
~ 3 – 100 pmol/l

Sensitivity
0.3 pmol/l

Specificity
No cross-reactivity has been observed:

Human Insulin	< 10 000 pmol/l
Human C-Peptide	50 000 pmol/l
Des (31,32) – Proinsulin	< 200 pmol/l
Split (32,33) – Proinsulin	5000 pmol/l
Des (64,65) – Proinsulin*	200 pmol/l
Split (65,66) – Proinsulin	1000 pmol/l

* not present in Serum and Plasma samples

Sample

Sample volume
50 µl

Sample type
Serum, EDTA / Heparin plasma, cell culture

Sample preparation

• Fasting blood sample collection.
• Due to higher stability, EDTA or heparin plasma samples are preferred to serum samples.
• Plasma: the sample collection can take place in HbA1C-tubes.
• These samples are stable at room temperature and should be centrifuged within 48 hours. Plasma should be used in the assay or can be stored in aliquots, stable > 2 years at -20 °C.
• Serum: centrifuge whole blood within 4 hours. Proteases degrade intact proinsulin in serum, do not store longer than 1 day at 2-8 °C.
• Serum should be used in the assay or can be stored in aliquots at -20 °C.
• Avoid repeated freeze/thaw cycles.

Incubation time
2.5 hours

Species
Human

Reference values

After fasting: mean 3.99 pmol/l +/- 1.58 SD

≤ 11 pmol/l (normal secretion)
> 11 pmol/l (dysfunction of secretion)

Background

Proinsulin is produced in the pancreatic ß-cells and is normally further processed to insulin and C-peptide. It is only seen in low concentrations in the plasma of healthy subjects. An increase in the insulin demand, as provided by insulin resistance in later stages of type 2 diabetes mellitus, can result in increased expression of proinsulin into the blood. Intact proinsulin is rapidly degraded, but is considered to be an independent cardiovascular risk factor. The intact molecule and its degradation products are known to block fibrinolysis because of plasminogenactivator inhibitor (PAI-1) stimulation.

Fasting morning intact proinsulin can be used as highly specific indicator of insulin resistance and to research the effect on ß-cell dysfunction. Subjects with type 2 diabetes mellitus and with elevated fasting intact proinsulin levels should be regarded as insulin resistant.

Elevated fasting intact proinsulin levels may also be seen in subjects with insulinoma, a benign insulin producing tumor of the pancreas.