TECO® Intact Proinsulin ELISA

The TECO® Intact Proinsulin ELISA enables the specific detection of human intact proinsulin in serum, plasma, and cell culture samples. Intact proinsulin is a biomarker of pancreatic beta cell stress and dysfunction for use in the research of type 1 diabetes (T1D), type 2 diabetes (T2D), insulinoma, insulin resistance, and diabetes drug development.

Proinsulin is produced in the specialized beta cells of pancreatic islets. Normally, proinsulin is processed into insulin and C-peptide, which are secreted into the bloodstream of healthy individuals at low concentrations. However, under conditions of beta cell stress, insulin processing enzymes become deficient in their ability to effectively cleave proinsulin. This results in the accumulation and subsequent release of proinsulin into circulation. Elevated levels of intact proinsulin in the bloodstream indicate beta cell dysfunction, which may stem from insulin resistance, autoimmune attack on beta cells, or other damaging effects on pancreatic islets.

TECO® Intact Proinsulin ELISA offers high specificity for intact proinsulin, des (64,65)- proinsulin and split (65,66)- proinsulin detection without cross-reactivity to human insulin, c-peptide, or the other intermediate proinsulin cleavage products. This specificity ensures a more accurate assessment of beta cell functionality compared to total proinsulin measurements, which can be ambiguous.

 

Due to its utility as an indicator of beta cell health, TECO® Intact Proinsulin ELISA has research applications in the following indications:

• Type 1 Diabetes: Intact proinsulin is often measured in ratio with C-peptide (PI:C) to assess beta cell function.

• Type 2 Diabetes: Fasting intact proinsulin can be measured alone or in combination with oral glucose tolerance testing (OGTT) to evaluate insulin production and sensitivity.
• Diabetes Drug Development:
  • The PI:C ratio, which includes intact proinsulin, has been used to predict response to teplizumab treatment in T1D.
  • Intact proinsulin has been used to predict risk of disease progression in prediabetes, T1D, and T2D.
• Staging of insulin resistance and beta cell dysfunction
• Coronary artery disease (CAD): Fasting intact proinsulin levels can identify subjects at high-risk for CAD
• Insulinoma: Elevated levels of intact proinsulin can indicate tumors of the pancreas.
• Polycystic ovary Syndrome (PCOS): Intact proinsulin can be used to evaluate insulin resistance in PCOS.

 

 

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 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 or bound enzyme activity is measured by adding a chromogenic substrate. The intensity of colour development is proportional to the concentration of proinsulin in the sample.

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 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)