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Insulinogen C peptide biological activity measuring method

A technology of biological activity and measurement method, which is applied in the field of chemiluminescence immunoassay to determine the biological activity of proinsulin C peptide, and can solve the problems of difficult operation control, inconsistent detection methods, and difficulty in establishing an in vitro activity measurement method.

Inactive Publication Date: 2008-03-05
SHANGHAI NEWSUMMIT BIOPHARMA +1
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  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

The results showed that the C-peptide effect on normal human red blood cell Na + -K + -ATPase activity has a certain promoting effect, but even when the concentration of C peptide is extremely high, the difference in the effect on the activity of red blood cell membrane sodium potassium ATPase is not obvious, indicating that normal human red blood cell Na + -K + -ATPase is not sensitive to the effect of C-peptide, there is no dose-dependent relationship, and the reproducibility is not good
Furthermore, RBC Na + -K + -ATPase must react on the red blood cell membrane, and the operation is difficult to control. It does not meet the requirements of the detection method for determining the stability between batches of products. Therefore, this method is not suitable for the activity detection method of C peptide.
However, there is no literature report on the direct detection of C-peptide activity using the method of endothelial NO synthase activity.
[0006] It can be seen that it is difficult to establish an in vitro activity assay method

Method used

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  • Insulinogen C peptide biological activity measuring method
  • Insulinogen C peptide biological activity measuring method

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0040] Embodiment 1. Method establishment

[0041] 1.1 Sample: self-made active reference substance (batch number 20040601, 0.8mg / bottle).

[0042] 1.2 Method introduction:

[0043] I. Grouping:

[0044] Group 1, model control group: normal saline 0.1ml / 100g (1ml / kg)

[0045] Group 2, high-dose group: C-peptide 0.8mg / kg, 0.1ml / 100g (1ml / kg), that is, 0.8mg / ml;

[0046] 3. Medium-dose group: C-peptide 0.4mg / kg, 0.1ml / 100g (1ml / kg), that is, 0.4mg / ml;

[0047] Group 4, low-dose group: C-peptide 0.2mg / kg, 0.1ml / 100g (1ml / kg), ie 0.2mg / ml.

[0048] II. Half an hour after the subcutaneous injection, the glomerular filtration rate was analyzed: for details, see Appendix 1 "Standard Operating Procedures for Bioactivity Detection of Recombinant Human Proinsulin C Peptide".

[0049] 1.2.1 Linear range:

[0050] Dose group

GFRml / min / 100gW

mouse number

Blank control group

low dose group

Middle dose group

high dose group

1

0.648

0.61 ...

Embodiment 2

[0055] Example 2. Method verification

[0056] 2.1 Sample: self-made active reference substance (batch number 20040601, 0.8mg / bottle).

[0057] 2.2 Method introduction:

[0058] I. Grouping:

[0059] Group 1, model control group: normal saline 0.1ml / 100g (1ml / kg)

[0060] Group 2, high-dose group: C-peptide 0.8mg / kg, 0.1ml / 100g (1ml / kg), that is, 0.8mg / ml;

[0061] Group 3, medium dose group: C-peptide 0.4mg / kg, 0.1ml / 100g (1ml / kg), that is, 0.4mg / ml;

[0062] Group 4, low-dose group: C-peptide 0.2mg / kg, 0.1ml / 100g (1ml / kg), ie 0.2mg / ml.

[0063] II. Half an hour after the subcutaneous injection, the glomerular filtration rate was analyzed: for details, see Appendix 1 "Standard Operating Procedures for Bioactivity Detection of Recombinant Human Proinsulin C Peptide".

[0064] 2.1 Linearity and measurement range:

[0065] Dose group

GFRml / min / 100gW

mouse number

Blank control group

low dose group

Middle dose group

high dose group

1 ...

Embodiment 3

[0077] Embodiment 3 finished product detection:

[0078] 3.1 Batch number of finished product: 20050501, 20050601; specification 0.7mg / bottle (700unit / bottle)

[0079] 3.2 Standard product: self-made active reference substance (batch number 20040601, 800 unit / bottle or 0.8mg / bottle).

[0080] 3.3 Method introduction: Prepare standard curves of 0unit / ml, 200unit / ml, 400unit / ml and 800unit / ml, dilute the finished product to the linear middle of the standard curve, and conduct activity detection; see Appendix 1 "Recombinant Human Proinsulin C Peptide Biological Activity Detection Standards Operating Procedures

[0081] 3.4 Test results:

[0082] 3.4.1 Standard curve:

[0083] standard curve line

unit / ml

0

200

400

800

average glomerular filtration

RateGFR ml / min / 100gW

0.527

0.31

0.28

0.116

r＝

0.952

linear equation

y=-0.0005x+0.473

[0084] See attached drawing 3.

[0085] 3.4.2 Finished prod...

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Abstract

The method comprises: a) establishing diabetes rat model; b) feeding the medicine; c) using the glomerular filtration rate (GFR) as the testing index of the biological activity to test the bioactivity of the reconstructed human proinsulin C peptide.

Description

technical field [0001] The invention relates to a chemiluminescent immunoassay for measuring the biological activity of proinsulin C-peptide, and the measuring range, accuracy and linearity of the method are verified. On this basis, we calibrate the specifications of self-made active standards. And establish the activity quality standard of the pilot product. Background technique [0002] C-peptide is the connecting peptide connecting insulin A chain and B chain in the proinsulin molecule. It consists of 31 amino acids and has a molecular weight of 3020kD. The basic plasma C-peptide level of normal people is about 0.4nmol / L, and the plasma C-peptide level of patients with type 1 diabetes is lower than normal or even undetectable. Early studies concluded that C-peptide has no biological activity other than assisting insulin formation. Since the 1990s, a research team headed by Swedish scientists Johansson and Professor Wahren found that C-peptide can improve the long-term ...

Claims

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Application Information

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IPC IPC(8): G01N33/15G01N33/68G01N33/53G01N21/76A61K49/00A61K38/28A61D99/00
Inventor 黄阳滨孙九如张翊任军徐晓燕应勖
Owner SHANGHAI NEWSUMMIT BIOPHARMA
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