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A method for the determination of free exenatide by competitive chemiluminescence

A technology of exenatide and chemiluminescence, which is applied in chemiluminescence/bioluminescence, analysis through chemical reaction of materials, biological testing, etc., to achieve the effect of improving sensitivity, increasing sensitivity, and increasing dilution factor

Active Publication Date: 2020-11-27
SHANGHAI MEDICILON INC +1
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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  • A method for the determination of free exenatide by competitive chemiluminescence
  • A method for the determination of free exenatide by competitive chemiluminescence

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0052] Main materials and equipment:

[0053] Molecular Device full-wavelength microplate reader;

[0054] Black bottom opaque chemiluminescence ELISA plate (Nunc, Cat# 437111).

[0055] Biological and chemical reagents:

[0056] Carbonate buffer at pH 9.6

[0057] Phosphate buffer at pH 7.4

[0058] Mouse mAb to Exendin (Abcam, Cat#: ab23407)

[0059] Streptavidin-HRP (21130, Thermo)

[0060] Biotin-conjugated exenatide (Biotin-Exendin 4, 0.1mg / mL)

[0061] Free Exenatide (Exendin-4)

[0062] SuperSignal TM ELISA Femto Substrate (Thermo, Cat #: 37074).

[0063] Method steps:

[0064] 1) Use carbonate buffer (pH9.6) to dilute the mouse anti-exenatide (Exendin-4 1mg / mL) solution to 1 μg / mL, add 100 μL to each well of a 96-well microwell plate, 2- Incubate overnight at 8°C;

[0065] 2) Shake off the liquid in the plate, wash 3 times with PBST (PBS+0.05% Tween-20), 300uL / well, shake for 30sec. Pat dry, add PBS buffer containing 5% skimmed milk, 200uL / well, 37°C, incu...

Embodiment 2

[0075] Standard curve: because the method of the present invention has used competitive reaction mode, so concentration and signal value are in inverse relationship, the proterties of standard curve also present anti-S type, and typical curve sees figure 1 . The standard curves of different analysis batches were verified and compared (see Table 1). The standard curves can be fitted robustly every time, and the differences between different batches are within the range of precision and accuracy requirements, and will not exceed 20% deviation;

[0076]Table 1 Standard curves of different analytical batches

[0077] .

Embodiment 3

[0079] Recovery rate comparison: Add free exenatide to PBS and cell culture medium respectively, the concentration is 8000, 4000, 300, 100, 10, 5pg / mL, measure the recovery rate with reference to the method of Example 1, the recovery rate is In the range of 80% to 120% (see Table 2);

[0080] Table 2 The recovery rate of exenatide in PBS, cell culture medium

[0081] .

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Abstract

The invention relates to a method of determining free exenatide according to a competitive chemiluminescence method. The method comprises the steps of coating a microporous plate with an anti-exenatide antibody to form a solid phase antibody, adding exenatide standards of different concentrations and a sample to be determined into pores of the coated microporous plate for incubation for a period of time, adding biotin-labeled exenatide into the pores for continuous incubation for a period of time and washing, adding horse radish peroxidase labeled streptavidin into the pores of the microporousplate for incubation for a period of time and washing, adding chemiluminescence substrates capable of being catalyzed by horse radish peroxidase into the pores of the microporous plate, detecting chemiluminescence signal values after reaction for a period of time, fitting a standard curve with the concentrations of the exenatide standards and the corresponding chemiluminescence signal values, inserting the chemiluminescence signal value of the sample to be determined into the standard curve, and calculating the concentration of the sample to be determined.

Description

technical field [0001] The invention belongs to the fields of biological analysis, pharmaceutical analysis and immunodetection, and in particular relates to a method for measuring free exenatide by a competitive chemiluminescent method. Background technique [0002] Exendin-4 is a GLP-1 analog. Its discovery stems from the study of a poisonous lizard. The animal eats 4 times a year. During fasting, the pancreas shuts down and stops secreting insulin. Substance related. Later studies have shown that the role of Exendin-4 is almost identical to that of GLP-1. Exendin-4 can also act through the GLP-1 receptor, also known as GLP-1 receptor agonist, but the affinity for the receptor It is also stronger than GLP-1. Moreover, the N-terminus of Exendin-4 is not easy to be degraded by DPP-IV, has a longer plasma half-life, has good clinical application value and provides ideas for the treatment of type 2 diabetes. Many pharmaceutical companies have developed Exendin-4-related biot...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): G01N33/68G01N33/58G01N21/76
CPCG01N21/76G01N33/58G01N33/68
Inventor 章登吉任朋亮常亚敏
Owner SHANGHAI MEDICILON INC
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