Method for detecting affinity indicators of reversible binding of insulin and serum protein and application thereof

A serum protein and measurement method technology, which is applied in the field of affinity index determination of the reversible binding between insulin and serum protein, and can solve the problem that the fasting blood sugar level cannot be maintained.

Active Publication Date: 2015-01-07
FUDAN UNIV
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  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

If serum albumin-insulin binding is increased, the level of free insulin available decreases; conversely, if serum albumin-insulin binding is decreased, the level of free insulin available after a meal is rises and falls at fasting levels; therefore, under either condition, the pancreas secretes more insulin to maintain normal levels of blood glucose in the fasting and postprandial state; if this compensatory mechanism is out of balance, normal Fasting blood sugar levels cannot be maintained

Method used

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  • Method for detecting affinity indicators of reversible binding of insulin and serum protein and application thereof
  • Method for detecting affinity indicators of reversible binding of insulin and serum protein and application thereof
  • Method for detecting affinity indicators of reversible binding of insulin and serum protein and application thereof

Examples

Experimental program
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Effect test

Embodiment 1

[0082] 1 Detection of insulin concentration

[0083] Insulin levels contained in serum and 2% albumin were determined using an insulin detection kit (Diagnostic System Laboratory, Webster, TX) according to the manufacturer's instructions. In order to determine the insulin content in albumin obtained by different preparation methods, we detected 4 kinds of albumin, respectively: 2% bovine serum albumin (BSA, Sigma-Aldrichcatalogue number A0281, BSA0281, St .Louis, MO), bovine serum albumin (Sigma-Aldrich catalog number A3803), human serum albumin (HSA, EMD Chemicals catalog number 12666, San Diego, CA), high-purity human serum albumin (EMD Chemicals catalog number 126658) . Other tests include:

[0084] ELISA was used to detect the insulin content of the supernatant serum,

[0085] ELISA detects the insulin content of the lower serum,

[0086] ELISA was used to detect the insulin content of total serum,

[0087] ELISA to detect the insulin content of albumin,

[0088] ELI...

Embodiment 2

[0104] Example 2 Cell Proliferation Test

[0105] Primary rat aortic endothelial cells (AEC) were isolated from 8-9-week-old male Wistar rats according to literature reports. Cells of passage 4 were used for the study. Mouse brain microvascular endothelial cells (bEnd.3) were purchased from the American ATCC Cell Bank (ATCC, Manassas, VA). Both AEC and bEnd.3 cells were cultured with DMEM (1000 mg / L D-glucose) containing 10% fetal bovine serum (Invitrogen, carlsbad, CA), 100 U / ml ampicillin, and 100 mg / ml streptomycin. After digestion with 0.25% trypsin-0.02% EDTA, 150 μl of serum-free culture medium containing 3000 cells was added to each well and allowed to recover for 12 hours. Then the common culture medium was replaced by the experimental culture medium prepared above. MTT (Sangon, Shanghai, China) method was used to detect changes in cell activity on days 1, 3, and 5. The absorbance at 490 nm was detected by a microplate reader (Molecular Devices, Sunnyvale, CA).

...

Embodiment 3

[0118] The influence of embodiment 3 lipid and medicine on insulin binding capacity

[0119] Add PBS, high concentration (5mmol / L) and low concentration (2mmol / L) triglyceride (sigma intralipids), high concentration (10mmol / L) and low concentration (5mmol / L) cholesterol (amresco) to 2% BSA0281 respectively , 2mmol / L free fatty acid (sigma FFAP), 5mmol / l LDL, 1mg / L glibenclamide (sigma), 15mg / L gliclazide, 5mg / L metformin, after standing at room temperature for 30 minutes, pass ultrafiltration A spin column (Microncon YM-30 Centrifugal ultrafiltration tube, Millipore Corporation, Billerica, MA) was used for separation, and the upper and lower layer solutions after ultrafiltration were used for insulin detection. In order to observe whether the albumin that removes free insulin can continue to release free insulin, add PBS to the upper layer solution after ultrafiltration in the PBS group to restore to the original capacity, and place it at room temperature for 30 minutes before...

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Abstract

The invention belongs to the technical field of biology and relates to detection of insulin / serum protein binding indictors and particularly relates to a method for detecting affinity indicators of reversible binding of insulin and serum protein and application thereof. The method includes detection of protein-binding insulin, free insulin and bound / free insulin ratio and determination of protein-capturing ability of insulin. Based on the in vitro biochemical study, the study in cultured cell model, the study of patient serum marker detection, the study on drugs and serum components affecting the affinity of reversible binding of insulin and serum protein, the study on impact of change of affinity of binding of insulin and serum protein on blood sugar level in healthy animal and diabetes animal models, it is proven that the detected indicators obtained by the inventive method can further provide experimental data and theoretical basis for prewarning, early diagnosis, prognosis, drug screening and prevention and treatment guidance of diabetes.

Description

technical field [0001] The invention belongs to the field of biological technology, and relates to the measurement of the binding index of insulin and serum protein, in particular to an affinity index measurement method and application of the reversible binding of insulin and serum protein. The method of the invention can be used for early warning, early diagnosis, prognosis judgment, drug screening, guidance of prevention and treatment of diabetes. Background technique [0002] The prior art discloses that diabetes is a common chronic disease characterized by blood sugar metabolism disorder, and more than 90% of them are type 2 diabetes (Type 2diabetes, T2DM). Insulin resistance plays an important role in the pathogenesis of type 2 diabetes and runs through the whole process of type 2 diabetes. The mechanism of insulin resistance is not completely clear. Various experimental research results show that the occurrence of insulin resistance is mostly caused by the joint actio...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): G01N33/74
Inventor 陈思锋陆超栾丽娟张雪晴侯彦强孟丹李晓波向萌王丽赵凤娣
Owner FUDAN UNIV
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