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Method and kit for detecting biological activity of apolipoprotein ApoA5 non-diagnostically

A technology of biological activity and kit, which is applied in the field of detection of biological activity of ApoA5, ​​and can solve the problem of detection of less ApoA5

Inactive Publication Date: 2015-05-06
THE SECOND XIANGYA HOSPITAL OF CENT SOUTH UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0005] At present, there are few methods for detecting the biological activity of ApoA5

Method used

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  • Method and kit for detecting biological activity of apolipoprotein ApoA5 non-diagnostically
  • Method and kit for detecting biological activity of apolipoprotein ApoA5 non-diagnostically
  • Method and kit for detecting biological activity of apolipoprotein ApoA5 non-diagnostically

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Experimental program
Comparison scheme
Effect test

Embodiment 1

[0043] The detection of embodiment 1 ApoA5 biological activity

[0044] Uptake of ApoA5: HL-1 cardiomyocytes were inoculated on laser confocal small dishes. After the cells were confluent, 1% serum claycomb containing 600ng / mL ApoA5 was added and incubated at 37°C for 12 hours. After the incubation, the culture supernatant of HL-1 cardiomyocytes was discarded and washed 3 times with PBS.

[0045] Fixing and punching: add 1 mL / well of 4% polyformic acid, fix the cells at room temperature for 20 minutes, and wash 3 times with PBS. Add 1 mL / well of PBS solution containing 0.5% Triton-Xl00 and incubate at room temperature for 10 minutes, then wash with PBS solution 3 times. Add 1 mL / well of PBS solution containing 5% BSA, and incubate at room temperature for 30 minutes.

[0046] Primary antibody incubation: Discard the supernatant, add ApoA5 primary antibody (1:200) diluted with 1% BSA to the test wells, and set the wells without ApoA5 primary antibody as negative control wells,...

Embodiment 2

[0050] The detection of embodiment 2ApoA5 biological activity

[0051] Uptake of ApoA5: HL-1 cardiomyocytes were inoculated on laser confocal small dishes. After the cells were confluent, 1% serum claycomb containing 1200ng / mL ApoA5 was added and incubated at 37°C for 18 hours. After the incubation, the culture supernatant of HL-1 cardiomyocytes was discarded and washed 3 times with PBS.

[0052] Fixing and punching: add 1 mL / well of 4% polyformic acid, fix the cells at room temperature for 15 minutes, and wash 3 times with PBS. Add 1 mL / well of PBS solution containing 0.5% Triton-Xl00 and incubate at room temperature for 5 minutes, then wash with PBS solution 3 times. Add 1 mL / well of PBS solution containing 5% BSA, and incubate at room temperature for 30 minutes.

[0053] Primary antibody incubation: Discard the supernatant, add ApoA5 primary antibody (1:200) diluted with 1% BSA, and set the wells without ApoA5 primary antibody as negative control wells, and incubate at 4°...

Embodiment 3

[0056] The detection of embodiment 3ApoA5 biological activity

[0057] ApoA5 uptake: HL-1 cardiomyocytes were inoculated on laser confocal small dishes. After the cells were full, 1% serum claycomb containing 3000ng / mL ApoA5 was added to culture at 37°C for 12 hours. The ApoA5 used was inactivated. Biological activity has been lost. After the incubation, the culture supernatant of HL-1 cardiomyocytes was discarded and washed 3 times with PBS.

[0058] Fixing and punching: Add 1 mL / well of 4% polyformic acid, fix the cells at room temperature for 25 minutes, and wash 3 times with PBS. Add 1 mL / well of PBS solution containing 0.5% Triton-Xl00 and incubate at room temperature for 15 minutes, then wash with PBS solution 3 times. Add 1 mL / well of PBS solution containing 5% BSA, and incubate at room temperature for 30 minutes.

[0059] Primary antibody incubation: Discard the supernatant, add ApoA5 primary antibody (1:200) diluted with 1% BSA, and incubate at 4°C for 12 hours. D...

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Abstract

The invention belongs to the field of protein activity detection and particularly relates to a method and kit for detecting the biological activity of an apolipoprotein ApoA5 non-diagnostically. The method comprises the following steps: incubating the apolipoprotein ApoA5 in an HL-1 cardiomyocyte, fixing, punching, incubating the first antibody of the apolipoprotein ApoA5 in the HL-1 cardiomyocyte, incubating the fluorescently-labeled second antibody of the apolipoprotein ApoA5 in the HL-1 cardiomyocyte, and observing the position of labeling fluorescence in the HL-1 cardiomyocyte to obtain the biological activity of the apolipoprotein ApoA5. The method provided by the invention can be used for detecting the biological activity of the apolipoprotein ApoA5 accurately by observing the uptake of the apolipoprotein ApoA5 by the HL-1 cardiomyocyte.

Description

technical field [0001] The invention relates to the field of protein activity detection, in particular to a method and kit for non-diagnostic detection of ApoA5 biological activity. Background technique [0002] With the improvement of modern living standards and changes in lifestyles, the incidence of obesity is increasing. At the same time, the increase in lipids and ectopic deposition associated with obesity makes the incidence of obesity-related diseases increase year by year. Therefore, effectively reducing the lipid deposition associated with obesity is an important challenge facing medicine today. [0003] Obesity is the result of excessive storage of triglyceride (TG) in adipocytes due to the imbalance between energy intake and energy consumption, and the result of abnormal increase in cell volume. The root cause is abnormal lipid metabolism. The essence of abnormal lipid metabolism refers to the abnormality of lipoprotein and apolipoprotein. The activity of key en...

Claims

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Application Information

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IPC IPC(8): G01N33/68
CPCG01N33/582G01N33/92
Inventor 赵水平罗俊郑小燕聂赛赵旺
Owner THE SECOND XIANGYA HOSPITAL OF CENT SOUTH UNIV
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