IL-6 detection kit based on bimolecular fluorescence complementary technology as well as preparation and use methods

A detection kit, IL-6 technology, applied in biological testing, fluorescence/phosphorescence, analytical materials, etc., can solve the problems of low detection sensitivity and narrow linear range

Inactive Publication Date: 2018-06-29
NANJING TZONE BIOLOGICAL SCI & TECH
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

The ELISA method uses horseradish peroxidase (horseradish peroxidase, HRP) or alkaline phosphatase to label the antibody, and catalyzes the color change of the substrate. It has the characteristics of simple operation and long stable period of the reagent, but the detection sensitivity of the ELISA method is low. , narrow linear range, currently mainly used in projects that require low detection sensitivity, such as infectious disease screening

Method used

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  • IL-6 detection kit based on bimolecular fluorescence complementary technology as well as preparation and use methods
  • IL-6 detection kit based on bimolecular fluorescence complementary technology as well as preparation and use methods
  • IL-6 detection kit based on bimolecular fluorescence complementary technology as well as preparation and use methods

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0027] The anti-IL-6 antibody is coupled to the fluorescent protein N-terminal fragment, taking the yellow fluorescent protein (YFP) 1-154 amino acid fragment YFPN as an example, the specific implementation process is as follows:

[0028] 1) Add 0.1mg of YFPN protein into a centrifuge tube, and prepare with 0.05mol / L pH9.5 carbonate buffer (CB) to dilute the YFPN protein to 1mg / mL.

[0029] 2) Add glutaraldehyde at a final concentration of 1.25% in a fume hood.

[0030] 3) Reaction in a water bath at 37°C for 2 hours.

[0031] 4) Dialyze with desalting column Sephadex G-25 or 0.05mol / L pH9.5 CB to remove excess glutaraldehyde.

[0032] 5) Take 0.1mg anti-IL-6 antibody, prepare 1mg / mL antibody with 0.05mol / L pH9.5 CB, and mix the activated YFPN protein and antibody.

[0033] 6) React overnight at 4°C.

[0034] 7) Blocking: add 50 μL of 0.2 mol / L lysine solution, and block for 2 hours at room temperature to block residual aldehyde groups and terminate the reaction.

[0035] ...

Embodiment 2

[0038] The anti-IL-6 antibody is coupled to the fluorescent protein C-terminal fragment, taking the yellow fluorescent protein (YFP) 155-238 amino acid fragment YFPC as an example, the specific implementation process is as follows:

[0039] 1) Add 0.1mg of YFPC protein into a centrifuge tube, and prepare with 0.05mol / L pH9.5 carbonate buffer (CB) to dilute the YFPC protein to 1mg / mL.

[0040] 2) Add glutaraldehyde at a final concentration of 1.25% in a fume hood.

[0041] 3) Reaction in a water bath at 37°C for 2 hours.

[0042] 4) Dialyze with desalting column Sephadex G-25 or 0.05mol / L pH9.5 CB to remove excess glutaraldehyde.

[0043] 5) Take 0.1mg of IL-6 antibody, prepare 1mg / mL antibody with 0.05mol / L pH9.5 CB, and mix the activated YFPC protein and antibody.

[0044] 6) React overnight at 4°C.

[0045] 7) Blocking: add 50 μL of 0.2 mol / L lysine solution, and block for 2 hours at room temperature to block residual aldehyde groups and terminate the reaction.

[0046] ...

Embodiment 3

[0049] The main components of the kit:

[0050] 1) N-terminal fragment of fluorescent protein coupled with anti-IL-6 antibody;

[0051] 2) Anti-IL-6 antibody-coupled fluorescent protein C-terminal fragment.

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Abstract

The invention provides an IL-6 diagnostic kit based on a bimolecular fluorescence complementary technology. The kit comprises an anti-IL-6 antibody coupled fluorescin N-terminal fragment and an anti-IL-6 antibody coupled fluorescin C-terminal fragment. The invention further discloses a preparation method of the IL-6 diagnostic kit based on the bimolecular fluorescence complementary technology. Themethod comprises the steps of preparing the anti-IL-6 antibody coupled fluorescin N-terminal fragment and preparing the anti-IL-6 antibody coupled fluorescin C-terminal fragment. Finally, the invention further discloses a use method of the kit. The kit provided by the invention has the advantages of simplicity in operation, wide linear range, good specificity, free from cleaning, high accuracy and the like, is convenient in clinical detection use and can be used for improving the accuracy of sepsis diagnosis when the kit is applied to infection monitoring, thereby being extensively popular tothe market and having a great market value.

Description

technical field [0001] The invention relates to a bimolecular fluorescence complementary technology used for in vitro immunodiagnosis to detect the content of IL-6 in a human body, belonging to the field of disease diagnosis and detection. Background technique [0002] Acute phase protein (APP) is a kind of specific protein closely related to infectious inflammation. In recent years, a large number of studies have shown that a variety of positive and negative acute phase proteins have a significant correlation with infectious inflammation, and are more accurate and reliable than detection of leukocytes, erythrocyte sedimentation rate, enzyme activity and content changes, etc. inflammatory markers. [0003] Interleukin-6 (IL-6) was first detected in mouse fibroblasts by Content et al. in 1982. It is a polypeptide cytokine composed of 184 amino acids and a molecular weight of 26kD, which is produced by a variety of tissue cells (mainly macrophages, T lymphocytes, and B lymph...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): G01N33/68G01N21/64
CPCG01N21/6486G01N33/68
Inventor 徐林
Owner NANJING TZONE BIOLOGICAL SCI & TECH
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