Heart-type fatty acid binding protein immunodetection reagent and preparation and detection method thereof

A technology for fatty acid binding and detection reagents, applied in measurement devices, instruments, scientific instruments, etc., can solve the problems of unsuitability for high-throughput detection, time-consuming, and high cost, and is conducive to large-scale clinical application, accurate results, and high cost. less demanding effects

Inactive Publication Date: 2018-05-15
太原瑞盛生物科技有限公司
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  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Immunoelectrophoresis is not suitable for high-throughput detection because it takes a long time; radioimmunoassay has been basically replaced by other methods due to radioactive contamination; enzyme-linked immunosorbent assay was not highly automated before, so the repeatability is not very good. There has been a great deal of improvement with the spread of automation
Chemical or electrochemiluminescence immunoassay and fluorescence immunoassay require special equipment, resulting in high cost and limited scope of application
Scattering turbidimetry must be performed on a special protein analyzer. Although the precision is good, its use is limited
[0004] At present, there is a lack of high-sensitivity and specificity heart-type fatty acid-binding protein detection reagents on the market, especially good-quality automated detection reagents

Method used

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  • Heart-type fatty acid binding protein immunodetection reagent and preparation and detection method thereof
  • Heart-type fatty acid binding protein immunodetection reagent and preparation and detection method thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0047] Example 1: Preparation of glucose dehydrogenase-enzyme-labeled antigen conjugate

[0048] 1) Preparation of glucose dehydrogenase (GDH) solution:

[0049]a. Weigh 15mg of GDH with a specification of 100KU, dissolve it in 12mL at room temperature containing 72.6mg (0.05M) PBS, 8mg MgCl 2 (3.3mM) and 100mg NaCl, the solution pH=8.0; this step is carried out in small beaker A.

[0050] 225 mg of reduced nicotinamide adenine dinucleotide NADH, 135 mg of glucose and 0.75 mL of carbitol were added to the above beaker A.

[0051] In the aforementioned beaker A, 2 mL of dimethyl sulfoxide (DMSO) was added dropwise.

[0052] 2) Activation of heart fatty acid binding protein

[0053] a. Weigh 10 mg heart-type fatty acid binding protein in anhydrous state and dissolve in 600 μL DMF;

[0054] b. The temperature of the above solution drops to 2-8°C;

[0055] c. Add 3 μL tributylamine;

[0056] d. Add 3 μL isobutyl chloroformate;

[0057] e. Stir at 2-8°C for 30 minutes;

[0...

Embodiment 2

[0061] Example 2: Preparation of heart-type fatty acid binding protein homogeneous enzyme immunoassay reagent

[0062] The homogeneous enzyme immunoassay reagent for heart-type fatty acid-binding protein includes: the above-mentioned enzyme-labeled heart-type fatty acid-binding protein, and an indicator reagent for detecting a heart-type fatty acid-binding protein antibody-enzyme-labeled heart-type fatty acid-binding protein complex. The indicator reagents are selected from enzyme reagents, radioisotope reagents, fluorescent reagents or chemiluminescent reagents. Preferably, the indicator reagent is an enzyme reagent, including: an enzyme-labeled conjugate and an enzyme substrate. Wherein, the enzyme-labeled conjugate includes glucose dehydrogenase-enzyme-labeled antigen conjugate, which is obtained by the above chemical synthesis method.

[0063] Before using the heart-type fatty acid binding protein homogeneous enzyme immunoassay reagent, in order to avoid the reaction betw...

Embodiment 3

[0073] Example 3: Heart-type fatty acid binding protein homogeneous enzyme immunoassay

[0074] 1. Obtain the standard curve: set the reaction parameters of the Olympus AU400 automatic biochemical analyzer. The operation process is: first add reagent 1, then add the standard, and finally add reagent 2. After adding reagent 2, measure the OD340 absorbance value at different time points, and calculate the reaction rate at different concentrations of standard substances. Ideal reaction standard curve graph, such as figure 1 shown.

[0075] 2. Detection of biochemical analyzer: Take the operation of Olympus AU400 automatic biochemical analyzer as an example: add 8 μL of sample, then add 160 μL of reagent 1, mix and incubate for 5 minutes, add 40 μL of reagent 2, mix and incubate for 30 seconds, and then start reading Take the absorbance A1, and after incubation for 5 minutes, read the absorbance A2, and calculate ΔA=A2-A1. Reaction conditions: dominant wavelength: 700nm; react...

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Abstract

The invention discloses a heart-type fatty acid binding protein immunodetection reagent and a preparation and a detection method thereof, and concretely relates to heart-type fat binding protein immunodetection reagent and a preparation and a detection method thereof. The heart-type fatty acid binding protein immunodetection reagent comprises enzyme-labeled heart-type fat binding protein, and an indication reagent used for detecting a heart-type fat binding protein antibody-enzyme-labeled heart-type fat binding protein compound. The heart-type fatty acid binding protein immunodetection reagenthas the advantages that the above homogeneous immunodetection agent can conveniently, accurately and rapidly determine the heart-type fat binding protein content in a sample, and several samples canbe simultaneously determined on an automatic biochemical analyzer, high-flux rapid determination on heart-type fatty acid binding protein is realized, accuracy is high, specificity is strong, stability is good, and accuracy and detection efficiency are greatly increased.

Description

technical field [0001] The invention relates to a heart-shaped fatty acid binding protein detection reagent and its preparation and detection method, in particular to a heart-shaped fatty acid binding protein homogeneous enzyme immunological detection reagent and its preparation and detection method. Background technique [0002] Fatty acid-binding protein (FABP) is a group of multi-source small molecular proteins, widely distributed in the cytoplasm of mammalian heart, brain, bone, intestine, liver, fat and other cells, according to its tissue specificity It is divided into nine types including heart type, brain type, kidney type, liver type and intestinal type. The heart-type fatty acid-binding protein (Heart-type FattyAcid-Binding Protein, H-FABP) is distributed in cardiomyocytes, accounting for about 4% to 8% of all soluble proteins in the heart, and is mainly involved in the regulation of myocardial energy metabolism. The differences in biology make it highly specific ...

Claims

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Application Information

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IPC IPC(8): G01N33/535G01N33/558
CPCG01N33/535G01N33/558
Inventor 赵慧敏严芳芳王亚盟丁兰艳杜爱铭徐兵
Owner 太原瑞盛生物科技有限公司
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