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Method for detecting hepatitis A virus antibodies, kit for detection through method and preparation method for kit

A hepatitis A virus and kit technology, applied in the field of biology, can solve the problems of decreased activity of markers, difficult to store, affect enzyme activity, etc., to eliminate false positive interference, tolerate a wide pH range, and reduce the cost of antibodies. Effect

Active Publication Date: 2014-08-20
QINGDAO HIGHTOP BIOTECH
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  • Abstract
  • Description
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AI Technical Summary

Problems solved by technology

However, this method has the following problems: (1) There are many steps in each batch of experiments, such as adding samples, incubating and washing plates, the operation is cumbersome, the time is long, and special operators and special instruments are required, so it is difficult to carry out in primary medical units
(2) The chromogenic components A and B need to be added separately during the detection, which increases the labor intensity and the cost of the kit to a certain extent
Patent application number 200710075306.3 discloses a color development system that can make chromogen and peroxide coexist within a certain period of time, but the key component in this system—chromogen protective agent sodium thiosulfate, invented here An acidic environment with a pH lower than 6.0 is not conducive to long-term stability. If there is CO in the solution system 2 , microorganisms or long-term exposure to light are not conducive to the stability of sodium thiosulfate, so it cannot effectively protect against oxidation of chromogenic substances such as TMB
Among them, Bassoon-1789, which is used to absorb ultraviolet rays, can protect against ultraviolet rays, but it turns red when it encounters metal ions, which interferes with the test results. Therefore, additional requirements for reagent purity and operation are added during the preparation process.
(3) The capture method for detection of hepatitis A IgM, etc. is susceptible to interference from RF (IgM type) and other non-specific IgM[1]
However, there are the following problems in this labeling process: (1) The pH needs to be adjusted repeatedly during the labeling process, and the pH value adjustment range is wide, which inevitably affects the enzyme activity; (2) The labeling process is cumbersome and time-consuming; (3) During the labeling process Cross-linking of the enzyme itself is inevitable; (4) The marker is not stable enough to be stored, and repeated freezing and thawing will reduce the activity of the marker
The storage buffer of some diagnostic kits does not give specific components for commercial reasons, which brings inconvenience to the development and application of the kits

Method used

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  • Method for detecting hepatitis A virus antibodies, kit for detection through method and preparation method for kit
  • Method for detecting hepatitis A virus antibodies, kit for detection through method and preparation method for kit
  • Method for detecting hepatitis A virus antibodies, kit for detection through method and preparation method for kit

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0051] Amination Modification of Horseradish Peroxidase

[0052] 1. Main raw materials: horseradish peroxidase HRP; ethylenediamine, EDC-HCl (1-ethyl-(3-dimethylaminopropyl) carbodiimide hydrochloride); sodium dihydrogen phosphate , disodium hydrogen phosphate, sodium chloride, benzoic acid, glycerin; trehalose.

[0053] 2. Main instruments: precision electronic balance; refrigerator; pH meter; oscillator; magnetic stirrer; freeze dryer.

[0054] 3. The steps are as follows:

[0055] Dissolve 1.0g of horseradish peroxidase in 25ml of 0.01M pH7.4 phosphate buffer, add 80.0mg of ethylenediamine, adjust the pH to 5.0 with HCl, mix the resulting solution with 120.0mg of EDC, and shake at room temperature for 1h , and then loaded into a dialysis bag and dialyzed with pre-prepared PBS for 2 hours.

[0056] Add 10% glycerin by volume, 3% trehalose by mass and 0.05% benzoic acid to the solution prepared above, and then store it in refrigeration for immediate use or after concentrat...

Embodiment 2

[0068] Preparation of enzyme-labeled hepatitis A antigen

[0069] 1. Raw materials: 1mg / ml hepatitis A antigen; 4.8mg / ml Sulfo-SMCC (4-(N-maleimidomethyl)cyclohexane-1-carboxylic acid sulfosuccinimidyl ester sodium salt) ;Ultrafiltration tube with molecular weight cut-off MWCO of 10K; Aminated HRP self-made or commercialized natural HRP; Sodium dihydrogen phosphate, disodium hydrogen phosphate, sodium chloride.

[0070] 2. Main instruments: high-speed centrifuge; refrigerator; pH meter.

[0071] 3. The operation steps are as follows:

[0072] (1) Dissolve a certain amount of amino-horseradish peroxidase in 1ml double-distilled water to make a 5mg / ml solution; at the same time, prepare 0.01M phosphate buffered saline (hereinafter referred to as PBS) for later use.

[0073] (2) Add 70 μl of cross-linking agent Sulfo-SMCC to 1 ml of newly prepared 5 mg / ml aminated HRP solution, mix well and react at room temperature for 30 min.

[0074] (3) Add the reactant to a MWCO 10K ultra...

Embodiment 3～5

[0101] Preparation of chromogenic solution

[0102] 1. Main raw materials: sodium acetate trihydrate, tetramethylbenzidine TMB, carbamide peroxide, ascorbic acid, chlorogenic acid; 2-cyano-3,3-diphenylisooctyl acrylate (octocrylene); Phosphoglycine.

[0103] 2. Preparation method:

[0104] (1) Weigh 8.3g of sodium acetate trihydrate crystals and add it to 600ml of ultrapure water, shake and mix well, adjust the pH of the solution to 5.0 with 0.1mol / L hydrochloric acid, and then prepare 0.05mol / L acetic acid-sodium acetate buffer liquid.

[0105] (2) Weigh 0.4-1.0g of tetramethylbenzidine (hereinafter referred to as TMB), dissolve it in 3ml of absolute ethanol, then add the dissolved TMB solution to the above-mentioned acetic acid-sodium acetate buffer solution, shake and mix well, spare.

[0106] (3) Weigh 80 mg of chlorogenic acid and 80 mg of ascorbic acid, add them to the buffer, shake and mix.

[0107] (4) Measure 10ml of isooctyl 2-cyano-3,3-diphenylacrylate (octocry...

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Abstract

The invention relates to the technical field of biology, in particular to a method for detecting hepatitis A virus antibodies, a kit for detection through the method and a preparation method for the kit. Antihuman IgM (immunoglobulin m) and antihuman IgG (immunoglobulin g) are fixed on a membrane respectively; blood serum to be detected are added; after the hepatitis A antibodies in the blood serum to be detected are combined with the antihuman IgM and the antihuman IgG fixed on the membrane, enzyme-labeled hepatitis A antigen solution is added; and finally, color development solution is added for color development so as to display the result. The invention has the advantages as follows: false positive interference in the result caused by rheumatoid disease factors in a sample can be eliminated, the process that enzyme labels hepatitis A antigen is enabled to be quicker and more efficient, and meanwhile, the antibody cost of the kit is lowered.

Description

technical field [0001] The invention relates to the technical field of biology, in particular to a method for detecting hepatitis A virus antibody, a kit for detection using the method, and a preparation method for the kit. Background technique [0002] Viral hepatitis A, referred to as hepatitis A and hepatitis A, is caused by hepatitis A virus (HAV), an infectious disease mainly caused by liver inflammation, and is mainly transmitted through the fecal-oral route. Clinically, it is characterized by fatigue and loss of appetite. , Hepatomegaly, abnormal liver function as the main manifestations, jaundice in some cases, mainly manifested as acute hepatitis, common in asymptomatic infection. The pathogen HAV is carried in the feces of patients with hepatitis A and can be transmitted through contaminated water and food. Testing for hepatitis A virus can therefore be used as a preventive measure to prevent the spread of the disease. [0003] At present, the main method for det...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): G01N33/576G01N33/535G01N33/558
CPCY02A50/30
Inventor 杨致亭杨金红武新清王爱法沈孝功刘济宁丁建华夏安春
Owner QINGDAO HIGHTOP BIOTECH
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