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5-hydroxyindoleacetic acid immunogen, antibody and detection reagent, and preparation methods thereof

A technology of oxindoleacetic acid and immunogen, which is applied in chemical instruments and methods, biological testing, immunoglobulin, etc., can solve the problems of inapplicability to the detection of 5-HIAA content, small detection scale, and high cost, and achieve favorable Clinical application, high accuracy, and easy operation

Inactive Publication Date: 2015-12-09
苏州博源医疗科技有限公司
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  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, these methods are time-consuming, costly, and small-scale, and are not suitable for the detection of 5-HIAA in the urine of clinical patients.
At present, there is a lack of 5-HIAA detection reagents with good stability, high sensitivity and strong specificity in the market, especially good quality automatic detection reagents

Method used

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  • 5-hydroxyindoleacetic acid immunogen, antibody and detection reagent, and preparation methods thereof
  • 5-hydroxyindoleacetic acid immunogen, antibody and detection reagent, and preparation methods thereof
  • 5-hydroxyindoleacetic acid immunogen, antibody and detection reagent, and preparation methods thereof

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Experimental program
Comparison scheme
Effect test

Embodiment 1

[0044] Synthesis of embodiment one 5-hydroxyindoleacetic acid immunogen

[0045] The 5-hydroxyindoleacetic acid immunogen is composed of bovine serum albumin (BovineSerumAlbumin, BSA) and 5-hydroxyindoleacetic acid represented by formula (II) The specific steps are as follows:

[0046] 1. Dissolve 200mg of bovine serum albumin in 50ml of 0.2M, pH8.5 phosphate buffer;

[0047] 2. Add the following chemicals into a small beaker and stir to dissolve: 200mg of 5-hydroxyindoleacetic acid, 3.5ml of dimethylformamide, 3.5ml of ethanol, 7.0ml of 10mM potassium phosphate buffer at pH 5.0, 200mg of 1-ethyl- 3-(-3-dimethylaminopropyl) carbodiimide, 50mg N-hydroxyl sulfosuccinimide, these chemicals were stirred and dissolved at room temperature for 30 minutes;

[0048] 3. Add the dissolved solution dropwise to the BSA solution, and stir overnight at 2-8°C to obtain the antigen; purify the synthesized antigen by dialysis to obtain the 5-hydroxyindoleacetic acid immunogen.

Embodiment 2

[0049] Example 2: Preparation of Anti-5-Hydroxyindoleacetic Acid Specific Antibody

[0050] The 5-hydroxyindoleacetic acid immunogen prepared in Example 1 was inoculated into experimental animal rabbits by conventional methods, and the antiserum was taken after booster immunization. The specific steps were as follows:

[0051] 1. Dilute the above-mentioned synthetic 5-hydroxyindoleacetic acid immunogen to 1.0 mg / ml with PBS to obtain an antigen solution, then mix 1.0 ml of the antigen solution with an equal amount of Freund's complete adjuvant, and inject the experimental animal rabbit.

[0052] 2. After 2-3 weeks, inject 1.0ml of the same antigen solution and the same amount of Freund's incomplete adjuvant to the above-mentioned experimental animal rabbit once, and then inject once every four weeks, a total of 4 injections.

[0053] 3. Take blood from the experimental animal rabbit in step 2, separate and purify to obtain anti-5-hydroxyindoleacetic acid specific antibody with...

Embodiment 3

[0054] Embodiment three: the ELISA test of 5-hydroxyindoleacetic acid

[0055] 1. Establishment of standard curve for ELISA detection of 5-hydroxyindoleacetic acid

[0056] (1) Preparation of standard products

[0057] 5-Hydroxyindoleacetic acid powder (purchased from Sigma Company) was dissolved in methanol solution to prepare a 1000 μmol / L stock solution. The stock solution was sequentially diluted with ELISA buffer to 80.00 μmol / L, 40.00 μmol / L, 20.00 μmol / L, 10.00 μmol / L, 5.00 μmol / L and 0.00 μmol / L standard solutions. Among them, the ELISA buffer contains 50.0mM Tris, 145mM NaCl and 0.25% BSA.

[0058] (2) Utilize the ELISA test method of 5-hydroxyindoleacetic acid to prepare standard curve

[0059] Dilute the anti-5-hydroxyindoleacetic acid specific antibody prepared in Example 2 with PBS to a final concentration solution of 1:8000, coat 100 μL / well on a 96-well enzyme-linked plate, and place at 4°C for 12-24 hours; After the 96-well ELISA plate coated with anti-5-hy...

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Abstract

The invention discloses a 5-hydroxyindoleacetic acid immunogen, antibody and detection reagent, and preparation methods thereof. The 5-hydroxyindoleacetic acid immunogen prepared in the invention has high immunogenicity, can prepare the specific antibody against 5-hydroxyindoleacetic acid through induction and has no cross reaction with common 62 drugs. The 5-hydroxyindoleacetic acid detection reagent prepared from the antibody can accurately and rapidly determine the content of 5-hydroxyindoleacetic acid immunogen in biological samples like urine. Compared with conventional detection reagents on the market, the detection reagent provided by the invention has the advantages of simple operation, high sensitivity, good specificity, accurate results, etc., can effectively reduce detection cost for 5-hydroxyindoleacetic acid immunogen and is favorable for large-scale clinical popularization and application.

Description

technical field [0001] The invention belongs to the field of biotechnology, and relates to a 5-hydroxyindoleacetic acid immunogen, an anti-5-hydroxyindoleacetic acid specific antibody, a 5-hydroxyindoleacetic acid detection reagent and a preparation method thereof. Background technique [0002] 5-Hydroxyindole-3-acetic acid (5-Hydroxyindole-3-acetic acid, 5-HIAA), its structural formula is as shown in formula (II): [0003] [0004] 5-Hydroxyindoleacetic acid (5-HIAA), as a metabolite of 5-hydroxytryptamine (5-HT), is catalyzed by monoamine oxidase through the conversion of 5-hydroxyindole acetaldehyde. 5-HIAA is metabolized in the liver and excreted in the urine. The level of 5-HT is highest at noon and lowest at midnight, so the content of its metabolite 5-HIAA is higher during the day than at night, so the determination of 24-hour urine can more accurately reflect the level of 5-HIAA. Clinically, elevated levels of 5-HIAA are common in diseases such as carcinoid tumo...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C07K14/765C07K14/795C07K14/435C07K1/34C07K16/06C07K16/44G01N33/68G01N33/535G01N33/534G01N33/533
CPCC07K19/00C07K14/435C07K14/765C07K14/795C07K16/06C07K16/44G01N33/533G01N33/534G01N33/535G01N33/68
Inventor 虞留明贾玫成志鹏
Owner 苏州博源医疗科技有限公司
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