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Method for marking salbutamol polyclonal antibody by fluorescent microsphere

A technology of polyclonal antibodies and fluorescent microspheres, which is applied in the direction of measuring devices, instruments, scientific instruments, etc., can solve the problems of low antibody labeling efficiency and label dispersion, and achieve good colloidal stability, good dispersibility and solubility, and binding strong effect

Inactive Publication Date: 2013-12-25
NANCHANG UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

This coupling method has the advantages of short coupling time and simple operation, but in this coupling method, some antigen-binding sites are shielded due to the random binding of antibodies to the label, which is the so-called steric hindrance, which leads to the efficiency of antibody labeling. and reduced dispersion of markers

Method used

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  • Method for marking salbutamol polyclonal antibody by fluorescent microsphere

Examples

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Comparison scheme
Effect test

Embodiment 1

[0020] Example 1: Preparation method of novel conjugates of fluorescent microsphere-labeled salbutamol polyclonal antibody in the present invention

[0021] 1) Preparation method of amino-modified fluorescent microspheres

[0022] Wash 10 mg of fluorescent microspheres and ultrasonically dissolve them in 1 mL of distilled water, add 20 μL of glacial acetic acid and 20 μL of (3-trimethoxysilylpropyl)-diethylethylenediamine for ultrasonic dissolution, and stir magnetically for 3 to 4 h, washed with 10 mM MES buffer (pH 5.5) and resuspended.

[0023] 2) Preparation method of active esterified biotin

[0024] Accurately weigh 4.5 mg biotin-PEG 5000 The complex was dissolved in 2 mL of DMF solution, and after adding 20.5 mg of dicyclohexylcarboimide and 10.5 mg of N-hydroxysuccinimide, the reaction was carried out under magnetic stirring at room temperature for 24 h, and then centrifuged at 4000 rpm for 5 min, and the precipitate was discarded. Actively esterified biotin was o...

Embodiment 2

[0028] Example 2: Application of a novel conjugate of fluorescent microsphere-labeled albuterol polyclonal antibody on fluorescent microsphere test strips

[0029] 1) Preparation of fluorescent microsphere immunochromatographic test strips

[0030]Preparation of fluorescent microsphere pads: After reconjugating the conjugate prepared in Example 1 with 0.01 M PNPB (which contains 5% sucrose and 0.05% Tween-20) to the initial volume, use BIODOT Dispensing System, according to 4 μL The amount of / cm sprayed onto the glass fiber membrane, vacuum dried at 25 ℃ for 1-2 hours, and placed in a dry environment for later use.

[0031] The albuterol-BSA conjugate and the donkey anti-mouse secondary antibody were coated on the NC membrane: adjust the coating concentration to 0.4 mg / mL and 0.4 mg / mL with 0.01 M PBS buffer respectively, and spray the membrane volume to 0.74 μL / cm, the detection line is coated with albuterol-BSA conjugate, the quality control line is coated with donkey a...

Embodiment 3

[0040] Example 3: Detection of salbutamol residues in pig liver on fluorescent microsphere test strips prepared by a novel conjugate of salbutamol polyclonal antibody labeled with fluorescent microspheres

[0041] The sample pretreatment method and test strip production are the same as in Example 2.

[0042] The qualitative and quantitative detection methods are the same as in Example 2, and a standard curve is drawn by adding a series of concentrations (0.5, 1, 2, 3, 4, 5, 6, 8, 10, 15 ng / mL) and corresponding fluorescence intensity values. Draw 100 μL of the extract and add it to the sample well. After 10 minutes, use a reader to detect. According to the data output of the test sample, the output values ​​are 2.90. Check the standard curve to find that the residual amount of salbutamol in the test sample is 0.

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Abstract

The invention belongs to the biotechnical field, and discloses a method for marking a salbutamol polyclonal antibody by a fluorescent microsphere. The method comprises the following steps: coupling an amino group modified fluorescent microsphere with actively esterified biotin to obtain a conjugate, mixing and reacting streptavidin with the conjugate according to a ratio of 1000:1 to obtain a fluorescent microsphere and biotin-streptavidin conjugate, and adding a biotinylated salbutamol polyclonal antibody into the fluorescent microsphere and biotin-streptavidin conjugate solution for a reaction to obtain a target product. The fluorescent microsphere marked salbutamol polyclonal antibody novel conjugate can be effectively used in salbutamol fluorescent microsphere test strips, so the specificity of the test strips is correspondingly improved, thereby the residual quantity of salbutamol in animal foods can be rapidly, accurately, qualitatively and quantitatively detected.

Description

technical field [0001] The present invention belongs to the β 2 - The field of detection of receptor agonist residues, specifically relating to a conjugate of salbutamol polyclonal antibody labeled with fluorescent microspheres and a preparation method thereof. Background technique [0002] Salbutamol (Salbutamol) also belongs to beta 2 -receptor agonist, and in recent years has been used as the main substitute of clenbuterol hydrochloride. It is well known that β 2 - Receptor agonist drugs used in animal husbandry can improve feed conversion rate and increase lean meat production. When humans eat beta 2 - Animal foods with high residues of receptor agonists may cause symptoms such as heart palpitations, headaches, dizziness, nausea, etc., and have certain damage to the liver and kidneys. my country and many other countries in the world have put such The drug is listed as a banned drug. However, due to the drive of economic interests and the lack of an effective detectio...

Claims

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Application Information

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IPC IPC(8): G01N33/533
Inventor 赖卫华彭涛
Owner NANCHANG UNIV
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