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A kind of biological probe, test strip for detecting clenbuterol hydrochloride and application thereof

A technology of clenbuterol hydrochloride and biological probe, which is applied in the field of rapid detection of clenbuterol hydrochloride, the probe field of bacterial enrichment of gold nanoparticles and labeling of monoclonal antibodies, which can solve the low sensitivity of immunochromatographic detection And other issues

Inactive Publication Date: 2019-08-09
NORTHWEST A & F UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0005] Aiming at the defects and deficiencies in the prior art, in order to solve the problem of low sensitivity of immunochromatographic detection, the present invention constructs a new type of carrier to enrich signal substances, while ensuring the strength of signal substances, it reduces the dosage of antibodies, thereby improving The sensitivity of the analysis system is of great significance and application value for monitoring clenbuterol hydrochloride in meat, eggs and dairy foods, including a biological probe, a test strip for detecting clenbuterol hydrochloride and its application

Method used

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  • A kind of biological probe, test strip for detecting clenbuterol hydrochloride and application thereof
  • A kind of biological probe, test strip for detecting clenbuterol hydrochloride and application thereof
  • A kind of biological probe, test strip for detecting clenbuterol hydrochloride and application thereof

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preparation example Construction

[0030] Preparation of signal carrier solution: Escherichia coli was cultured in liquid medium at 37°C for 24h, inactivated with 0.5% formalin (w / v) for 24h, and washed with ultrapure water by centrifugation (5000rpm, 10min). Afterwards, the bacterial concentration was adjusted to 10 8 CFU / mL.

[0031] Colloidal gold is usually used as the label of test strips, because it is bright wine red, has stable chemical properties and good biocompatibility, and is widely used in biological labels, so this paper chooses nano gold as the signal substance, specifically , the signal substance is gold nanoparticles obtained by reducing chloroauric acid with trisodium citrate. Gold nanoparticles (AuNPs) were prepared according to a previously reported method, and gold nanoparticles were obtained by reducing chloroauric acid with sodium citrate, and the particle size of gold nanoparticles was 15-20 nm.

[0032] Preparation of signal substance solution: In an Erlenmeyer flask, 100 mL of ultra...

Embodiment 1

[0041] Embodiment 1: The preparation method of new probe Bacteria@Au-Ab

[0042] The preparation method of new probe Bacteria@Au-Ab comprises the following steps:

[0043] 1. Preparation of Probe Raw Materials

[0044] Escherichia coli was cultured in liquid medium at 37°C for 24h, inactivated with 0.5% formalin (w / v) for 24h, and washed with ultrapure water by centrifugation (5000rpm, 10min). Afterwards, the bacterial concentration was adjusted to 10 8 CFU / mL.

[0045] For the production of anti-clenbuterol monoclonal antibody (mAb), the artificial antigen of clenbuterol hydrochloride, that is, the conjugate of commercially available clenbuterol hydrochloride CL-bovine serum albumin (CL-BSA) was immunized with female Balb / c mice were induced to produce anti-clenbuterol antiserum, and then the spleen cells of the mice were taken for cell fusion with SP2 / 0 myeloma cells, and hybridoma cells capable of producing the target antibody were screened by indirect ELISA and indirec...

Embodiment 2

[0054]Example 2: Preparation of immunochromatographic test strips for rapid detection of clenbuterol hydrochloride

[0055] The preparation method of the high-sensitivity immunochromatographic test paper strip that detects clenbuterol hydrochloride rapidly comprises the following steps:

[0056] The immunochromatographic test strip sensor consists of five parts, including substrate, nitrocellulose (NC) membrane, binding pad, sample pad and absorbent pad. First, soak the bond pad and sample pad made of glass fiber in a solution containing 2% BSA, 1% sucrose and 0.02% NaN 3 PBS to block the microwells and dry overnight at 37 °C. Next, spray the probe solution onto the conjugate pad at a rate of 8 μL / cm and dry it with a vacuum freeze dryer to prevent antibody inactivation. Then 0.5 mg / mL antigen (CL-BSA) and 0.5 mg / mL goat anti-mouse immunoglobulin were coated on the NC membrane at a streaking rate of 0.8 μL / cm as test (T) line and control (C) line Wire. These parameters are...

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Abstract

The invention discloses a biological probe, a test strip for detection of clenbuterol hydrochloride and application thereof. Through construction of a novel probe using bacteria as the signal carrier, labeling of an extremely limited amount of antibody (Ab) with rich nanogold (AuNPs) can be achieved, which is the key of improving the competitive immunodetection sensitivity. The immunochromatographic test strip constructed by the invention has a visual detection limit (VDL) of 0.1ng / mL on clenbuterol, and the sensitivity is improved by 20 times than that of the traditional test strip. Benefiting from rich active sites on the surface and unique biocompatibility, bacteria can become a promising carrier of signal enrichment, and the work opens up a new way for signal amplification and performance improvement of immunochromatographic assay, and is beneficial to further expansion of the immunosensor application.

Description

technical field [0001] The invention belongs to the field of biological detection, and relates to a biological probe, a test strip for detecting clenbuterol hydrochloride and its application, in particular to a probe for enriching gold nanoparticles with bacteria and marking monoclonal antibodies, using the A test strip for the probe, and its application for rapid detection of clenbuterol hydrochloride. Background technique [0002] Clenbuterol hydrochloride is a synthetic β-adrenergic receptor agonist with bronchodilator effect and is used to treat asthma, bronchospasm, emphysema and other lung diseases. Later, it was found that when the dosage is 5-10 times of the therapeutic dosage, it can redistribute nutrients to the animal body, promote muscle growth and inhibit fat synthesis. Therefore, it is often used as a feed additive in animal husbandry in the past few years. The study found that the drug can accumulate in animals for a long time due to its stable chemical prop...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): G01N33/74G01N33/577G01N33/558G01N33/532
CPCG01N33/532G01N33/558G01N33/577G01N33/74
Inventor 张道宏黄琼王建龙补彤闫灵芝赵兵欣窦磊娜赵东阳
Owner NORTHWEST A & F UNIV
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