Reagent for quantitative detection of Beta-receptor stimulant through Europium chelate latex time-resolved immunochromatographic assay

A time-resolved fluorescence and receptor agonist technology, applied in the field of β-receptor agonists and veterinary drug residues, can solve the problems of high sensitivity, expensive, complicated process, etc., to overcome low sensitivity, easy storage and transmission, quantitative detection The effect of the method

Inactive Publication Date: 2014-10-08
ROHI BIOTECH
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Instrumental analysis requires expensive instruments and equipment, specialized laboratories and well-trained professionals, and has disadvantages such as high requirements for sample pretreatment, complicated process, and slow speed.
Enzyme-linked immunosorbent assay technology has the characteristics of high sensitivity and low analysis cost, but it is time-consuming and requires professionals and laboratories, so it is not suitable for on-site screening
Colloidal gold chromatography and immunofluorescence chromatography are fast and simple, and are suitable for rapid on-site screening and detection. Since colloidal gold detection is based on visual judgment, for items with low detection limits, such as items below 1ppb or items that cannot be detected, the current technology difficult to realize
In addition, the color of colloidal gold particles is slightly different in different media, so it is difficult to quantify according to the color depth, because these defects limit its vertical development
The principle of fluorescence detection is similar to the colloidal gold method, and the sensitivity is higher than that of colloidal gold. Because the nitrocellulose membrane in the reagent composition and the background of the detected sample (such as urine samples, tissue samples, etc. contain proteins and carbohydrates, these substances have fluorescence effects. ) interference, affecting the accuracy of the test results, especially for items with low detection limits, this kind of noise signal interference is particularly prominent. In addition, this method needs to be equipped with instruments, and the current application area is far less than that of colloidal gold.

Method used

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  • Reagent for quantitative detection of Beta-receptor stimulant through Europium chelate latex time-resolved immunochromatographic assay
  • Reagent for quantitative detection of Beta-receptor stimulant through Europium chelate latex time-resolved immunochromatographic assay
  • Reagent for quantitative detection of Beta-receptor stimulant through Europium chelate latex time-resolved immunochromatographic assay

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0037] Example 1 Preparation of chromatography detection reagent strip / card (eg clenbuterol hydrochloride)

[0038] 1) Packed with Eu 3+ Chelate fluorescent latex-labeled clenbuterol hydrochloride antibody: take 100ul of 10% 200nm europium latex microspheres with carboxyl groups on the surface (Thermo Fisher Company), add 900ul of 0.1M MES pH6.1 for washing, centrifuge at 13000rpm for 30 minutes, discard clear. Add 1ml 0.1MMES pH6.1 for ultrasonic resuspension, add NHS (N-hydroxysuccinimide, Thermo Fisher) 1.2mg, EDC.HCl (1-ethyl-(3-dimethylaminopropyl) carbonyl Diimine hydrochloride, Thermo Fisher) 2 mg, activated reaction at room temperature for 1 hour, centrifuged at 13000 rpm for 30 minutes, and discarded the supernatant. Ultrasonic resuspend with 1ml 0.1M MES buffer pH6.1, add 0.2mg clenbuterol hydrochloride antibody and mix well, react at room temperature (25-30°C) for 2 hours. The labeled latex was centrifuged at 13,000 rpm for 30 minutes, and the supernatant was dis...

Embodiment 2

[0050] Example 2 sample detection

[0051] A. Standard curve establishment

[0052] Prepare 1.0ml of 0.01M pH7.2 PBS solution of clenbuterol hydrochloride with a concentration of 6.4ng / ml, take 500ul and carry out doubling dilution with PBS to obtain different concentrations of clenbuterol hydrochloride solutions (3.20, 1.60, 0.80, 0.40, 0.20, 0.10, 0.05ng / ml), and PBS was used as blank control. Take 100ul standard product directly into the labeled microporous container, dissolve the labeled fluorescent latex particles attached to the wall of the container, mix well, add dropwise on the sample pad of the test strip or test card, react at room temperature for 5 minutes, and then put the test strip Insert a fluorescence reader to detect the fluorescence values ​​in the T and C areas, and the results are shown in the table below.

[0053]

[0054] B. Data processing

[0055] After obtaining the fluorescence values ​​of the T-line and C-line, calculate the T / C ratio, take th...

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PUM

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Abstract

Disclosed is a reagent for quantitative detection of a Beta-receptor stimulant through Europium chelate latex time-resolved immunochromatographic assay. The reagent includes test card or test strip which includes a nitrocellulose membrane, absorbent paper, a sample pad and a PVC bottom plate, and a microporous container. A test sample firstly dissolves out Eu3+ fluorescent latex particles which are attached in the microporous container and marked with an anti-Beta-receptor-stimulant small-molecule antibody and after sufficient mixing, the test sample reacts completely with the marker and then a reaction liquid is dropped to the test card or test strip to carry out immunochromatography and at the same time, an immune competition reaction with a Beta-receptor-stimulant small molecule and BSA conjugate which envelopes the nitrocellulose membrane is carried out and five minutes later, the test card or the test strip is inserted into a fluorescent reading meter to measure a fluorescent value so as o obtain a test result. The method is high in sensitivity and quantitative and integrates the advantages of simple and convenient operation and rapidness, the method is applied to rapid detection of veterinary drug residuals such as the Beta-receptor stimulant and the like in food and raw materials on sites of production fields such as plantation, cultivation, animal husbandry and food processing and the like.

Description

technical field [0001] The present invention applies the principle of immunology to mark specific antibodies on the latex particles coated with europium chelate, through the immunochromatography technology and the immune reaction of the corresponding coated antigen, and then achieves the quantitative detection of low-concentration β by means of a time-resolved fluorescence instrument. - Receptor agonists, used for rapid quantitative detection of β-receptor agonists and veterinary drug residues in food and feed. Background technique [0002] People pay more and more attention to food safety, especially through the "3.15 Incident" in 2011, the exposure of illegal addition of clenbuterol in animal husbandry, and the frequent occurrence of food safety accidents in recent years, such as pork clenbuterol poisoning, melamine poisoned milk powder, malachite Green cancer-causing fish and so on have made my country's food safety the focus of global attention. [0003] Although my coun...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): G01R33/02G01N21/64G01N33/558G01N33/577
Inventor 王学生杜森垚黄华胡小龙
Owner ROHI BIOTECH
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