Fluorescent microsphere-colloidal gold double-color-development qualitative and quantitative immunochromatographic test strip for detecting ractopamine, and preparation method of test strip

An immunochromatographic test paper and ractopamine technology, which is applied in the field of veterinary drug residue detection in food safety, can solve the problems of large sample background interference, long time consumption, and narrow detection linear range, so as to eliminate false positive results, avoid matrix interference, The effect of saving inspection time

Active Publication Date: 2018-12-21
江西中德生物工程股份有限公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0005] ⑴General test strips are qualitatively analyzed by visual observation, and accurate quantitative detection cannot be achieved
[0006] (2) The matrix effect of different materials is obvious, and the sample background interference is large, which is easy to produce false positive results
[0007] (3) The positive result of the test cannot be saved, usually after the judgment time is exceeded, the result is no longer accurate and reliable
[0009] ⑴In the actual test, the proportion of negative results is very high, but all test strips of this method need to be read by an instrument, otherwise the results cannot be obtained, and it takes a long time to test a large number of samples!
[0010] ⑵In quantitative detection, the detection department still needs a threshold to distinguish negative and positive, so the amount of data processing is relatively large;
[0011] (3) The sensitivity of a single fluorescent microsphere test strip is high, but in practical applications, the detection linear range of the higher sensitivity is narrower, and the quantitative concentration range of the sample is not suitable for the actual detection requirements

Method used

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  • Fluorescent microsphere-colloidal gold double-color-development qualitative and quantitative immunochromatographic test strip for detecting ractopamine, and preparation method of test strip
  • Fluorescent microsphere-colloidal gold double-color-development qualitative and quantitative immunochromatographic test strip for detecting ractopamine, and preparation method of test strip
  • Fluorescent microsphere-colloidal gold double-color-development qualitative and quantitative immunochromatographic test strip for detecting ractopamine, and preparation method of test strip

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0055] Example 1: Preparation of Fluorescent Microsphere-Colloidal Gold Double Chromogenic Qualitative and Quantitative Immunochromatography Test Strip for Detection of Ractopamine (Optimal Ratio of Fluorescent Microsphere and Colloidal Gold Antibody Labeling Complex)

[0056] 1. Preparation process of immunochromatographic test strips

[0057] 1. Preparation of nitrocellulose membrane;

[0058] (1) Preparation of ractopamine artificial antigen (RAC-BSA)

[0059] The coupling method is 1,4-butylene ether method, the coupling protein is bovine serum albumin, and the coupling ratio is 1:10-1:100. After coupling, dialysis is purified to obtain RAC-BSA.

[0060] (2) Preparation of detection line and quality control line

[0061] RAC-BSA conjugates and goat anti-mouse antibodies were coated onto nitrocellulose membranes: Dilute the RAC-BSA conjugates with 0.02M pH 7.5 PBS (phosphate buffered saline) to make the concentration 0.5mg / mL, The resulting solution was sprayed on the me...

Embodiment 2

[0079] Example 2: Preparation of fluorescent microspheres-colloidal gold dual-color qualitative and quantitative immunochromatographic test strips for detection of ractopamine (the proportion of fluorescent microsphere-labeled complexes increases)

[0080] The difference with Example 1 is:

[0081] (1) After the preparation of fluorescent microsphere antibody complex and colloidal gold antibody complex is completed, calculate according to the amount of antibody on different markers in each test strip, and the accurate amount of colloidal gold antibody is 20ng / strip, The amount of antibody was sprayed on the conjugate pad at a marker concentration of 12ng / strip. Spray it onto a 30×0.8cm glass fiber membrane, dry it in vacuum at 25°C for 1-2 hours, and put it in a drying cabinet for later use. The quantitative detection results of fluorescent microspheres are: under this condition, the concentration of the standard curve is: 0, 0.1, 0.3, 0.9, 1.5, 2.7ng / mL, R 2 0.8977, IC 50 ...

Embodiment 3

[0085] Example 3: Preparation of fluorescent microspheres-colloidal gold dual-color qualitative and quantitative immunochromatographic test strips for detection of ractopamine (the proportion of colloidal gold-labeled complexes increases)

[0086] The difference with Example 1 is:

[0087] (1) After the preparation of fluorescent microsphere antibody complex and colloidal gold antibody complex is completed, calculate according to the amount of antibody on different markers in each test strip, and the accurate amount of colloidal gold antibody is 40ng / strip, The amount of antibody was sprayed on the conjugate pad at a marker concentration of 6 ng / strip. Spray it onto a 30×0.8cm glass fiber membrane, dry it in vacuum at 25°C for 1-2 hours, and put it in a drying cabinet for later use. The quantitative detection results of fluorescent microspheres are: under this condition, the concentration of the standard curve is: 0, 0.1, 0.3, 0.9, 1.5, 2.7ng / mL, R 2 0.9012, IC 50 0.46ng / mL...

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Abstract

The invention discloses a fluorescent microsphere-colloidal gold double-color-development qualitative and quantitative immunochromatographic test strip for detecting ractopamine, and a preparation method of the test strip. A bottom plate is lapped and pasted with filter paper, a sample pad, a glass fiber mat sprayed with a fluorescent microsphere-ractopamine monoclonal antibody complex and a colloidal gold-ractopamine monoclonal antibody complex, a nitrocellulose membrane and water absorption paper in sequence; and the nitrocellulose membrane is coated with a ractopamine coupled antigen as a detection line and is coated with an anti-mouse antibody as a quality control line. When the color development degree of the detection line is lower than that of the quality control line, the content of the ractopamine in a sample is greater than 0.5ng/ml, and the test strip is qualitatively judged to be positive. The test strip qualitatively judged to be positive is directly inserted into a fluorescence reader, and the reader performs numeralization on a fluorescent signal of a fluorescent microsphere color development system so as to realize quantitative detection. The test strip is mainly used for qualitative and quantitative detection of the ractopamine in food safety detection.

Description

technical field [0001] The invention belongs to the field of detection of veterinary drug residues in food safety, and in particular relates to a time-resolved fluorescent microsphere-colloidal gold dual-color qualitative and quantitative immunochromatographic test strip capable of qualitatively and quantitatively detecting ractopamine in a sample and a preparation method thereof. Background technique [0002] Ractopamine (RAC) is a phenolamine β-2 adrenergic receptor agonist, which, like clenbuterol hydrochloride, can promote nutrient redistribution and growth, so it is used as a new type of additive to promote body growth. Because ractopamine has the characteristic of accumulating residues in animals, animals fed with ractopamine additives will cause people to have varying degrees of toxic reactions, specifically manifested as increased skeletal muscle contraction and muscle tremors. In animal husbandry production, some illegal users add it as a growth promoter to animal f...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): G01N33/94G01N33/58G01N33/558G01N33/543G01N33/533G01N33/532
CPCG01N33/532G01N33/533G01N33/54313G01N33/558G01N33/58G01N33/582G01N33/9413
Inventor 陈媛赖卫华罗凯刘文娟伍燕华
Owner 江西中德生物工程股份有限公司
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