Colloidal gold detection card for rapidly detecting melamine and preparing method thereof

A melamine, detection card technology, applied in the direction of measuring devices, instruments, scientific instruments, etc., can solve the problems of no large-volume sample detection, inability to achieve rapid detection, and high detection costs, and achieve low cost, rapid detection, and high detection accuracy. Effect

Inactive Publication Date: 2009-04-22
江苏省苏微微生物研究有限公司
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  • Abstract
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  • Claims
  • Application Information

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Problems solved by technology

Therefore, it is necessary to detect the content of melamine in feed and food. At present, there are many high-performance liquid chromatography methods. This method has good sensitivity and specificity, but the operation is cumbersome and time-consuming, and it is not suitable for detection. The detection of large quantities of samples requires expensive instruments and equipment, and the detection cost is high; it can also be detected by enzyme-linked immunosorbent assay. Although this method is relatively simple to operate, does not require expensive It needs to be washed many times, and it needs to be measured with the help of microplate reader reading, so it cannot realize the real one-step rapid detection

Method used

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  • Colloidal gold detection card for rapidly detecting melamine and preparing method thereof
  • Colloidal gold detection card for rapidly detecting melamine and preparing method thereof
  • Colloidal gold detection card for rapidly detecting melamine and preparing method thereof

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preparation example Construction

[0028] A preparation method of a colloidal gold detection card for rapid detection of melamine, comprising the following steps:

[0029] (1) Coupling melamine hapten and ovalbumin by carbodiimide method to form conjugate melamine-ovalbumin as artificial antigen;

[0030] (2) hybridoma cells were hybridized and fused with immunogen-immunized mouse spleen cells and mouse myeloma cells, and a cell line capable of stably secreting anti-melamine monoclonal antibodies was screened to prepare anti-melamine monoclonal antibodies;

[0031] (3) prepare colloidal gold solution;

[0032] (4) labeling anti-melamine monoclonal antibody with prepared colloidal gold solution;

[0033] (5) Treat the nitrocellulose membrane with the prepared coating antigen and goat anti-mouse IgG;

[0034] (6) process the gold standard pad with the prepared gold standard anti-melamine monoclonal antibody;

[0035] (7) Assemble gold standard test strips.

Embodiment 1

[0038] Preparation of melamine-ovalbumin: weigh melamine, dissolve it in pyridine solution, add 10% succinic acid solution and sodium cyanoborohydride respectively, stir and react the mixture for 1 hour at room temperature, then concentrate to dryness at low temperature to obtain Hapten, take this hapten, dissolve it in N,N-dimethylformamide (DMF) solution, add 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide (EDC) dropwise , after stirring and reacting for 20 minutes, the reaction solution was added dropwise to ovalbumin (OVA) solution, N-hydroxyl sulfosuccinimide (Sulfo-NHS) was added immediately, stirred overnight at room temperature, phosphate buffered saline ( PBS) dialyzed for 72 hours, freeze-dried and stored at -40°C; the ratio of the above reaction components is: melamine: succinic acid: sodium cyanoborohydride = 1000 mg: 4000 μl: 6 mg, hapten: EDC: OVA: NHS = 2000 mg : 5000mg: 10mg: 2500mg.

[0039] Preparation of melamine-bovine serum albumin conjugated antigen: Weigh...

Embodiment 2

[0046] The preparation of the melamine-ovalbumin: weigh melamine, dissolve it in pyridine solution, add 20% succinic acid solution and sodium cyanoborohydride respectively, stir and react the mixture at room temperature for 3 hours, then concentrate to dryness at low temperature, To obtain the hapten, take the hapten, dissolve it in N,N-dimethylformamide (DMF) solution, add 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide (EDC) dropwise ), after stirring and reacting for 20 minutes, the reaction solution was added dropwise to the ovalbumin (OVA) solution, and N-hydroxysulfosuccinimide (Sulfo-NHS) was added immediately, stirred overnight at room temperature, and phosphate buffer saline (PBS) dialysis for 48 hours, freeze-dried and stored at -30°C; the ratio of the above reaction components is: melamine: succinic acid: sodium cyanoborohydride = 10 mg: 8000 μl: 300 mg, hapten: EDC: OVA: NHS = 10mg: 10000mg: 1000mg: 5000mg.

[0047] Preparation of the melamine-bovine serum albumin c...

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Abstract

The invention provides a colloidal gold cassette for rapid detection of melamine, the colloidal gold cassette has rapid detection, convenient carry and simple operation, the invention also provides a preparation method of the cassette, the preparation process thereof is simple, and the cost is low. The colloidal gold cassette comprises a test strip, the test strip is packaged in a shell body of the cassette, a sample adding hole and an observation hole are arranged on the shell body of the cassette, the test strip comprises a bottom plate, the colloidal gold cassette is characterized in that water absorbent paper, a nitrocellulose membrane, a gold-labeled pad and a sample pad are sequentially arranged on the bottom plate of the test strip, the water absorbent paper and the gold-labeled pad are respectively arranged at the two ends of the nitrocellulose membrane, small sections of the water absorbent paper and the gold-labeled pad are respectively overlapped on the nitrocellulose membrane at the junctions at the two ends of the nitrocellulose membrane, the sample pad is arranged at the other end of the gold-labeled pad, a small section of the sample pad is overlapped on the gold-labeled pad; a gold-labeled anti-melamine monoclonal antibody is sprayed on the gold-labeled pad; a detection line with the material of a melamine-ovalbumin coupled conjugate and a quality control line with the material of goat anti-mouse IgG are sequentially sprayed on the nitrocellulose membrane.

Description

(1) Technical field [0001] The invention relates to the technical field of rapid detection of melamine content in feed and food, in particular to a colloidal gold detection card for rapid detection of melamine and a preparation method thereof. (2) Background technology [0002] Melamine is a triazine-type nitrogen-containing heterocyclic organic compound, an important nitrogen-heterocyclic organic chemical raw material, referred to as triamine, also known as melamine, melamine, and cyanuronamide. Melamine is a basic organic chemical intermediate product with a wide range of uses, and its main use is as a raw material for the production of melamine-formaldehyde resin (MF). Because the melamine molecule contains a lot of nitrogen, it is often artificially added to animal feed and plant protein to increase the protein content in the product. Long-term or repeated consumption of feed or food containing melamine in large quantities will cause damage to the reproductive and urinar...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): G01N33/577G01N33/543
Inventor 赵晓联赵春城杨婷婷蔡建荣叶进秦海萍张凌裳龚燕吴杰沈雯琰王文静
Owner 江苏省苏微微生物研究有限公司
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