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Ractopamine-clenbuterol fluorescent microsphere detection test strip and preparation method and application

A technology of ractopamine and fluorescent microspheres, applied in measurement devices, instruments, scientific instruments, etc., can solve the problems of carcinogenicity, low sensitivity, and expensive microplate readers.

Inactive Publication Date: 2012-12-19
JINAN UNIVERSITY
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

High-performance liquid chromatography (HLCP), liquid chromatography-mass spectrometry (LC-MC), and gas chromatography-mass spectrometry (GC-MC) are physical and chemical detection methods. These methods have high sensitivity and good specificity, but the operation Complicated, long sample pretreatment process, expensive detection equipment and professional operators
Enzyme-linked immunosorbent assay (ELISA) and colloidal gold immunochromatography are immunological detection techniques. The ELISA method has high detection sensitivity, and there are many steps of adding samples and washing. The process is relatively cumbersome and time-consuming. The substrates used for color development are Carcinogenicity, the price of the microplate reader used to read the value is relatively expensive; the colloidal gold immunochromatography technology is easy and fast to operate, but the sensitivity is low, and it is only suitable for the primary screening of a large number of samples and it needs to be exposed to heavy metal ions

Method used

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  • Ractopamine-clenbuterol fluorescent microsphere detection test strip and preparation method and application
  • Ractopamine-clenbuterol fluorescent microsphere detection test strip and preparation method and application
  • Ractopamine-clenbuterol fluorescent microsphere detection test strip and preparation method and application

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0103] (1) Preparation of ractopamine protein conjugates: Weigh 50 mg of ractopamine hydrochloride and 15 mg of succinic anhydride and dissolve them in anhydrous pyridine, stir and react at room temperature for 24 hours, and drain with a centrifugal concentrator to obtain derivative A; dissolve derivative A In N,N'-dimethylformamide (DMF), add N-hydroxysuccinimide (NHS) 11mg, dicyclohexylcarbodiimide (DCC) 20mg and react overnight at room temperature; centrifuge at 1000rpm, Take the supernatant to obtain solution B; weigh 250mg of bovine serum albumin and dissolve it in 0.015M, pH=7.4 phosphate buffer, pre-cool at 4°C; add solution B to the bovine serum albumin dropwise under the condition of stirring in an ice bath In the solution, stir and react overnight at 4°C; centrifuge at 5000rpm at 4°C for 5 minutes, and take the supernatant to obtain liquid C; place liquid C at 4°C, dialyze in 0.015M, pH=7.4 phosphate buffer for three days to obtain ractopamine protein conjugates.

...

Embodiment 2

[0114] Carry out following test, verify the detection specificity of the detection test strip prepared with the present invention:

[0115] a. Prepare serial gradient standard solutions of ractopamine (RAC), clenbuterol (CL) and salbutamol (SAL) with phosphate buffer (concentrations are 0, 0.5, 1, 2, 3, 5, 20ng respectively) / ml).

[0116] b. Use a dropper to absorb 3 drops (about 100 μl) of the standard solution obtained in step a, drop them on the sample absorption pad of the test strip, and start timing after adding the sample;

[0117] c. Interpret the results after 10 minutes under the irradiation of 450nm excitation light;

[0118] By carrying out the specific cross-reaction test to the prepared ractopamine-clenbuterol fluorescent microsphere detection test strip, the results are as follows:

[0119] Table 1 shows the cross-reactivity results of ractopamine, clenbuterol and other analogues

[0120]

[0121]

[0122] The results showed that the ractopamine-clenbu...

Embodiment 3

[0124] Carry out following test, detect the stability of the test strip prepared by embodiment 1:

[0125] Every 5 days, take out the ractopamine-clenbuterol fluorescent microsphere detection test strips prepared in Example 1 from the aluminum foil bag in airtight storage, detect the same sample with different detection test strips of the same batch, and use different Batch detection test strips measure the same sample, and the color development time and fluorescence intensity of the quality control line and detection line under the irradiation of 450nm excitation light and the final result interpretation are the same. The results showed that the ractopamine-clenbuterol fluorescent microsphere test strip had good stability in detecting ractopamine and clenbuterol.

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Abstract

The invention discloses a ractopamine-clenbuterol fluorescent microsphere detection test strip and a preparation method and an application. The test strip comprises a sample absorption pad, a combined pad, a chromatography membrane and a water absorption pad, which are adhered onto a soleplate and sequentially and closely connected with one another; the combined pad comprises fluorescent polystyrene microsphere labeled protein; the chromatography membrane is provided with two detection lines and one quality control line, the detection lines are close to the combined pad, and the quality control line is close to the water absorption pad; one detection line is coated with ractopamine protein conjugate, the other detection line is coated with clenbuterol protein conjugate, the quality control line is coated with protein conjugate which can be combined with proteins in the fluorescent polystyrene microsphere labeled protein and is not combined with the ractopamine and clenbuterol; and the fluorescent polystyrene microsphere labeled protein is ractopamine monoclonal antibody and a clenbuterol monoclonal antibody labeled by the fluorescent polystyrene microsphere. The test strip can simultaneously detect the ractopamine and the cleubuterol and is rapid, simple, convenient, flexible and accurate in detection.

Description

technical field [0001] The invention belongs to the field of food safety inspection and relates to a ractopamine-clenbuterol fluorescent microsphere detection test strip and its preparation and application. Background technique [0002] Ractopamine (RAC) and clenbuterol (CL) are both β-stimulant drugs, which can strengthen heart contraction, expand skeletal muscle blood vessels and bronchial smooth muscle, and are used in veterinary medicine and medicine to treat shock and bronchospasm. Because these two drugs can promote animal skeletal muscle hyperplasia, some farmers illegally use them as feed additives to increase the lean meat rate of poultry and livestock products and increase economic benefits. my country has banned the production, sale and use of these two substances. After being ingested by animals, ractopamine (RAC) and clenbuterol (CL) will remain in the muscle, liver, and lung tissues of animals. Humans will have a series of toxic and side effects after eating t...

Claims

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Application Information

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IPC IPC(8): G01N33/577G01N33/558
Inventor 唐勇崔浩
Owner JINAN UNIVERSITY
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