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Enzyme-linked immunosorbent assay (ELISA) detection kit for detecting ractopamine as well as preparation method and application of detection kit

An enzyme-linked immunoassay and ractopamine technology, applied in the direction of measuring devices, instruments, scientific instruments, etc., can solve the problems of high detection limit, complicated instrument operation, expensive instruments, etc., achieve strong biocompatibility, simple synthesis steps, The effect of simple separation

Active Publication Date: 2015-05-27
NINGBO UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, they all have problems such as complicated instrument operation, expensive instruments, and long time consumption, which make them difficult to be used for on-site rapid screening
Although the rapid detection test strip is convenient, it can only be used qualitatively, not quantitatively.
At the same time, the traditional ractopamine ELISA detection kit generally uses the competitive method, although it is fast and effective, there are still shortcomings such as too high detection limit and false positives.
At present, there is no published information on the use of Fe 3 o 4 β-CD as a capture probe and PtNPs / PV complex as an enzyme-labeled antibody for the detection of ractopamine in an immunocolorimetric assay

Method used

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  • Enzyme-linked immunosorbent assay (ELISA) detection kit for detecting ractopamine as well as preparation method and application of detection kit
  • Enzyme-linked immunosorbent assay (ELISA) detection kit for detecting ractopamine as well as preparation method and application of detection kit
  • Enzyme-linked immunosorbent assay (ELISA) detection kit for detecting ractopamine as well as preparation method and application of detection kit

Examples

Experimental program
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Effect test

specific Embodiment 1

[0032] An ELISA kit for detecting ractopamine, comprising a microtiter plate and a signal probe, the microtiter plate is immobilized with a ractopamine antigen and a capture probe Fe 3 o 4 β-CD, the signal probe is a complex of PtNPs / PV loaded with a large amount of PtNPs, the standard product is ractopamine standard product, the antibody working solution is anti-ractopamine antibody monoclonal antibody, chromogenic solution A and chromogenic solution B are Chromogenic reagent DAB, stop solution is 2M HCl or 2M H 2 SO 4 , the washing solution is PBST buffer.

[0033] The colorimetric method is mainly composed of Fe 3 o 4 β-CD magnetic nanoparticles are used as capture probes to capture ractopamine (RAC) in the solution, and a non-competitive immunological method is used to make RAC on the capture probes specifically bind to the added antibody, which can specifically bind to PtNPs / PV Antibodies on the complex, incubated and magnetically separated, leaving an immune complex...

specific Embodiment 2

[0034] A preparation method for an enzyme-linked immunoassay kit for detecting ractopamine, the specific steps are as follows:

[0035] (1) Fe 3 o 4 -NH 2 preparation of

[0036] Anhydrous sodium acetate, hexamethylenediamine (1,6-hexanediamine) and FeCl 3 ?6 H 2 Add O to ethylene glycol after mixing, stir at 42-60°C to obtain iron source solution, transfer the iron source solution to a muffle furnace, react at 195-205°C for 6-8 hours, and separate with a magnet , and then washed with water and ethanol for 2-3 times to remove the incompletely reacted solvent to obtain Fe with a particle size of 15-25 nm 3 o 4 -NH 2 Magnetic nanoparticles; of which anhydrous sodium acetate, hexamethylenediamine (1,6-hexanediamine), FeCl 3 ?6 H 2 The mixing ratio of O and ethylene glycol is 4g: 13g: 2g: 30ml; figure 1 For the prepared Fe 3 o 4 -NH 2 The scanning electron microscope image; The prepared magnetic Fe 3 o 4 -NH 2 The particle size is about 17nm, and its surface is...

specific Embodiment 3

[0046] A method for detecting the concentration of ractopamine by applying the above enzyme-linked immunoassay kit for detecting ractopamine, using Fe 3 o 4 β-CD magnetic nanoparticles are used as capture probes to capture RAC in the solution. Non-competitive immunological methods are used to make RAC on the capture probe specifically bind to the added antibody, and the free antigen-antibody conjugates are removed by washing the plate and magnetic separation. and other free matter; then add colloidal platinum-labeled PV to specifically bind to the antibody, and after incubation and plate washing, an immune complex consisting of ractopamine antigen, antibody, and colloidal platinum-labeled PV remains. The amount of this immune complex decreases with increasing ractopamine in the sample. Finally, DAB was added for color development and colorimetric analysis. Determine the concentration of ractopamine in the sample to be tested with the results of colorimetric analysis (princip...

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Abstract

The invention discloses an enzyme-linked immunosorbent assay (ELISA) detection kit for detecting ractopamine as well as a preparation method and the application of the detection kit. The detection kit is characterized in that Fe3O4@beta-CD is taken as a capture probe, and a compound of colloidal platinum particles and polymerase chelate PV is taken as a signal probe; the preparation method of the detection kit comprises the steps of preparing Fe3O4-NH2; preparing the capture probe Fe3O4@beta-CD; preparing PtNPs; preparing a PtNPs / PV signal label; and preparing an immune complex. The application comprises the following steps: feeding 50mu L of liquid A and 50mu L of liquid B of diaminobenzidine (DAB) into the obtained immune complex; carrying out light-avoiding incubation for 15-30 minutes; measuring the absorbance of the obtained DAB developed product by a microplate reader; drawing a standard curve according to the relation between corresponding light absorption values and concentrations of the ractopamine; and determining the concentration of the ractopamine in a sample to be tested according to the standard curve. The detection kit has the advantages of being high in sensitivity, selectivity and accuracy as well as rapid in detection speed.

Description

technical field [0001] The invention relates to ractopamine detection technology, in particular to a method based on Fe 3 o 4 An enzyme-linked immunoassay kit for detecting ractopamine using β-CD as a capture probe and PtNPs / PV as a signal probe, as well as its preparation method and application. Background technique [0002] beta 2 Receptor agonists (β-adrenergic agonists) are often used as veterinary medicine because they can relieve asthma symptoms; however, they are also often added to feed because they can increase muscle and reduce fat accumulation. Ractopamine (RAC) as beta 2 One of the receptor agonists is a synthetic Clonbaan, which is being used as a new type of clenbuterol in some pig farms. The "Catalogue of Drugs Prohibited for Use in Feed and Animal Drinking Water" jointly issued by the Ministry of Agriculture, the Ministry of Health, and the State Food and Drug Administration stipulates that ractopamine is prohibited from being used as a growth promoter ...

Claims

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Application Information

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IPC IPC(8): G01N33/577G01N33/543
CPCG01N33/543G01N33/9413
Inventor 潘道东陈淑贤孙杨赢曹锦轩曾小群吴振
Owner NINGBO UNIV
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