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Ractopamine residual time resolution immunoassay kit and detection method thereof

A ractopamine and detection kit technology, which is applied in the field of immunodetection and can solve the problem of lack of a ractopamine time-resolved immunoassay detection kit and the like

Inactive Publication Date: 2013-12-25
SOUTH CHINA AGRI UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0010] However, there is currently no related technical report on the detection of ractopamine by time-resolved immunoassay, and there is no kit suitable for time-resolved immunoassay detection of ractopamine in order to achieve high-sensitivity, simple-to-operate large-scale rapid detection

Method used

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  • Ractopamine residual time resolution immunoassay kit and detection method thereof
  • Ractopamine residual time resolution immunoassay kit and detection method thereof
  • Ractopamine residual time resolution immunoassay kit and detection method thereof

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Experimental program
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Effect test

Embodiment 1

[0080] The preparation of embodiment 1 hapten

[0081] The synthetic route to the ractopamine hapten is shown below:

[0082]

[0083] Dissolve ractopamine hydrochloride and succinic anhydride in anhydrous pyridine and stir at room temperature. After the reaction was completed, pyridine and unreacted raw materials were removed by extraction, and evaporated to dryness on a vacuum rotary evaporator under reduced pressure to obtain the crude product of the hapten ractopamine-succinate. The above post-reaction treatment process was monitored in real time by TLC. The crude product was purified by column chromatography to obtain a relatively pure hapten, which was identified by ESI-MS.

[0084] (+)ESI-MS full scan mass spectrum of ractopamine hapten see figure 1 As shown, the molecular ion peak of m / z 400 appears in the figure, which corresponds to the molecular mass of the hapten. In order to further confirm its structure, (+) ESI-MS2 analysis was carried out on the molecula...

Embodiment 2

[0085] The preparation of embodiment 2 antigen

[0086] Preparation of ractopamine antigen:

[0087] a. Dissolve 50 μmol / L ractopamine hapten in 1 mL of dimethylformamide (DMF), then add equimolar dicyclohexylcarbodiimide (DCC) and N-hydroxysuccinimide to the solution Amine (NHS), let it react overnight at room temperature;

[0088] b. Centrifuge, take 800 μL of supernatant, slowly add to 4 mL of 15 mg / mL BSA or OVA carrier protein carbonic acid buffer solution, and then react for 4 hours under magnetic stirring;

[0089] c. After the reaction is completed, put it into a dialysis bag, first dialyze twice with distilled water, and then dialyze with 0.8% normal saline to obtain the product;

[0090] d. The binding ratio was determined by ultraviolet scanning (Chen Xinxin et al., 1998), and finally the antigen was concentrated or lyophilized to obtain the ractopamine immunogen and the coating agent, which were stored in a refrigerator at -20°C.

Embodiment 3

[0091] The preparation of embodiment 3 antibody

[0092] Preparation of monoclonal antibodies:

[0093] Animal immunization procedure: Balb / c mice were used as immunized animals, the ractopamine hapten and bovine serum albumin conjugate was used as the immunogen, the immunization dose was 60 μg / mouse, and the immunogen was mixed with the same amount of Freund’s The complete adjuvant was mixed to make an emulsifier, which was injected intraperitoneally. The same dose of immunogen and the same amount of Freund's incomplete adjuvant were mixed and emulsified at intervals of 3 weeks, and a booster immunization was given.

[0094] Cell fusion and cloning: Splenocytes from immunized Balb / c mice were fused with SP2 / 0 myeloma cells at a ratio of 4:1. Cell supernatants were measured by indirect competitive time-resolved immunoassay, and positive wells were screened. The positive wells were cloned by microcloning until a hybridoma cell line stably secreting the monoclonal antibody was ...

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Abstract

The invention provides a ractopamine residual time resolution immunoassay kit and a detection method thereof. The kit of the invention contains an ELISA plate of ractopamine antigen, lanthanide labelled goat anti rabbit or goat anti mouse antibody and ractopamine. The invention also discloses a method for detecting ractopamine residue by applying the kit. The kit for detecting ractopamine provided by the invention adopts indirect competitive time resolution immunoassay technology, sensitivity is high, stability is good, operation steps are greatly simplified and reaction time is reduced, error caused by complex operation is reduced, and cost is reduced, thus being applicable to screening of massive samples and having important reality significance.

Description

technical field [0001] The invention belongs to the technical field of immune detection, and relates to a detection kit for chemical pollution residues and a preparation method thereof, in particular to a time-resolved immunoassay kit for detecting ractopamine residues and a preparation method thereof. Background technique [0002] Food safety is a major event related to the daily life of the broad masses of people. Food safety issues mainly include several factors such as physical hazards, chemical hazards and microbial hazards in food, among which chemical hazards mainly include veterinary drug residues, pesticide residues, heavy metals, and environmental hazards. [0003] In the past few decades, with the development of intensive farming, veterinary drugs have been widely used as additives and drugs in animal husbandry, and the resulting problem of veterinary drug residues in animal foods has become increasingly prominent. It mainly refers to the accumulation of drugs in...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): G01N33/543G01N33/577G01N1/28C07K16/44C07K16/06C12N15/12C12N15/70
Inventor 孙远明徐振林王弘雷红涛李丽华沈玉栋杨金易李振峰
Owner SOUTH CHINA AGRI UNIV
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