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Testing kit for enrofloxacin and testing method thereof

A technology of enrofloxacin and reagent kits, applied in the direction of measuring devices, instruments, scientific instruments, etc., can solve the problems of limited application, expensive, cumbersome sample processing, etc., and achieve the effects of high sensitivity, simple structure, and convenient use

Inactive Publication Date: 2010-05-19
JIANSGU INST OF MICROBIOLOGY
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Chromatographic detection is sensitive and accurate, requires expensive instruments and specialized staff, and sample processing is cumbersome, so its application is limited, especially in large-scale screening and on-site testing at the grassroots level

Method used

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  • Testing kit for enrofloxacin and testing method thereof
  • Testing kit for enrofloxacin and testing method thereof
  • Testing kit for enrofloxacin and testing method thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0012] Example 1: Preparation of kit and detection of food of animal origin

[0013] Preparation of immune antigen:

[0014] The artificial immune antigen of the present invention combines enrofloxacin (ENR) with a macromolecule carrier protein by a mixed anhydride method, and uses synthetic ENR-BSA as an immune antigen to immunize animals.

[0015] The carrier protein is bovine serum albumin (BSA). Weigh 30mg~300mg bovine serum albumin, dissolve in 6mL~10mL of 50% dioxane aqueous solution, adjust the pH value to 9~10, and make it A liquid; weigh 10mg~40mg ENR, dissolve in 2mL dioxane Set it at 4°C for 10 minutes, add 20 μL of tri-n-butylamine and 10 μL of isobutyl chloroformate in sequence, stir at 4°C for 30 minutes, and use it as liquid B. Add liquid A dropwise to liquid B at 4°C, and stir overnight. The reaction solution was dialyzed against distilled water for 72 hours, during which the distilled water was changed 5 times. Or purified by Sephadex G50 column and eluted...

Embodiment 2

[0056] Implementation example 2 preparation kit

[0057] ENR-BSA antigen preparation:

[0058] Weigh 12mg ENR, 40mg N-hydroxysuccinamide (NHS), 35mg carbodiimide (EDC) and dissolve in N,N′-dimethylformamide (DMF), stir at room temperature for 24h to prepare A solution; Dissolve 50mg of BSA in 3mL of 0.01mol / mL pH7.4 PBS to prepare solution B. Add solution A dropwise to solution B, stir while adding, react at room temperature for 3 hours, dialyze the reaction solution in distilled water for 72 hours, and change distilled water 5 times during the period. Or purified by Sephadex G50 column and eluted with 0.05mol / L pH8.0 carbonate buffer solution. Collect the dialysis purified liquid or the column eluate of the first peak, aliquot and store at -20°C for immunogen use.

[0059] Preparation of polyclonal enrofloxacin antibody: same as in Example 1, omitted.

[0060] Eu 3+ -Preparation of goat anti-rabbit antibody: same as Example 1, omitted.

[0061] Preparation of solid-phas...

Embodiment 3

[0068] The reagent provided by the kit is the same as that in Example 1, and is used to detect honey samples.

[0069] Reagents that should be prepared by the laboratory:

[0070] (1) 0.02mol / L PB buffer solution: weigh Na 2 HPO 4 12H 2 O 5.16g, NaH 2 PO 4 0.87g into the volumetric flask, add water to dissolve and make the volume to 1L;

[0071] (2) dichloromethane solution;

[0072] (3) 0.02mol / L PB buffer solution: weigh Na 2 HPO 4 12H 2 O 5.16g, NaH 2 PO 4 0.87g into the volumetric flask, add water to dissolve and make the volume to 1L;

[0073] (4) Phosphate buffer working solution: pH7.2, take 1.45g disodium hydrogen phosphate (containing 12 crystal water), 0.1g potassium dihydrogen phosphate (anhydrous), 8.0g sodium chloride, dissolve in water and set Dissolve to 1000mL.

[0074] Matters needing attention before determination: same as Example 1.

[0075] The specific detection steps are as follows:

[0076] Accurately weigh 1.0g±0.01g of honey sample into ...

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Abstract

A testing kit for enrofloxacin and a testing method thereof belong to the TRFIA field, used for testing the ENR content in animal-based food, blood, urine and forage. The kit prepared in the invention uses RRFIA to detect ENR, wherein the testing base is marking immunization response. A micro-hole plate is coated by ENR-carrier protein, is added with ENR standard or sample and ENR antibody. Dissociative ENR competes against ENR-carrier protein on the micro-hole plate for ENR antibody. The unconnected ENR antibody is washed off and then Eu3+- goat antibody to rabbit is added. Unconnected Eu3+- goat antibody to rabbit is washed off after marking immunization response. Fluorescence intensity cps is tested by using a time-resolved fluoroimmunoassay instrument after adding enhancing liquid. The fluorescence intensity has inverse ratio to the ENR concentration in sample. The ENR content in the tested sample can be immediately confirmed by comparing standard curve. The kit for testing ENR of the invention has simple structure, convenient use, low cost, and high sensitivity which can reach to 0.01ng / ml.

Description

technical field [0001] The invention relates to a detection kit for detecting Enrofloxacin (Enrofloxacin, ENR) and a detection method thereof, belonging to the technical field of time resolved fluoroisnmunoassay (TRFIA), and used for animal-derived food, blood and urine And the detection of enrofloxacin drug residues in feed. Background technique [0002] Enrofloxacin (Enrofloxacin, ENR) is a synthetic quinolone antibacterial drug, because of its high efficiency and broad spectrum, few drug-resistant bacteria, fast absorption in animals, wide distribution, high bioavailability, and fast metabolism, it is a low toxicity. , low-accumulation drugs, and no cross-resistance with other antibacterial drugs, it is widely used in the prevention and treatment of infectious diseases in livestock, poultry and aquaculture. Enrofloxacin is widely used in livestock and poultry breeding and aquaculture. It will enter the soil and water environment with the excrement of livestock and poultr...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): G01N33/543G01N33/533
Inventor 陆茂林李利东宓晓黎袁建兴黄丽俊陈蕴
Owner JIANSGU INST OF MICROBIOLOGY
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