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Immunofluorescence detection method for detection of enrofloxacin based on quantum dots and special kit thereof

A technology for immunofluorescence detection and enrofloxacin, applied in fluorescence/phosphorescence, measuring devices, instruments, etc.

Inactive Publication Date: 2013-11-06
SHENZHEN GRADUATE SCHOOL TSINGHUA UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

[0004] Since the quantum dot labeling materials suitable for immunodiagnostic reagents must meet the requirements of high quantum yield, strong fluorescence, good biocompatibility, high stability and low cost, the quantum yield of the current foreign commercialized quantum dots is mostly in the range of 40 % or less, its size is relatively small, and it needs to be irradiated and excited by laser optical instruments. At present, it can only be used in cell immunofluorescence detection and flow cytometry detection and sorting. Therefore, there is no quantum dot immunoassay reagent on the international market.

Method used

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  • Immunofluorescence detection method for detection of enrofloxacin based on quantum dots and special kit thereof
  • Immunofluorescence detection method for detection of enrofloxacin based on quantum dots and special kit thereof
  • Immunofluorescence detection method for detection of enrofloxacin based on quantum dots and special kit thereof

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Experimental program
Comparison scheme
Effect test

Embodiment 1

[0048] Embodiment 1, composition and preparation of detection kit

[0049] Enrofloxacin was purchased from Beijing Hengyuan Qitian Chemical Technology Research Institute, the product catalog number is 30235CDCT-C13170000. Its chemical name is 1-cyclopropyl-7-(4-ethyl-1-piperazinyl)-6-fluoro-1,4-dihydro-4-oxo-3-quinolinecarboxylic acid. Enrofloxacin is a quinolone drug, the molecule itself has a carboxyl group and can be directly coupled to proteins.

[0050] 1. Enrofloxacin coated original

[0051] The conjugate of enrofloxacin and carrier protein BSA, enrofloxacin and BSA are connected through the peptide bond formed by the carboxyl group in enrofloxacin and the amino group in BSA.

[0052] The preparation method of enrofloxacin coating former is as follows:

[0053] 1) Mix 35.9mg of enrofloxacin, 3ml of N,N-dimethylformamide (DMF) and 3ml of dioxane (dioxane itself is a solution) to obtain a mixture a, and the mixing temperature is 25°C , the mixing time is 30min, and th...

Embodiment 2

[0074] Embodiment 2, the preparation method of standard curve

[0075] Add the enrofloxacin standard solution that concentration is 0,0.001,0.005,0.01,0.05,0.1,0.5,1,5,10ug / L in the prepared enrofloxacin microwell strip, 50ul / hole, will Dilute quantum dot-labeled ENR antibody with PBS-T diluent 1:50 [25ug labeled antibody: 5ml diluent], add 50ul to each microwell, shake at room temperature for 1 hour, wash with washing solution 3 times and use a fluorescent microplate reader Check its fluorescence intensity value. The fluorescent microplate reader was set to an excitation wavelength of 345 nm and an emission wavelength of 620 nm.

[0076] When the detection of competition detection is limited to B0 / B=1.2 (B0 is 0 standard sample detection value, B is the mass concentration of the competition drug of testing sample detection value), according to the curve regression equation ( figure 2 ) to determine the sensitivity of the detection system. The detection results are shown ...

Embodiment 3

[0079] Embodiment 3, the mensuration of cross reaction

[0080] Choose ciprofloxacin (Beijing Hengyuan Qitian Chemical Technology Research Institute, 30233 CDCT-C11668500), dafloxacin (Sigma-aldrich, 33700-100MG-R), norfloxacin (Beijing Hengyuan Qitian Chemical Technology Research Institute, 30234 CDCT-C15648000), enoxacin (Beijing Hengyuan Qitian Chemical Technology Research Institute, 29641NIC-130453), ofloxacin (Beijing Hengyuan Qitian Chemical Technology Research Institute, 30237 CDCT-C15717000) and Penoxacin (Sigma-aldrich, 33899-100MG-R) was formulated into a series of concentrations, and detected with quantum dot kit strips. Calculate the IC50 of each competitor, and use the following formula to calculate the cross-reaction rate of these five drugs and the enrofloxacin quantum dot kit respectively. The calculation formula is: cross-reaction rate (%)=[IC50 (enrofloxacin) / IC50 (drug to be tested)]×100.

[0081] The measurement and calculation results are shown in Table ...

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Abstract

The invention discloses an immunofluorescence detection method for the detection of enrofloxacin based on quantum dots and a special kit thereof. The immunofluorescence detection kit for the detection of enrofloxacin, which is provided in the invention, comprises an enrofloxacin coating antigen and a quantum dot labeled enrofloxacin antibody. The method of the invention, which can realize the quantitative detection of enrofloxacin residues and has the advantages of low detection limit, high detection sensitivity and good specificity, is also suitable for the detection of various samples. So the method and the kit thereof have wide application prospects in the enrofloxacin detection fields.

Description

technical field [0001] The invention relates to a method for detecting enrofloxacin by immunofluorescence based on quantum dots and a special kit. Background technique [0002] Enrofloxacin (ENR) is a quinolone antibacterial drug, because it can inhibit bacterial DNA helicase, has a wide antibacterial spectrum, high efficiency, low toxicity, and strong tissue penetration. One of the important anti-infective drugs, it is widely used for treatment, prevention and growth promotion. Due to its drug resistance and potential carcinogenicity, the European Union, Japan and my country have all established maximum residue limits in tissues. At present, the gold standard for the quantitative detection of enrofloxacin residues is chromatography / mass spectrometry, but its sample pretreatment process is tedious and time-consuming, and the detection cost is high. The ELISA method based on the principle of competitive enzyme-linked immunosorbent reaction does not require complicated sampl...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): G01N33/577G01N21/64
Inventor 马岚袁航吴峰
Owner SHENZHEN GRADUATE SCHOOL TSINGHUA UNIV
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