Fluorescence polarization immunoassay detection method for sarafloxacin

A technology of fluorescence polarization and immunoassay, which is applied in the direction of analyzing materials, measuring devices, instruments, etc., and can solve the problems of complex and time-consuming operations

Inactive Publication Date: 2012-08-01
NANKAI UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

[0005] The purpose of the present invention is to solve the problem of complex and time-consuming operation of traditional analysis methods, to provide a preparation method of sarafloxacin fluorescent markers and a fluorescent polarization immunoassay method for sarafloxacin established by using fluorescent markers and highly specific antibodies

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  • Fluorescence polarization immunoassay detection method for sarafloxacin
  • Fluorescence polarization immunoassay detection method for sarafloxacin
  • Fluorescence polarization immunoassay detection method for sarafloxacin

Examples

Experimental program
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Embodiment 1

[0057] The preparation of embodiment 1 sarafloxacin marker:

[0058] a. Weigh 3-4 mg of SAR and dissolve it in a mixed solvent consisting of 100 μL of water, 500 μL of methanol, and 50 μL of triethylamine to form liquid A;

[0059] b. Weigh 6 mg FITC and dissolve it in 600 μL methanol to form solution B;

[0060] c. Take 200 μL of solution B and add it dropwise to solution A, shake, and keep at room temperature overnight in the dark.

[0061] d. The next day with the mixed solvent CHCl 3 : MeOH = 2: 1 (volume ratio) as the developing agent, carry out thin-layer chromatography to the overnight reaction solution at room temperature, separate and purify, observe under the ultraviolet projector, select the band of Rf = 0.1, extract with 120 μ L MeOH to obtain salsa Star fluorescent marker.

Embodiment 2

[0062] The determination of embodiment 2 sarafloxacin fluorescent marker working concentration:

[0063] Doubling dilution with 2.5mmol / L, pH=8.0 borate buffer solution to obtain different concentrations (1 / 400, 1 / 800, 1 / 1600, 1 / 3200, 1 / 6400, 1 / 12800, 1 / 25600) The sarafloxacin marker is used to detect the fluorescence polarized light intensity, and the polarized light intensity of about 5 times that of the blank solution (the blank polarized light intensity is 1861) is used as its working concentration; the concentration of 1 / 12800 dilution is determined to be selected as the working concentration, and the blank solution The solution is 2.5mmol / L borate buffer solution with pH=8.0.

[0064]

Embodiment 3

[0065] The determination of the working concentration of the sarafloxacin polyclonal antibody of embodiment 3:

[0066] Add 500 μL of 2.5mmol / L, pH=8.0 borate buffer to each test tube and dilute to different concentrations (1 / 100, 1 / 200, 1 / 400, 1 / 800, 1 / 1600, 1 / 3200) polyclonal antibody against sarafloxacin, and sequentially add 500 μL working concentration of sarafloxacin fluorescent markers, mix well and incubate at room temperature for 5 minutes, detect the intensity of fluorescence polarized light, and the antibody corresponding to about 70% of the maximum polarization value (14243) The concentration is taken as the optimal working concentration of the antibody; the 1 / 1600 dilution concentration is selected as the working concentration of the antibody.

[0067]

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Abstract

The invention discloses a fluorescence polarization immunoassay detection method for sarafloxacin (SAR), which takes a synthetic sarafloxacin fluorescent label as basis, belonging to the technical field of fluorescence polarization immunoassay detection. According to the method, the sarafloxacin is taken as hapten coupling fluorescein isothiocyanate (FITC), the fluorescent label is synthesized, the sarafloxacin is taken as a standard substance, and the sarafloxacin polyclonal antibody is taken as the antibody, so that the fluorescence polarization immunoassay detection method for the sarafloxacin is established.The invention provides a quick and high-efficiency analysis means for analyzing the content of the sarafloxacin. The method provided by the invention is easy, simple and quick to operate, and low in cost. The IC50 is 43.23ng / mL, and the linear range is 5.7-327.6ng / mL. Due to the high specificity and affinity of the immunoassay, the fluorescence polarization immunoassay method is extremely high in selectivity and sensitivity.

Description

technical field [0001] The invention relates to a fluorescence polarization immunoassay method for sarafloxacin, belonging to the technical field of fluorescence polarization immunoassay detection. technical background [0002] Sarafloxacin (SAR) belongs to the special fluoroquinolone drugs for animals. Its hydrochloride is used in medicine. It has the characteristics of broad-spectrum antibacterial, strong activity, and small toxic and side effects. It is mainly used to treat sensitive bacteria and mycoplasma in chickens and pigs. Diseases caused by infection. According to reports, sarafloxacin hydrochloride has a long half-life, and the residues in food animals will cause drug resistance, and excessive drug residues will directly endanger the health of animals and humans. my country stipulates that the maximum residue limit of veterinary drug sarafloxacin in animal food shall not exceed 80 μg / kg. [0003] At present, there are four main methods for the detection of (fluo...

Claims

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Application Information

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IPC IPC(8): G01N33/542G01N33/533
Inventor 郗日沫宋佩尹永梅孟萌张太昌田溪薛虎寅
Owner NANKAI UNIV
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