Fluorescence polarization immunoassay method for detecting polymyxin

A polymyxin and fluorescence polarization technology, applied in the direction of material analysis by optical means, analyzing materials, fluorescence/phosphorescence, etc., can solve the limitation of effective supervision of polymyxin B and colistin, high operator requirements, Problems such as long detection time, to achieve the effect of satisfying the monitoring of polymyxin blood concentration

Pending Publication Date: 2022-03-11
CHINA AGRI UNIV
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Problems solved by technology

[0004] At present, the detection methods of polymyxin B and colistin are mainly liquid chromatography and liquid chromatography-mass chromatography. However, due to the high price of large-scale instruments, high requirements for operators, and long detection time, etc., the detection method is severely limited. Effective regulation of polymyxin B and colistin use

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  • Fluorescence polarization immunoassay method for detecting polymyxin
  • Fluorescence polarization immunoassay method for detecting polymyxin
  • Fluorescence polarization immunoassay method for detecting polymyxin

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Embodiment 1

[0058] Preparation of embodiment 1 fluorescent marker

[0059] 1. Preparation of fluorescent marker Hapten-M-FITC

[0060] Weigh 2mg of Hapten and 1.56mg of 5-maleimide fluorescein powder, add to 400μL of DMF and shake until dissolved; add 50uL of triethylamine, and react in the dark for 3 hours at room temperature; take 50uL of the reaction solution and use thin layer chromatography ( Separation by TLC, the developing solvent is methanol; the yellow band with R=0.2 is scraped off the silica gel plate, eluted with methanol:water (v:v, 1:1), and detected for later use.

[0061] Mass Spectrometry Identification: The chemical formula of the fluorescent marker Hapten-M-FITC is: C 47 h 57 N 7 o 14 S, the theoretical molecular weight is 976.07. The measured value of mass spectrum: 976.1, consistent with the molecular weight of the target product, see the mass spectrometry results figure 2 .

[0062] 2. Preparation of fluorescent markers Hapten-2NM-FITC, Hapten-4NM-FITC, Hapt...

Embodiment 2

[0073] Example 2 Screening of the best fluorescent marker and antibody combination

[0074] 1. Combine the 12 synthetic fluorescent markers with polymyxin monoclonal antibodies (5D10, 11D5, 14G6 and 17D11) and polymyxin polyclonal antibody pAb respectively. Set to the concentration (10nM) of the corresponding fluorescent marker when the fluorescence intensity is 10 times the background fluorescence intensity of the buffer solution, each antibody is used in borate buffer according to 1 / 100, 1 / 200, 1 / 400, 1 / 800 , 1 / 1600, 1 / 3200, l / 6400, 1 / 12800, 1 / 25600 and 1 / 51200 dilutions, draw the antibody binding curve, and obtain the maximum change value of signal intensity δmP (δmP=mP max -mP min ). For each combination, the antibody dilution concentration with a δmP of 80-120 was selected as the working concentration of the antibody.

[0075] Among them, the polymyxin monoclonal antibody is obtained through cell fusion and hybridoma preparation methods.

[0076] Polymyxin polyclonal ...

Embodiment 3

[0082] The establishment of embodiment 3 FPIA method

[0083] 1. Competing for FPIA

[0084] Use borate buffer (50mmol / L, pH8.0) to configure different concentration gradients of 2000ng / mL, 400ng / mL, 80ng / mL, 16ng / mL, 3.2ng / mL, 0.64ng / mL and 0.128ng / mL polymyxin standard solution. Add 70uL polymyxin standard substance, 70uL fluorescent marker with a working concentration of 10nM and 70uL polymyxin polyclonal antibody solution with a working concentration of 8μg / mL in a 96 black low-binding microwell plate, and incubate at room temperature in the dark After 5 min, the fluorescence polarization value was measured.

[0085] 2. Draw a standard curve

[0086] After the competition reaction, the measured fluorescence polarization value of each well was plotted on the ordinate, and the concentration of the polymyxin standard solution was plotted on the abscissa, and the four-parameter model of Origin 8.0 was used to fit the standard curve.

[0087] Fluorescence polarization stand...

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Abstract

The invention provides a fluorescence polarization immunoassay method for detecting polymyxin. The method comprises the following steps: 1) preparing polymyxin standard substance solutions with different concentrations, respectively mixing and incubating the polymyxin standard substance solutions with a fluorescent marker solution and a polymyxin monoclonal antibody solution, carrying out a competitive reaction, and measuring the fluorescence polarization value of the obtained system; drawing a standard curve by taking the measured fluorescence polarization value as a vertical coordinate and the concentration of the polymyxin standard substance solution as a horizontal coordinate; and (3) replacing the polymyxin standard substance in the step (1) with a to-be-detected sample, determining the fluorescence polarization value of the to-be-detected sample according to the method in the step (1), and calculating the concentration of the polymyxin in the to-be-detected sample according to the standard curve in the step (1). The polymyxin fluorescence polarization immunoassay technology provided by the invention is a homogeneous immune method without separation and washing, and has the advantages of high throughput, convenience, reliability and the like.

Description

technical field [0001] The invention relates to the technical field of immunoassay technology and veterinary drug residue detection, in particular to a fluorescence polarization immunoassay method for detecting polymyxin. Background technique [0002] Polymyxin B (Polymyxin B, PMB) and colistin (colistin or Polymyxin E, PME) are a class of cationic polypeptide antibiotics isolated from Bacillus polymyxa. It was discovered in the 1950s and was used clinically, but was replaced by other antibiotics because of its toxic side effects. However, in order to deal with Gram-negative multidrug-resistant bacterial infections, it was reapplied to human clinical practice around 2000. In the field of animal husbandry and veterinary medicine, colistin has been widely used as a growth-promoting agent added to feed, but in 2016, China banned the use of colistin as a feed additive, but allowed it to be used as a therapeutic drug. [0003] In human clinical practice, the therapeutic window ...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): G01N21/64
CPCG01N21/6445
Inventor 温凯王战辉沈建忠江海洋余文博于雪芝柯跃斌张英杰
Owner CHINA AGRI UNIV
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