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Method for simultaneously detecting multiple aminoglycoside antibiotics

A technology of aminoglycosides and antibiotics, applied in the direction of measuring devices, instruments, scientific instruments, etc., can solve the problems of expensive instruments, high test costs, and large sample volume required for detection, and achieve rapid simultaneous detection, wide application range, and detection Efficient effect

Inactive Publication Date: 2019-04-19
JIANGSU UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

The commonly used AGs detection methods are mainly instrumental analysis methods, including HPLC, LC-MS and other methods, which have the advantages of good accuracy and high sensitivity, but because the instruments used are expensive, and there are high test costs, long analysis time, and detection requirements. The large sample volume and cumbersome pretreatment steps make it impossible to achieve rapid and sensitive detection of a large number of samples

Method used

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  • Method for simultaneously detecting multiple aminoglycoside antibiotics
  • Method for simultaneously detecting multiple aminoglycoside antibiotics
  • Method for simultaneously detecting multiple aminoglycoside antibiotics

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0026] Preparation of enzyme-labeled aminoglycoside antibiotic monoclonal antibody of the present invention:

[0027] Monoclonal antibodies to aminoglycoside antibiotics were labeled with horseradish peroxidase. Take the preparation method of labeled kanamycin antibody as an example: dissolve 10mg of HRP in 2mL PBS (0.01mol / L, pH 7.4) buffer solution, add 2~5mg of kanamycin monoclonal antibody, and place in an ice bath Stir slowly to avoid the generation of bubbles as much as possible. After completely dissolving, slowly add 4 mL of 1% glutaraldehyde solution dropwise. The final concentration of glutaraldehyde is 0.2%. For two days, the medium was changed 3 times a day, and after aliquoting, it was frozen at -20°C for later use, and the horseradish peroxidase-labeled kanamycin antibody was obtained.

[0028] The preparation process of other horseradish peroxidase-labeled antibiotics (gentamycin, streptomycin, neomycin) antibodies is the same as that of enzyme-labeled kanamyci...

Embodiment 2

[0032] In this example, the kanamycin and streptomycin standards were detected by the following steps:

[0033] (1) Preparation of kanamycin coater and streptomycin coater: weigh 5 mg kanamycin and 10 mg ovalbumin (OVA) and dissolve them in 3 mL phosphate-buffered saline (PBS), Stir on a magnetic stirrer to form a mixed solution, dissolve 10 mg of EDC in 2 mL of PBS and add dropwise to the above mixed solution, stir overnight at 4°C, dialyze with PBS (0.01mol / L, pH 7.4) for two days, change the solution every day Three times, after the dialysis was completed, it was subpackaged and frozen at -20°C to prepare the kanamycin coating agent. Weigh 10 mg streptomycin and 10 mg ovalbumin and dissolve in 2 mL PBS, stir on a magnetic stirrer to form a mixed solution, take 20 mg EDC and dissolve it in 1 mL PBS, add dropwise to the above mixed solution, 4°C Stir overnight, dialyze with PBS for two days, and change the medium three times a day. After the dialysis is completed, aliquot a...

Embodiment 3

[0041] In this embodiment, the color development under different concentrations is photographed, the detection result of embodiment 2 is carried out grayscale analysis based on application software Quantity One, by grayscale analysis value, on the logarithmic coordinate paper with standard product concentration as Abscissa, B / B 0 (%) draw the standard curves of kanamycin and streptomycin respectively for the ordinate, where B is the gray value corresponding to a certain concentration of the standard, and B 0 is the corresponding gray value when the standard product is 0ng / mL; image 3 Is the standard curve figure of kanamycin and streptomycin; Wherein, a is the streptomycin standard curve, and b is the kanamycin standard curve; as image 3 As shown, with the increase of kanamycin / streptomycin concentration, B / B 0 Decrease in turn, where B is the gray value corresponding to a certain concentration of the standard, B 0 is the corresponding gray value when the standard is 0ng / ...

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Abstract

The invention belongs to the field of detection of small molecular substances, in particular to a method for simultaneously detecting multiple aminoglycoside antibiotics. According to the method, a nitrocellulose membrane is utilized to solidify aminoglycoside antibiotic dox-ova, enzyme labeling is carried out on the aminoglycoside antibiotics, the aminoglycoside antibiotics is solidified on the membrane through specific binding of antigen-antibody, horse radish peroxidase or alkaline phosphatase reacts with a substrate to generate a product with a certain color, the product can exist stably for a long time, therefore, detection results can be directly read by naked eyes within 5-20 minutes; the rapid and simultaneous detection of multiple aminoglycoside antibiotics in a to-be-tested sample is achieved. Compared with a traditional immunoassay method, the method for simultaneously detecting the multiple aminoglycoside antibiotics has the advantages that the detection is efficient, the cost is low, and the time consumption is short; meanwhile, the detection results can be read by naked eyes, the method has convenience and practicability, is wider in application range, and is especially suitable for rapid screening of aminoglycoside antibiotics in liquid samples.

Description

technical field [0001] The invention belongs to the field of detection of small molecular substances, and in particular relates to a method for simultaneously detecting multiple aminoglycoside antibiotics. Background technique [0002] Aminoglycoside antibiotics (Aminoglycosides, AGs) bind to the 30s subunit of the bacterial ribosome with a sedimentation coefficient of 70s, inhibit the formation of the initiation complex, hinder the function of the termination factor, and hinder the release of the synthesized protein, thereby inhibiting the bacteria Protein synthesis in the body, thereby inhibiting bacterial growth. In the field of animal medicine, AGs are mainly used to prevent bovine mastitis, enteritis, metritis, peritonitis and sepsis, etc., and can also promote the growth of animals. However, AGs have obvious nephrotoxicity, ototoxicity and vestibular nerve function damage, and even cause shock and even death in severe cases. my country, the European Union, Japan and ...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): G01N33/577G01N33/535
CPCG01N33/535G01N33/577
Inventor 曾昆韦达理孟辉黄哲张旭芸
Owner JIANGSU UNIV
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