Time-resolved fluorescence immunochromatographic test strip for detecting vancomycin and preparation method and application thereof

A time-resolved fluorescence and vancomycin technology, which is applied in testing pharmaceutical preparations, analytical materials, biological testing, etc., can solve problems such as being unsuitable for large-scale clinical promotion, high cost, and inability to meet clinical monitoring needs, reducing costs. The effect of bottom fluorescence intensity, improving stability and eliminating interference

Inactive Publication Date: 2019-05-28
BEIJING DIAGREAT BIOTECH CO LTD
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  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0003] At present, there are thin layer chromatography (thin layerchromatography, TL3), capillary electrophoresis (Capillary Electrophoresis, CE), high performance liquid chromatography (HPL3), biochemical method at home and abroad to monitor the method of vancomycin drug concentration, but These methods are not suitable for large-scale clinical application
Although there are immunoturbidimetric assays (Tμrbidimetric inhibition immμno assay, TI1), homogeneous enzyme immunoassay, chemiluminescence and fluorescence polarization methods on the market for the determination of vancomycin, but their cost is high, all of which are monopolized by foreign importers. Unable to meet growing clinical monitoring needs

Method used

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  • Time-resolved fluorescence immunochromatographic test strip for detecting vancomycin and preparation method and application thereof
  • Time-resolved fluorescence immunochromatographic test strip for detecting vancomycin and preparation method and application thereof
  • Time-resolved fluorescence immunochromatographic test strip for detecting vancomycin and preparation method and application thereof

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Experimental program
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preparation example Construction

[0026] The invention provides a kind of preparation method of vancomycin-bovine serum albumin conjugate, comprising the following steps:

[0027] 1) mixing an aqueous solution of sodium periodate and an aqueous solution of vancomycin to perform a redox reaction to obtain a vancomycin pre-conjugate;

[0028] 2) mixing bovine serum albumin with a carbonate buffer to obtain a bovine serum albumin solution;

[0029] 3) mixing the vancomycin pre-conjugate described in step 1) with the bovine serum albumin solution described in step 2), and performing a coupling reaction to obtain a vancomycin-bovine serum albumin conjugate;

[0030] The steps 1) and 2) are not limited in time sequence.

[0031] In the present invention, the reason for the preparation of vancomycin-bovine serum albumin conjugates is that vancomycin is a small molecular substance, which has only immunoreactivity but no immunogenicity, and cannot induce the body to produce an immune response. Therefore, vancomycin T...

Embodiment 1

[0091] Example 1 A preparation method of a time-resolved fluorescent immunochromatographic test strip for detecting vancomycin

[0092] 1. Synthesis and identification of vancomycin hapten-carrier protein conjugates

[0093] (1) Preparation of vancomycin-bovine serum albumin conjugate:

[0094] 10 mg / mL NaIO in 1 mL 4 To the solution, 0.5 mL of ultrapure water in which 5 mg of vancomycin was dissolved was added, and stirred at room temperature in the dark for 1 h. The reaction mixture was added to 1mL of 5mg / mL bovine serum albumin solution (50mmol / L carbonate buffer, pH9.6), with 1mol / L Na 2 CO 3 The solution was adjusted to a pH of about 9, stirred for 12 hours, dialyzed against water for 5 times, and stored at -70°C for later use.

[0095] (2) Preparation of vancomycin-chicken ovalbumin conjugate:

[0096] Dissolve 10mg of chicken ovalbumin in 2mL of 0.1M pH7.4 phosphate buffer; add 10mg of vancomycin to the above solution and stir evenly, then add 3mg of 1-ethyl-3-(-3...

Embodiment 4

[0132] Embodiment 4 Correlation Analysis of Test Strip Measurement Results of Embodiment 1 of the present invention

[0133] The test strip of Example 1 of the present invention is compared with the method for determining vancomycin in human serum by high performance liquid chromatography, wherein the human serum is a clinical serum sample. High performance liquid chromatography according to the following scheme: adopt Waters 2695 type high performance liquid chromatography, C18 column (150mm * 4.6mm, 5 μ m), mobile phase: the 50mmol / L potassium dihydrogen phosphate buffer solution of pH3.2-acetonitrile (90 : 10); detection wavelength: 230nm; flow rate: 1mL / min; 400 μl of serum samples were added with 50 μl of 10% zinc sulfate to precipitate protein, after centrifugation at 5000r / min, 20 μl of samples were taken to measure the peak height of vancomycin. The results are shown in Table 2. Correlation curve see figure 1 , it can be seen that the two fit well.

[0134] The test...

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Abstract

The invention provides a time-resolved fluorescence immunochromatographic test strip for detecting vancomycin and a preparation method and application thereof, and belongs to the technical field of detection of drug concentration in in-vitro diagnostic reagents. The test strip comprises a bottom plate, a sample absorption pad, a fluorescent microsphere pad, a nitrocellulose membrane and a water absorption pad, wherein the sample absorption pad, the fluorescent microsphere pad, the nitrocellulose membrane and the water absorption pad are in sequential lap joint and adhere to the bottom plate; the nitrocellulose membrane is coated with vancomycin-carrier protein conjugate; a vancomycin monoclonal antibody with a fluorescent microsphere marker is sprayed on the fluorescent microsphere pad; the vancomycin monoclonal antibody is prepared with vancomycin-bovine serum albumin conjugate as an immunogen. The test strip is free of a cross reaction with various vancomycin analogues, and has the advantages of being high in detection accuracy and high in specificity.

Description

technical field [0001] The invention relates to the technical field of drug concentration detection in in vitro diagnostic reagents, in particular to a time-resolved fluorescent immunochromatographic test strip for detecting vancomycin and a preparation method and application thereof. Background technique [0002] Vancomycin, a tricyclic glycopeptide antibiotic, is clinically used to treat methicillin-resistant Staphylococcus aureus (MRS1), methicillin-resistant Staphylococcus epidermidis (MRSE) and penicillin-resistant Streptococcus pneumoniae (PRSP ) antibiotics of choice for severe infections. It has a strong bactericidal effect on Gram-positive bacteria, but has a narrow therapeutic index, significant differences in individual metabolism, and significant ototoxicity and nephrotoxicity. Therefore, it is necessary to monitor its blood concentration in clinical practice to adjust the dosage. [0003] At present, there are thin layer chromatography (thin layerchromatography...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): G01N21/64G01N33/15G01N33/53G01N33/533G01N33/543G01N33/577
CPCG01N33/54313C07K14/765C07K9/008G01N33/558G01N33/5306G01N33/533G01N33/5432G01N33/54366G01N33/9446G01N33/54388
Inventor 许秀丽王艳新常缘荣周建平
Owner BEIJING DIAGREAT BIOTECH CO LTD
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