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Tobramycin immunoassay reagent and preparing and detecting method thereof

A tobramycin and immune detection technology, applied in the field of medical testing, can solve the problems of low measurement accuracy, complicated tobramycin detection process and operation, and achieve the effects of high sensitivity, accurate quantification and strong specificity

Inactive Publication Date: 2018-09-07
太原瑞盛生物科技有限公司
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  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0005] The purpose of the present invention is to solve the problems of complex operation and low measurement accuracy in the tobramycin detection process in the prior art. Tobramycin homogeneous enzyme immunoassay reagent and preparation method thereof

Method used

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  • Tobramycin immunoassay reagent and preparing and detecting method thereof
  • Tobramycin immunoassay reagent and preparing and detecting method thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0038] Example 1: Preparation of glucose dehydrogenase-antigen conjugate

[0039] 1) Coupling of glucose dehydrogenase (GDH) to tobramycin

[0040] a. Dissolve 20 mg of tobramycin in 250 μL of dimethylformamide (DMF), shake slightly to dissolve; at the same time, dissolve 25.0 mg of GDH with a specification of 100 KU in 3 mL of 0.01mol / L PBS (pH7 .4) In the buffer solution, shake and dissolve evenly;

[0041] b. Slowly add the tobramycin solution into the GDH solution under stirring, and slowly add 20 μL of 25% glutaraldehyde solution dropwise at the same time. After the dropwise addition, place the solution at 4°C and stir overnight.

[0042] 2) Purify the conjugated enzyme-labeled antigen

[0043] The conjugated enzyme-labeled antigen was purified by G-25 gel chromatography column to obtain glucose dehydrogenase-tobramycin conjugate, which was stored at 2-8°C.

Embodiment 2

[0044] Example 2: Preparation of Tobramycin Homogeneous Enzyme Immunoassay Reagent

[0045] Homogeneous enzyme immunoassay reagents for tobramycin, including: enzyme-labeled tobramycin, indicator reagent for detecting tobramycin antibody-enzyme-labeled tobramycin complex. The indicator reagent is selected from enzyme reagents, radioisotope reagents, fluorescent reagents and chemiluminescent reagents. Preferably, the indicator reagent is an enzyme reagent, including: an enzyme-labeled conjugate and an enzyme substrate. Wherein, the enzyme-labeled conjugate includes glucose dehydrogenase-antigen conjugate, which can be obtained by chemical synthesis.

[0046] Tobramycin Homogeneous Enzyme Immunoassay Reagent Before use, in order to avoid the reaction between the enzyme-labeled conjugate in the indicator reagent and the enzyme substrate, the enzyme-labeled conjugate and the enzyme substrate are placed separately, so properly The Butomycin Homogeneous ELISA Reagent consists of t...

Embodiment 3

[0055] Example 3: Tobramycin Homogeneous Enzyme Immunoassay Reagent Reaction Calibration Curve.

[0056] 1) Preparation of calibrator: Dissolve commercially available human tobramycin recombinant protein in a solution similar to human serum matrix (NaCl 0.9%, BSA 0.2%, NaN 3 0.1%, Tris-HCl pH 7.4), and made different concentrations of calibrators. Taking the tobramycin calibrator of Xiamen Huijia Biotechnology Co., Ltd. as the original standard, use its tobramycin kit to test the calibrator of different concentrations 10 times, calculate the average value, and obtain the concentration of the tobramycin calibrator : 0.5, 1, 5, 10, 20, 40 ng / mL.

[0057] 2) Detection of biochemical analyzer: Take the operation of Hitachi 7170 as an example: the measurement wavelength is 340 nm, take different concentrations of calibrator solutions (15 μL), add tobramycin R 1 Reagent (200 μL), mix well, then add Tobramycin R 2 Reagent (50 μL), after mixing, measure the OD at different time po...

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Abstract

The invention discloses a tobramycin immunoassay reagent and a preparing and detecting method thereof. The tobramycin immunoassay reagent comprises enzyme-labelled tobramycin and an indication reagentfor detecting a tobramycin antibody-enzyme-labelled tobramycin compound. The enzyme-labelled tobramycin is formed by coupling tobramycin and glucose dehydrogenase. By means of the tobramycin immunoassay reagent, the content of tobramycin in human blood and other samples can be precisely and rapidly determined. Compared with existing detecting reagents on the market, the immunoassay reagent has the advantages of being convenient and rapid to use, high in sensitivity, high in specificity, accurate in quantifying and the like, and is beneficial for clinical use and popularization.

Description

technical field [0001] The invention relates to the field of medical testing, in particular to a tobramycin immunodetection reagent and a preparation and detection method thereof. Background technique [0002] Tobramycin (Tobramycin, TOB) is the second-generation aminoglycoside antibiotics. Compared with the first-generation aminoglycoside antibiotics, it has better efficacy and lower toxicity. It is usually used for injection to treat severe Gram It has become one of the most widely used aminoglycoside antibiotics in clinical practice in the world. Tobramycin has also been widely used in the field of veterinary medicine, so it also exists in foods commonly used by people such as meat and milk. Since its safe therapeutic concentration is 4 mg / L-8 mg / L, a long-term blood drug concentration exceeding 8 mg / L may cause severe nephrotoxicity and ototoxicity. Therefore, it is necessary to quickly and sensitively determine tobramycin in related food samples. [0003] At present,...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): G01N33/53G01N33/531
CPCG01N33/53G01N33/531
Inventor 丁兰艳康志禹张轩程月萍
Owner 太原瑞盛生物科技有限公司
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