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Netilmicin immunoassay reagent and preparation and detection method thereof

A technology for netilmicin and immunoassay, which is applied in the direction of biological testing, material inspection products, etc., can solve the problems of complex operation and low measurement accuracy in the detection process of netilmicin, and achieve high sensitivity, strong specificity, and quantitative accurate effect

Inactive Publication Date: 2018-12-07
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

[0006] The purpose of the present invention is to solve the problems of complex operation and low measurement accuracy in the detection process of netilmicin in the prior art. Netilmicin homogeneous enzyme immunoassay reagent and preparation method thereof

Method used

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  • Netilmicin immunoassay reagent and preparation and detection method thereof
  • Netilmicin immunoassay reagent and preparation and detection method thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0043] Example 1: Preparation of glucose dehydrogenase-antigen conjugate

[0044] 1) Preparation of glucose dehydrogenase (GDH) solution:

[0045] a. Weigh 25 mg of GDH with a specification of 150 KU, dissolve it in 8 mL of 10 mM PBS solution at room temperature, pH=7.4;

[0046] 2) Activation of Netilmicin

[0047] Add the following chemicals to a beaker and stir to dissolve: 20 mg netilmicin, 30 mg 1-ethyl-3-(-3-dimethylaminopropyl)carbodiimide, 15 mg N-hydroxysulfosuccinate imide, dissolved in 800 μL morpholineethanesulfonic acid (MES) solution, stirred and dissolved at room temperature, and reacted for 30 minutes;

[0048] 3) Coupling of GDH and netilmicin

[0049] a. adding the above-mentioned activated netilmicin solution dropwise to the above-mentioned dissolved GDH solution;

[0050] b Stir overnight at 2-8°C;

[0051] 4) Purify the conjugated enzyme-labeled antigen

[0052] The conjugated enzyme-labeled antigen was purified by G-25 gel chromatography column to o...

Embodiment 2

[0053] Example 2: Preparation of Netilmicin Homogeneous Enzyme Immunoassay Reagent

[0054] Netilmicin homogeneous enzyme immunoassay reagents, including: enzyme-labeled netilmicin, indicator reagent for detecting netilmicin antibody-enzyme-labeled netilmicin 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.

[0055]Before using the netilmicin homogeneous enzyme immunoassay reagent, in order to avoid the reaction between the enzyme-labeled conjugate and the enzyme substrate in the indicator reagent, the enzyme-labeled conjugate and the enzyme substrate are placed separately, so the netilmicin The tilmicin homogeneous enzyme immunoassay re...

Embodiment 3

[0064] Example 3: Netilmicin Homogeneous Enzyme Immunoassay Reagent Reaction Calibration Curve.

[0065] 1) Preparation of calibrator: Dissolve commercially available human netilmicin 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. Using the netilmicin calibrator of Shanghai Yansheng Industrial Co., Ltd. as the original standard, the netilmicin kit was used to test the calibrator with different concentrations 10 times, and the average value was obtained to obtain the concentration of the netilmicin calibrator : 0.5, 1, 5, 10, 20, 40 μg / mL.

[0066] 2) Biochemical analyzer detection: Take the operation of Hitachi 7170 as an example: the measurement wavelength is 340 nm, take different concentrations of calibrator solutions (15 μL), add netilmicin R 1 Reagent (200 μL), mix well, then add Netilmicin R 2 Reagent (50 μL), after mixing, measure the OD at differen...

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Abstract

The invention discloses a netilmicin immunoassay reagent and preparation and detection method thereof. The netilmicin immunoassay reagent includes enzyme-labeled netilmicin and an indicator reagent for detection of a netilmicin antibody-enzyme-labeled netilmicin complex; the enzyme-labeled netilmicin is conjugated by netilmicin and glucose dehydrogenase. The netilmicin immunoassay reagent can accurately and rapidly determine the content of the netilmicin in human blood and other samples. Compared with the existing detection reagents on the market, the netilmicin immunoassay reagent has the advantages of convenience, rapidity, high sensitivity, strong specificity, accurate quantification and so on, and is conducive to clinical popularization and use.

Description

technical field [0001] The invention relates to the field of biological detection, in particular to a netilmicin immunodetection reagent and a preparation and detection method thereof. Background technique [0002] Netilmicin (Amikacin) is a new generation of semi-synthetic broad-spectrum aminoglycoside antibiotics. It has good bacteriostatic and bactericidal effects on Gram-negative bacteria and some Gram-positive bacteria. It is mainly used clinically for urinary tract, gram-negative bacilli resistant to kanamycin or gentamicin Infection of the lower respiratory tract, abdominal cavity, soft tissue, bone and joints, reproductive system, etc., and sepsis, etc. Compared with other aminoglycoside antibiotics, netilmicin molecules have fewer sites attacked by enzymes, so the adverse reactions are less than similar drugs. [0003] However, if the concentration is too high, it will also produce adverse reactions such as tinnitus, hearing loss and even death, so it is very nece...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): G01N33/53
CPCG01N33/53
Inventor 严芳芳马运乐张轩王亚盟
Owner 太原瑞盛生物科技有限公司
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