Fluoroquinolones drug artificial immunity antigen, preparing method, enzyme-labeled antigen, competitive ELISA kit and application

A technology of fluoroquinolones and artificial immunity, which is applied in the field of enzyme-linked immunoassay to achieve the effect of low cost, high efficiency and low detection limit

Inactive Publication Date: 2016-10-12
HENAN UNIVERSITY OF TECHNOLOGY
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0007] However, judging from the current literature reports, there is no kit with good detection effec

Method used

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  • Fluoroquinolones drug artificial immunity antigen, preparing method, enzyme-labeled antigen, competitive ELISA kit and application
  • Fluoroquinolones drug artificial immunity antigen, preparing method, enzyme-labeled antigen, competitive ELISA kit and application
  • Fluoroquinolones drug artificial immunity antigen, preparing method, enzyme-labeled antigen, competitive ELISA kit and application

Examples

Experimental program
Comparison scheme
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Example Embodiment

[0033] Example 1 Preparation of artificial immune antigen of fluoroquinolones

[0034] The artificial immune antigen of the fluoroquinolone drugs of the present invention adopts a carbodiimide one-step method to couple the carboxyl group of bovine serum albumin with the amino group on the piperazine ring at the 7th position of norfloxacin to make the parent ring structure of norfloxacin With sufficient exposure, the fluoroquinolone drug artificial immune antigen (NOR-BSA) with the parent ring structure is prepared. The specific preparation method includes the following steps:

[0035] (1) Dissolve 6.6 mg bovine serum albumin (BSA) and 3 mg 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide (EDC) in 2 ml ultrapure water to obtain liquid A;

[0036] (2) Dissolve 3.6 mg norfloxacin in 200 μl dimethylformamide (DMF) to obtain liquid B;

[0037] (3) Add solution B to solution A, stir overnight at room temperature and avoid light with magnetic force, dialyze with pH7.2PBS for 3 days, change the ...

Example Embodiment

[0038] Example 2 Preparation of enzyme-labeled hapten of fluoroquinolones

[0039] The preparation method of the fluoroquinolone drug enzyme-labeled hapten (NOR-HRP) of the present invention includes the following steps:

[0040] (1) Dissolve 4mg horseradish peroxidase (HRP) and 3mg EDC in 2ml ultrapure water to obtain liquid C;

[0041] (2) Dissolve 3.6 mg norfloxacin in 200 μl DMF to obtain D solution;

[0042] (3) Add solution D to solution C, stir overnight at room temperature and avoid light, magnetically stir overnight, dialyzed with pH7.2PBS for 3 days, change the medium 3 times a day, collect the dialysate, centrifuge at 3000r / min for 5min, collect the supernatant, and freeze-dry in vacuum , Keep at -20℃ for later use.

Example Embodiment

[0043] Example 3 Preparation of polyclonal antibodies against fluoroquinolones

[0044] The preparation method of the polyclonal antibody against fluoroquinolones of the present invention includes the following steps:

[0045] New Zealand white rabbits were immunized with artificial immunization antigens of fluoroquinolone drugs, 3 rabbits in each group, the immunization dose was 500μg each time, subcutaneous injection on the back: the first immunization, the artificial immunization antigen of fluoroquinolone drugs was dissolved in PBS, and the same amount FCA mixed emulsification; boost immunization, dissolve fluoroquinolone drugs in PBS to artificially immunize the antigen, and mix with the same amount of FIA for emulsification; 15 days after the first immunization, a total of 8 immunizations, each 15 days apart, 10 days after the last immunization Blood was collected from the ear vein, the serum was separated, and the anti-fluoroquinolone polyclonal antibody was purified by the ...

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Abstract

The invention discloses a fluoroquinolones drug artificial immunity antigen, a preparing method, an enzyme-labeled antigen, a competitive ELISA kit and application. The kit comprises an enzyme-labeled plate coated by goat anti-rabbit IgG, a fluoroquinolones drug resisting specific antibody, fluoroquinolones drug enzyme-labeled hapten, confining liquid, scrubbing liquid, substrate liquid, stop buffer and a fluoroquinolones drug standard solution. By using A carbodiimide one-step method, a carboxyl group of bovine serum albumin and an amino group on a norfloxacin 7-site piperazine ring are coupled, the parent ring structure of norfloxacin is fully exposed, and the fluoroquinolones drug artificial immunity antigen with the parent ring structure is prepared. Meanwhile, norfloxacin and horse radish peroxidase conjugate (NOR-HRP) is synthesized by adopting the same method, multi-resistant and goat anti-rabbit IgG are combined to set up a mode of goat anti-rabbit IgG-multi-resistant-NOR-HRP and fluoroquinolones drug competitive ELISA, and multiresidue detection of the fluoroquinolones drug is achieved.

Description

technical field [0001] The invention relates to a fluoroquinolone drug artificial immune antigen, a preparation method, an enzyme-labeled antigen, a competitive ELISA kit and an application thereof, belonging to the technical field of enzyme-linked immunoassay. Background technique [0002] Fluoroquinolones (FQs) are a class of bactericidal antibacterial drugs with a 6-fluoro-7-piperazine-4-norone ring structure (mother core structure), including norfloxacin (NOR), ciprofloxacin , enrofloxacin, pefloxacin, etc. have the advantages of broad antibacterial spectrum and strong bactericidal effect, especially high antibacterial activity against Gram-negative bacilli; in addition, fluoroquinolones also have fast absorption and good distribution in the body. Wide range of benefits for the treatment of infectious diseases of various systems. [0003] However, with the abuse of fluoroquinolones, the residues of fluoroquinolones have seriously endangered human health. In 2002, my co...

Claims

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Application Information

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IPC IPC(8): C07K14/765C07K1/113C07K16/44G01N33/53
CPCC07K14/765C07K16/44G01N33/53
Inventor 赵银丽苏兰利王金荣赵红月刘珍黄进
Owner HENAN UNIVERSITY OF TECHNOLOGY
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