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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 effect on more than ten fluoroquinolones. The detection of multiple drug residues is of great significance

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
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Effect test

Embodiment 1

[0033] Example 1 Preparation of fluoroquinolone artificial immune antigen

[0034] The artificial immunization antigen of fluoroquinolone drugs of the present invention adopts the carbodiimide one-step method to couple the carboxyl group of bovine serum albumin with the amino group on the 7-position piperazine ring of norfloxacin to make the parent ring structure of norfloxacin Fully expose, make the fluoroquinolone artificial immune antigen (NOR-BSA) with parent ring structure, concrete preparation method comprises the following steps:

[0035] (1) Dissolve 6.6mg of bovine serum albumin (BSA) and 3mg of 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide (EDC) in 2ml of 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 liquid B to liquid A, stir overnight at room temperature and avoid light, dialyze with pH7.2 PBS for 3 days, change the liquid 3 times a day, collect the dialysate, cen...

Embodiment 2

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

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

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

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

[0042] (3) Add liquid D into liquid C, stir overnight at room temperature and avoid light, dialyze with pH7.2 PBS for 3 days, change the liquid 3 times a day, collect the dialysate, centrifuge at 3000r / min for 5min, collect the supernatant, and vacuum freeze-dry , Store at -20°C for later use.

Embodiment 3

[0043] Example 3 Preparation of polyclonal antibodies against fluoroquinolones

[0044] The preparation method of the anti-fluoroquinolone polyclonal antibody of the present invention comprises the following steps:

[0045] Immunize New Zealand white rabbits with artificial immune antigens of fluoroquinolones, 3 rabbits in each group, with an immunization dose of 500 μg / rat each time, and subcutaneously inject points on the back: first immunization, dissolve the artificial immune antigens of fluoroquinolones in PBS, and the same amount FCA mixed emulsification; booster immunization, dissolve fluoroquinolone artificial immune antigen in PBS, mix and emulsify with the same amount of FIA; 15 days after the first immunization, a total of 8 immunizations, each interval of 15 days, 10 days after the last immunization Blood was collected from the ear vein, the serum was separated, polyclonal antibodies against fluoroquinolones were purified by saturated ammonium sulfate salting-out m...

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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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