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Monoclonal antibody, enzyme-linked immunosorbent assay method and kit for detecting residues of fluoroquinolones

A fluoroquinolone, monoclonal antibody technology, applied in the fields of detection analysis and immunology, can solve the problems of difficult to control accuracy, inability to identify difloxacin and sarafloxacin, and large differences in the cross-reaction rate of individual drugs, To achieve the effect of good accuracy, simple sample processing method and wide application range

Inactive Publication Date: 2014-06-04
HUAZHONG AGRI UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0006] The above-mentioned antibodies and corresponding ELISA detection methods can meet the needs of fluoroquinolone drug residue detection to a certain extent, but because they cannot recognize difloxacin and sarafloxacin commonly used in veterinary medicine, and the cross-reactivity rate of individual drugs is relatively different. Large, making it difficult to control the accuracy of detection

Method used

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  • Monoclonal antibody, enzyme-linked immunosorbent assay method and kit for detecting residues of fluoroquinolones
  • Monoclonal antibody, enzyme-linked immunosorbent assay method and kit for detecting residues of fluoroquinolones
  • Monoclonal antibody, enzyme-linked immunosorbent assay method and kit for detecting residues of fluoroquinolones

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0031] Example 1 Preparation of Immunogen and Coating Original

[0032] Synthesis of immunogen (NOR-BSA conjugate): Accurately weigh 32.4mg of norfloxacin and dissolve it in 1.5mL N,N-dimethylformamide (DMF), and cool the solution in an ice bath at 4°C for 25min After that, 192.42 mg of carbodiimide (EDC) and 16.05 mg of N-hydroxysuccinimide (NHS) were added, followed by overnight reaction at 4°C (solution A). Accurately weigh 57.86mg of BSA and dissolve it in 15ml of carbonate buffered saline (CBS) (solution B). Then, liquid A was slowly dripped into liquid B drop by drop, and after reacting overnight at 4°C, the reaction mixture was transferred into a treated dialysis bag, and mixed with 0.01mol phosphate buffered saline (PBS, pH7.4) at 4°C. During dialysis, the liquid was changed continuously to remove free norfloxacin small molecule substances. The resulting product was lyophilized and stored at -20°C for future use.

[0033] Synthesis of the original coating (NOR-OVA c...

Embodiment 2

[0034] Example 2 Preparation of Monoclonal Antibody

[0035] 2.1 Immunization of mice

[0036] The NOR-BSA conjugate prepared in Example 1 was used as the immunogen to immunize Balb / C female mice. The immunization program adopts one basic immunization and several booster immunizations. For the first immunization, a protein emulsion containing 100 μg of immunogen emulsified with an equal volume of Freund’s complete adjuvant was injected subcutaneously on the back of the neck of the mouse for basic immunization, and then every 15 days with protein emulsion containing 100 μg of immunogen emulsified with Freund’s incomplete adjuvant Immunogen protein emulsion for booster immunization. From the third immunization, the tail blood was collected on the 8th day after each immunization, the serum was separated, and the serum antibody titer was detected by indirect ELISA method. Immunization qualified mice (high titer, good sensitivity) stop immunization to prepare for fusion.

[003...

Embodiment 3

[0041] Example 3 Establishment of Indirect Competitive ELISA Detection Method

[0042] 3.1 Reagent preparation

[0043] Carbonate buffer (pH9.6): Accurately weigh Na 2 CO 3 1.59g, NaHCO 3 2.93g, dissolved in a small amount of ultrapure water, and adjusted to 1000mL.

[0044] Washing solution (pH7.4): Accurately weigh 8.00g of NaCl, KH 2 PO 4 0.20g, Na 2 HPO 4 12H 2 O 2.90g, KCl 0.20g, dissolved in a small amount of ultrapure water, Tween 20 0.50mL was added, and the volume was adjusted to 1000mL.

[0045] Phosphate buffer (pH7.4): Accurately weigh 8.00g of NaCl, KH 2 PO 4 0.20g, Na 2 HPO 4 12H 2 Dissolve 2.90g of O, 0.20g of KCl in a small amount of ultrapure water, and dilute to 1000mL.

[0046] Blocking solution: Accurately weigh 10.00 g of ovalbumin, add 1000 mL of phosphate buffer, stir and mix until the protein is completely dissolved.

[0047] Substrate mixture: Accurately absorb 10mL of substrate B solution (purchased from Wuhan Feiyuan Technology Co....

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Abstract

The invention discloses a specific monoclonal antibody capable of recognizing fluoroquinolones. The monoclonal antibody is secreted by a hybridoma cell 5B11, which is preserved in the China Center for Type Culture Collection, and has a preservation number of CCTCC NO:C201149. The invention also discloses an enzyme-linked immunosorbent assay method and a kit for detecting, and application thereof to detection of residues of fluoroquinolones. Compared with prior art, the monoclonal antibody provided by the invention can identify 15 fluoroquinolones, especially can simultaneously identify Difloxacin and Sarafloxacin, thus the application range is wide. The enzyme-linked immunosorbent assay method and the kit established by the present invention have high sensitivity, high precision and good accuracy.

Description

technical field [0001] The invention belongs to the technical field of detection analysis and immunology, and in particular relates to a monoclonal antibody capable of detecting fluoroquinolone drug residues, an enzyme-linked immunosorbent method (ELISA) and a kit. Background technique [0002] Fluoroquinolones are a family of artificially synthesized antibacterial drugs, which are widely used in animal husbandry and aquaculture because of their broad antibacterial spectrum and strong antibacterial activity. However, due to irrational drug use and drug abuse, the adverse reactions of this type of drug and the emergence of drug-resistant strains have brought potential harm to human health. Therefore, the European Union and the Ministry of Agriculture of my country have respectively stipulated maximum residue limits (MRL) for this type of drug. . [0003] The establishment of residue detection methods is an important means to control drug residues. The currently reported metho...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C07K16/44G01N33/577
Inventor 袁宗辉曹光彩彭大鹏王玉莲陈冬梅陶燕飞刘振利
Owner HUAZHONG AGRI UNIV
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