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Monoclonal antibody, enzyme-linked immunosorbent assay method and kit for detecting cephalosporin antibiotics

A monoclonal antibody and cephalosporin technology, which is applied in the field of drug residue analysis and immunology, can solve the problems of insufficient drug types and failure to identify cephalosporin antibiotics, etc., and achieve simple sample processing methods, high accuracy, and time saving and cost effects

Active Publication Date: 2015-04-29
HUAZHONG AGRI UNIV
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  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0004] CN 101571539A discloses an enzyme-linked immunosorbent assay kit for detecting cephalosporins, which uses ceftiofur n-hexanoate obtained by reacting ceftiofur and 6-amino-n-caproic acid as a hapten, and the prepared monoclonal antibody can recognize cephalosporins Thifur, ceftriaxone, and cefotaxime, but other cephalosporin antibiotics cannot be identified, and the types of drugs identified are still not enough

Method used

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  • Monoclonal antibody, enzyme-linked immunosorbent assay method and kit for detecting cephalosporin antibiotics
  • Monoclonal antibody, enzyme-linked immunosorbent assay method and kit for detecting cephalosporin antibiotics
  • Monoclonal antibody, enzyme-linked immunosorbent assay method and kit for detecting cephalosporin antibiotics

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Experimental program
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Embodiment 1

[0031] The preparation of embodiment 1 immunogen and coating former

[0032] 1.1 Preparation of immunogen

[0033] Dissolve 50 mg of ceftiofur in 1 mL of dimethylformamide (DMF) to obtain liquid A. Weigh 66mg of HSA and dissolve it in 10mL of PBS to make solution B. Add 20mg of N,N'-dicyclohexylcarbodiimide (DCC) and 10mg of N-hydroxysuccinimide (NHS) to solution A respectively, and react at room temperature for 24 hours. After the reaction is completed, centrifuge to remove the precipitate. Drop into solution B, and react in ice bath for 24h. After the reaction is completed, transfer the reaction solution into a dialysis bag, dialyze in PBS at 4°C for 3 days, replace the dialysate every 4-6 hours, and freeze-dry the sample after the dialysis to obtain the conjugate ceftiofur-HSA. Store at 20°C.

[0034] 1.2 Preparation of coating agent

[0035] Dissolve 120 mg of OVA in 8 mL of carbonate buffered saline (CBS) to form A liquid. Ceftiofur 50mg is dissolved in 2mL CBS, whi...

Embodiment 2

[0036] The preparation of embodiment 2 monoclonal antibody

[0037] Preparation of hybridoma cells: referring to "Animal Immunology" by Yang Hanchun, Balb / C mice (purchased from the Experimental Animal Center of Hubei Academy of Medical Sciences) were immunized with the immunogen ceftiofur-HSA conjugate prepared in Example 1. The immunization procedure is as follows: for basic immunization, a protein emulsion containing 100 μg of immunogen emulsified with an equal volume of Freund’s complete adjuvant is injected subcutaneously at the back of the neck of the mouse; Protein emulsion of immunogen 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 in preparation for fusion. At the time of fusion, take a Balb / C mouse that has undergone the f...

Embodiment 3

[0042] The establishment of embodiment 3 indirect competition ELISA detection method

[0043] 3.1 Preparation of reagents (the reagents used in this example were prepared by the following methods unless otherwise specified)

[0044] Phosphate buffer: NaCl 8.0g, KH 2 PO 4 0.2g, Na 2 HPO 4 12H 2 O 2.9g, KCl 0.2g, add double distilled water to 1000mL, adjust pH to 7.4;

[0045] Coating solution: Take Na 2 CO 3 1.59g, NaHCO 3 2.93g, add triple distilled water to 1000mL, adjust the pH value to 9.6;

[0046] Washing liquid: NaCl 8.0g, KH 2 PO 4 0.2g, Na 2 HPO 4 12H 2 O 2.9g, KCl 0.2g, Tween 200.5mL, add double distilled water to 1000mL, adjust pH to 7.4;

[0047] Blocking solution: Ovalbumin 1g dissolved in 100mL phosphate buffer;

[0048] Substrate solution A: 3,3',5',5-tetramethylbenzidinediamine (TMB) 200mg, absolute ethanol 100mL, add double distilled water to 1000mL;

[0049] Substrate B: Na 2 HPO 4 14.6g, citric acid 9.3g, 0.75% urea hydrogen peroxide 6.4mL, ...

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Abstract

The invention discloses a specific monoclonal antibody capable of resisting various cephalosporin antibiotics such as ceftiofur, ceftriaxone and the like. The invention further discloses an enzyme-linked immunosorbent assay method and kit for detecting the various cephalosporin antibiotics such as the ceftiofur, the ceftriaxone and the like. According to the invention, the monoclonal antibody is secreted by a hybridoma cell 4D5 of which the preservation number is CCTCC No. C201341. Compared with the prior art, the monoclonal antibody, prepared by the invention, can be used for distinguishing the various cephalosporin antibiotics such as the ceftiofur, the ceftriaxone and the like at the same time. The enzyme-linked immunosorbent assay method and kit disclosed by the invention have the advantages of high detection efficiency, high sensitivity, high precision, high accuracy and the like.

Description

technical field [0001] The invention belongs to the technical field of drug residue analysis and immunology, and specifically relates to a monoclonal antibody against various cephalosporin antibiotics such as ceftiofur and ceftriaxone, and the invention also relates to a method for detecting ceftiofur and ceftriaxone Enzyme-linked immunosorbent assay (ELISA) and kits for various cephalosporin antibiotics such as pine. Background technique [0002] Ceftiofur, ceftriaxone and other cephalosporin antibiotics have the basic structure of β-lactam and have a wide antibacterial spectrum. They are often used to prevent and treat bacterial infections, and have been widely used in veterinary clinics and aquaculture. People abuse drugs in pursuit of economic benefits, resulting in a large amount of drug residues in animal food. After humans eat food containing drugs, it will cause a series of damage to the body, such as allergic reactions, shock and even death in severe cases. After t...

Claims

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Application Information

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IPC IPC(8): C07K16/44C12N5/20G01N33/577C12R1/91
Inventor 袁宗辉彭大鹏朱永利戴梦红王玉莲潘源虎刘振利
Owner HUAZHONG AGRI UNIV
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