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Monoclonal antibody for detecting thiamphenicol and florfenicol, ELISA (enzyme-linked immunosorbent assay) method and kit

An enzyme-linked immune reagent and monoclonal antibody technology, which is applied in the field of veterinary drug residue analysis and immunology, can solve problems such as low sensitivity, and achieve the effects of low detection cost, simple detection method, and little harm to health.

Active Publication Date: 2016-04-27
HUAZHONG AGRI UNIV
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  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Florfenicol IC 50 1.02μg / L, p-thiamphenicol IC 50 2.55 μg / L, this method can recognize florfenicol and thiamphenicol at the same time, but thiamphenicol is less sensitive than florfenicol, the cross-reactivity rate is 40%, and the method prepared is a polyclonal antibody
Jin Yinzhen et al. (2012) used florfenicol amine as the hapten, synthesized the immunogen by the carbodiimide method, used thiamphenicol succinic anhydride as the hapten, and used the active ester method to synthesize the coating agent for detection. IC of mycin 50 The cross-reactivity rate of florfenicol amine and florfenicol was 10.8% and 10.4%, respectively, so the antibody can only detect thiamphenicol with low sensitivity

Method used

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  • Monoclonal antibody for detecting thiamphenicol and florfenicol, ELISA (enzyme-linked immunosorbent assay) method and kit
  • Monoclonal antibody for detecting thiamphenicol and florfenicol, ELISA (enzyme-linked immunosorbent assay) method and kit
  • Monoclonal antibody for detecting thiamphenicol and florfenicol, ELISA (enzyme-linked immunosorbent assay) method and kit

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

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

[0032] 1.3 Preparation of florfenicol amine hemisuccinate and hemocyanin conjugate (FFA-HS-KLH)

[0033] Weigh florfenicol amine (FFA) 247mg (1mmol), BOC anhydride 218mg (1mmol), Na 2 CO 3 106mg (1mmol) in a 50mL round bottom flask, add 10mL of dichloromethane, and react overnight at room temperature. Filter to remove Na 2 CO 3 , and then 200 mg (2 mmol) of succinic anhydride and 1 mL of triethylamine were added, and the mixture was refluxed for 1d in an oil bath at 60°C. Evaporate to dryness to remove dichloromethane, add 2 mL of trifluoroacetic acid, react in a water bath at room temperature for 2-4 hours, add triethylamine dropwise to adjust neutrality after the reaction is complete, wash with ethyl acetate 2-3 times, combine organic phases, anhydrous sulfuric acid After magnesium dehydration, it was evaporated to dryness to obtain a yellow oil, namely the hapten florfenicol amine hemisuccinate (FFA-...

Embodiment 2

[0040] The preparation of embodiment 2 monoclonal antibody

[0041] 2.1 Animal immunity

[0042] Balb / C mice (purchased from Experimental Animal Center of Hubei Academy of Medical Sciences) were immunized with the immunogen (FFA-HS-KLH) prepared by the inventor's National Laboratory for Veterinary Drug Residues. The immunization procedure is to take a protein solution containing 50-100 μg of FFA-HS-KLH conjugate, mix it with an equal amount of adjuvant, and then inject it into the mouse body to make it produce specific serum.

[0043] 2.2 Cell fusion and cloning

[0044] With reference to Yang Hanchun's "Animal Immunology", the FF-HS-KLH immunogen prepared by the inventor's National Laboratory for Veterinary Drug Residues was used to immunize Balb / C mice (purchased from the Experimental Animal Center of the Hubei Provincial Center for Disease Control and Prevention), and the immunization procedure was as follows: : For basic immunization, the immunogen was emulsified with an...

Embodiment 3

[0049] The establishment of embodiment 3 thiamphenicol indirect competition ELISA detection method

[0050] 3.1 Preparation of reagents (the reagents used in this example were prepared by the following methods unless otherwise specified)

[0051] Phosphate buffer: NaCl8.0g, KH 2 PO 4 0.2g, Na 2 HPO 4 12H 2 O2.9g, KCl0.2g, add double distilled water to 1000mL, adjust pH to 7.4;

[0052] 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;

[0053] Washing solution: NaCl8.0g, KH 2 PO 4 0.2g, Na 2 HPO 4 12H 2 O2.9g, KCl0.2g, Tween200.5mL, add double distilled water to 1000mL, adjust pH to 7.4;

[0054] Blocking solution: Ovalbumin 1g dissolved in 100mL phosphate buffer;

[0055] Substrate solution A: 3,3',5',5-tetramethylbenzidinediamine (TMB) 200mg, absolute ethanol 100mL, add double distilled water to 1000mL;

[0056] Substrate B: Na 2 HPO 4 14.6g, citric acid 9.3g, 0.75% urea hydrogen peroxid...

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Abstract

The invention discloses a monoclonal antibody for detecting thiamphenicol and florfenicol. The monoclonal antibody is secreted by a hybridoma cell strain TAP / 5F4, the hybridoma cell strain TAP / 5F4 is preserved in the CCTCC (China Center for Type Culture Collection), and the collection number is CCTCC NO: C201549. The invention further discloses an ELISA (enzyme-linked immunosorbent assay) method for detecting thiamphenicol and florfenicol and a kit. Compared with the prior art, the prepared monoclonal antibody has higher sensitivity, high detection accuracy and good precision and has the advantages of simplicity, convenience, quickness, easiness in operation and the like.

Description

technical field [0001] The invention belongs to the technical field of veterinary drug residue analysis and immunology, and specifically relates to a monoclonal antibody capable of recognizing thiamphenicol and florfenicol, and an enzyme-linked immunosorbent method for detecting thiamphenicol and florfenicol (ELISA) and kits. Background technique [0002] Florfenicol and thiamphenicol belong to chloramphenicol compounds. Chloramphenicol has been used as a preventive and therapeutic drug in edible animals for more than 50 years. However, due to its serious harm to humans, many countries will use chlorine Mycin is listed as a prohibited drug. Therefore, there is a need for a compound that has similar antibacterial activity to chloramphenicol and has no potential harm to the human body to replace chloramphenicol. Florfenicol and thiamphenicol are mainly used to treat respiratory and intestinal infections in cattle, poultry and pigs. Although it is not as toxic as chloramphen...

Claims

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Application Information

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IPC IPC(8): C07K16/44C12N5/20G01N33/577C12R1/91
CPCC07K16/44G01N33/577G01N2430/00
Inventor 袁宗辉彭大鹏安玲玲王玉莲潘源虎陈冬梅周琪冯亮
Owner HUAZHONG AGRI UNIV
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