Hapten and artificial antigen of florfenicol, monoclonal antibody, and enzyme-linked immunosorbent assay (ELISA) method of florfenicol

A technology of florfenicol and artificial antigen, applied in immunoglobulin, chemical instruments and methods, biological testing, etc., can solve the problems of low sample throughput and expensive equipment

Inactive Publication Date: 2018-06-29
SOUTH CHINA AGRI UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0003] At present, the method for detecting florfenicol is mainly the instrument method. This method is accurate and reliable, and is often used as a confirmation method. However, it has disadvantages such as expensive equipment, the need for professionals, and low sample throughput.

Method used

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  • Hapten and artificial antigen of florfenicol, monoclonal antibody, and enzyme-linked immunosorbent assay (ELISA) method of florfenicol
  • Hapten and artificial antigen of florfenicol, monoclonal antibody, and enzyme-linked immunosorbent assay (ELISA) method of florfenicol
  • Hapten and artificial antigen of florfenicol, monoclonal antibody, and enzyme-linked immunosorbent assay (ELISA) method of florfenicol

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Experimental program
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Effect test

Embodiment 1

[0082] The preparation of embodiment 1 hapten

[0083] Add 580 mg of florfenicol and 240 mg of succinic anhydride to 5 ml of dichloromethane, then add 680 uL of triethylamine dropwise to the above mixed solution, and heat the reaction in a water bath at 50°C for 3 hours. After the reaction, the product mixed solution is washed with ethyl acetate The ester was extracted 3 times, and the volume ratio of each extraction mixture to ethyl acetate was 1:2; the extract was spin-dried and purified through a silica gel column, and after evaporation to dryness, a light yellow powder was obtained, which was the hapten FFD. figure 1 It is the identification map of FFD mass spectrum. The preparation of embodiment 2 artificial carrier

Embodiment 2

[0083] Add 580 mg of florfenicol and 240 mg of succinic anhydride to 5 ml of dichloromethane, then add 680 uL of triethylamine dropwise to the above mixed solution, and heat the reaction in a water bath at 50°C for 3 hours. After the reaction, the product mixed solution is washed with ethyl acetate The ester was extracted 3 times, and the volume ratio of each extraction mixture to ethyl acetate was 1:2; the extract was spin-dried and purified through a silica gel column, and after evaporation to dryness, a light yellow powder was obtained, which was the hapten FFD. figure 1 It is the identification map of FFD mass spectrum. The preparation of embodiment 2 artificial carrier

[0084] 1. Preparation of immunogen FFD-BSA

[0085] Active lipid method: Dissolve 9.6mg FFD hapten in 0.21mL N,N-dimethylformamide, then add 6.60mg N,N-dicyclohexylcarbodiimide and 3.62mg N-hydroxysuccinimide , stirred and reacted at 4°C overnight, and the supernatant was taken after centrifugation and ...

Embodiment 3

[0091] Example 3 Preparation of Florfenicol Monoclonal Antibody

[0092] 1. Animal immunity

[0093] When 6-week-old female Balb / C mice were immunized for the first time, the amount of immunogen injected was 0.1 mL / mouse, and 0.1 mL of immunogen with a concentration of 1 mg / mL was added to the same volume of Freund's complete adjuvant, and after emulsification was sufficient, the Inject subcutaneously at multiple points on the back and abdomen of the mice, about 40uL per point; perform the second immunization 21 days later, with an immunization dose of 0.1 mL / mouse, emulsified with Freund's incomplete adjuvant; after that, booster immunization once every 14 days, generally a total of Immune 5 times. After the 3rd immunization and the 5th immunization, blood was collected for antiserum titer determination. Three days before the cell fusion, the mice were boosted by direct intraperitoneal injection of 0.1 mL of the immunogen (without adjuvant). The mice should be numbered, ma...

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Abstract

The invention discloses a hapten and an artificial antigen of florfenicol and analogues thereof, a monoclonal antibody, and an enzyme-linked immunosorbent assay (ELISA) method of the florfenicol and analogues thereof. The hapten of the florfenicol is firstly prepared by deriving succinic acid half-ester coupling arm from benzyl hydroxyl of florfenicol molecules; the hapten is further coupled withcarrier protein, so that the artificial antigen of the florfenicol is obtained; after that, an animal is immunized with the artificial antigen, so that the monoclonal antibody for the florfenicol is obtained; the monoclonal antibody can be used for detecting the florfenicol and the analogues such as florfenicol amine and thiamphenicol. The ELISA method of the florfenicol is established by using the monoclonal antibody and can be used for detecting the specificity of florfenicol residues; the IC50 of the method for the florfenicol is equal to 5.85ng/mL, and the linear detection range is 1.67-20.49ng/mL, so that the method has the remarkable advantages of being high in sensitivity, good in specificity, and the like; the method can be used for rapidly detecting the residual florfenicol in food, and has a wide application prospect.

Description

technical field [0001] The invention relates to the technical field of florfenicol detection, in particular to a florfenicol hapten, an artificial antigen, a monoclonal antibody and an ELISA detection method thereof. Background technique [0002] Florfenicol (Florfenicol, C 12 h 14 Cl 2 FNO 4 S) belongs to chloramphenicol broad-spectrum antibiotics, and is widely used in fields such as aquaculture, animal husbandry, and treatment of poultry diseases. Compared with chloramphenicol, which can cause serious harm to human health and is listed as a banned drug in many countries, thiamphenicol can cause symptoms such as aplastic anemia in animals, florfenicol, chloramphenicol and thiamphenicol are antibacterial Similar activity and safer to use. However, it has been reported that florfenicol also has certain immunotoxicity and embryotoxicity. At present, many countries have stipulated the maximum residue limit of florfenicol in animal tissues. Improper use and residues of fl...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C07C317/40C07C315/04C07K14/795C07K14/765C07K14/77C07K16/44G01N33/94
CPCC07C317/40C07K14/765C07K14/77C07K14/795C07K16/44C07K19/00G01N33/9446G01N2430/00
Inventor 沈玉栋李然王弘徐振林杨金易韦晓群孙远明
Owner SOUTH CHINA AGRI UNIV
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