Florfenicol and thiamphenicol antigens and antibodies as well as enzyme-linked immunosorbent assay method for detecting florfenicol and thiamphenicol simultaneously

A technology of florfenicol and thiamphenicol, which is applied in the field of antibody and its simultaneous detection of enzyme-linked immunoassay, florfenicol and thiamphenicol antigen, and can solve the problems of expensive equipment and low sample throughput.

Inactive Publication Date: 2018-07-06
SOUTH CHINA AGRI UNIV
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  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0003] At present, the analytical methods of florfenicol and thiamphenicol are mainly instrumental detection methods. This method is accurate and reliable, and is often used as a confirmation method. However, there are disadvantages such as expensive equipment, the need for professionals, and low sample throughput.

Method used

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  • Florfenicol and thiamphenicol antigens and antibodies as well as enzyme-linked immunosorbent assay method for detecting florfenicol and thiamphenicol simultaneously
  • Florfenicol and thiamphenicol antigens and antibodies as well as enzyme-linked immunosorbent assay method for detecting florfenicol and thiamphenicol simultaneously
  • Florfenicol and thiamphenicol antigens and antibodies as well as enzyme-linked immunosorbent assay method for detecting florfenicol and thiamphenicol simultaneously

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Experimental program
Comparison scheme
Effect test

Embodiment 1

[0082] The preparation method of embodiment 1 hapten

[0083] 1. Preparation of Hapten FFD

[0084] Add 580 mg of florfenicol and 240 mg of succinic anhydride into 5 ml of dichloromethane, then add 680 μL of triethylamine dropwise to the above mixture, and heat the reaction in a water bath at 50°C for 3 hours. After the reaction, the product mixture 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. 2. Preparation of Hapten FFM

[0085] Add 580 mg of florfenicol and 240 mg of maleic anhydride to 5 ml of dichloromethane, then add 680 μL of triethylamine dropwise to the above mixture, and heat the reaction in a water bath at 50°C for 3 hours. After the reaction, the produc...

Embodiment 2

[0086] The preparation of embodiment 2 immunogen and coating former

[0087] 1. Preparation of Immunogen FFD-KLH

[0088] Active lipid method: Dissolve 5mg FFD hapten in 0.2mL N,N-dimethylformamide, then add 4.78mg N,N-dicyclohexylcarbodiimide and 2.65mg N-hydroxysuccinimide , stirred and reacted at 4°C overnight, and the supernatant was taken after centrifugation and recorded as solution A. Dissolve the carrier protein (14.6mg KLH) in 4mL 0.01mol / L PBS, record it as solution B, drop solution A into solution B under stirring, react at 4°C for 12h, centrifuge after the reaction, take the supernatant, put in PBS at 4°C The solution was dialyzed for 3 days to obtain the florfenicol artificial immunogen, which was dispensed into 1 mL centrifuge tubes at a concentration of 1 mg / mL, and frozen in a -20°C refrigerator for later use.

[0089] 2. Preparation of coated raw FFM-OVA

[0090] Active lipid method: Dissolve 10mg FFM hapten in 0.2mL N,N-dimethylformamide; add 9.56mg N,N-di...

Embodiment 3

[0094] Example 3 Antibody Preparation and Identification

[0095] When 2.5kg female New Zealand white rabbits were immunized for the first time, the amount of immunogen injected was 0.5mL / rat. Take 0.5mL 1mg / mL immunogen plus an equal volume of Freund's complete adjuvant. Spot injection, about 200 μl per spot; the second immunization was carried out four weeks later, the amount of injected immunogen was 0.5mL / monkey, emulsified with an equal volume of Freund's incomplete adjuvant; the third immunization was carried out three weeks later, the fourth There was also an interval of 3 weeks between immunization and the third time, and a total of 4 times of immunization. 4 One week after immunization, blood was collected from the heart to collect antibody serum. The antiserum was purified by caprylic acid-ammonium sulfate precipitation method to obtain polyclonal antibody, and stored at -20°C for future use.

[0096] On the seventh day after the third booster immunization, 200 μL o...

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Abstract

The invention discloses florfenicol and thiamphenicol antigens and antibodies as well as an enzyme-linked immunosorbent assay method for detecting florfenicol and thiamphenicol simultaneously. One hapten FFM is obtained by deriving a semiester maleate coupled arm on hydroxybenzyl of a florfenicol molecule structure, the other hapten FFD is obtained by deriving a semiester succinate coupled arm onhydroxybenzyl of a florfenicol molecule, and the hapten FFM and the hapten FFD are coupled with carrier protein respectively to obtain florfenicol artificial antigens to serve as a coated antigen andimmunogen; and a detection antibody is prepared, the enzyme-linked immunosorbent assay method is established, the IC50 on the florfenicol is equal to 1.32 ng / mL and the linear detection range is 0.29to 5.91 ng / mL, the IC50 on the thiamphenicol is equal to 2.13 ng / mL and the linear detection range is 0.41 to 11.20 ng / mL, remarkable advantages of high sensitivity, high specificity and the like areachieved, the invention can be used for rapidly detecting the residual florfenicol and thiamphenicol in food, and wide application prospect is achieved.

Description

technical field [0001] The invention relates to the technical field of florfenicol and thiamphenicol detection, more specifically, relates to an enzyme-linked immunoassay method for simultaneous detection of florfenicol and thiamphenicol antigens, antibodies and the same. Background technique [0002] Florfenicol (Florfenicol, C 12 h 14 Cl 2 FNO 4 S) and Thiamphenicol (Thiamphenicol, C 12 h 15 Cl 2 NO 5 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, florfenicol and thiamphenicol have similar antibacterial activities to chloramphenicol and are safer to use. However, it has been reported that florfenicol has certain immunotoxicity and embryotoxicity, and thiamphenicol can cause aplastic anemia in animals. At present, many countries h...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C07C317/32C07K14/795C07K14/765C07K14/77C07K16/44G01N33/535G01N21/33
CPCC07C317/32C07K14/765C07K14/77C07K14/795C07K16/44C07K19/00G01N21/33G01N33/535
Inventor 沈玉栋李然王弘徐振林林泽佳杨金易韦晓群孙远明
Owner SOUTH CHINA AGRI UNIV
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