Florfenicol and florfenicol amine antigen, antibody and simultaneous detection of enzyme-linked immuno sorbent assay analytical method thereof

A technology of florfenicol and florfenicol, which is applied in the field of antibody and its simultaneous detection enzyme-linked immunoassay, florfenicol and florfenicol antigen, which can solve the problem of expensive equipment, the need for professionals and samples problems such as low throughput, to achieve the effect of good accuracy and high sensitivity

Active Publication Date: 2018-09-21
SOUTH CHINA AGRI UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

At present, the analytical methods of florfenicol and florfenicol amine 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 florfenicol amine antigen, antibody and simultaneous detection of enzyme-linked immuno sorbent assay analytical method thereof
  • Florfenicol and florfenicol amine antigen, antibody and simultaneous detection of enzyme-linked immuno sorbent assay analytical method thereof
  • Florfenicol and florfenicol amine antigen, antibody and simultaneous detection of enzyme-linked immuno sorbent assay analytical method thereof

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Experimental program
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Effect test

Embodiment 1

[0070] The preparation method of embodiment 1 hapten

[0071] The preparation of hapten FFD is as follows:

[0072] 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.

Embodiment 2

[0073] The preparation method of embodiment 2 artificial antigen

[0074] 1. Preparation of Immunogen FFD-KLH

[0075] 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 / LPBS, record it as solution B, drop solution A into solution B under stirring, react at 4°C for 12h, centrifuge after the reaction and take the supernatant, put it in PBS solution at 4°C After dialysis for 3 days, the florfenicol artificial immunogen was obtained, 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.

[0076] 2. Preparation of coated raw FF-OVA

[0077] N,N-carbonyldiimidazole method: Dissolve 10.67mg of FF hapten in 0.25mL of N,N-dimethylforma...

Embodiment 3

[0081] Example 3 Antibody Preparation and Identification

[0082] 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, and the antiserum was purified by octanoic acid-ammonium sulfate precipitation to obtain polyclonal antibody, which was stored at -20°C for future use.

[0083] On the seventh day after the third booster immunization, 200 μ...

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Abstract

The invention discloses a florfenicol and florfenicol amine antigen, antibody and a simultaneous detection enzyme-linked immuno sorbent assay analytical method thereof. The simultaneous detection enzyme-linked immuno sorbent assay analytical method utilizes florfenicol as a hapten to be coupled with a carrier protein to obtain an artificial antigen, and the artificial antigen is used as a coatingantigen; meanwhile, the hydroxyl group of the florfenicol molecule derives the succinic acid half ester to couple with arm to obtain the florfenicol hapten, and the florfenicol artificial antigen is obtained as an immunogen by coupling with the carrier protein; the detection antibody is prepared, and the enzyme-linked immuno sorbent assay analytical method is established. The IC50 of florfenicol is 37.10ng / mL, the linear detection range is 12.03-114.49ng / mL, the IC50 of florfenicol amine is 45.3ng / mL, and the linear detection range is 10.76-190.69ng / mL. The method can be used for quickly detecting the residual florfenicol and florfenicol amine in the food, and has wide application prospect.

Description

technical field [0001] The invention relates to the technical field of detection of florfenicol and florfenicol amine, and more specifically relates to an enzyme-linked immunoassay method for simultaneous detection of florfenicol and florfenicol amine antigens, antibodies and the same. 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 aplastic anemia in animals, etc. Florfenicol has similar antibacterial activity to chloramphenicol and thiamphenicol And it is safer to use. However, it has been reported that florfenicol has certain immunotoxicity and embryotoxicity, and many countries have stipulated the maximum residue limit of florfenico...

Claims

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Application Information

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IPC IPC(8): C07C317/40C07K14/795C07K14/765C07K14/77G01N33/531G01N33/94
CPCC07C317/40C07K14/765C07K14/77C07K14/795C07K19/00G01N33/531G01N33/9446
Inventor 沈玉栋李然王弘杨金易徐振林韦晓群孙远明
Owner SOUTH CHINA AGRI UNIV
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