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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AI-Extracted 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...

Method used

Experimental result shows, florfenicol polyclonal antibody has 82% intersection with florfenicol amine, almost does not have intersection with structural analog thiamphenicol and chloramphenicol, illus...

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.

Application Domain

OvalbuminSerum albumin +3

Technology Topic

ChemistryImmunogen +12

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Examples

Experimental program(5)

Example Embodiment

[0070] Example 1 Preparation method of hapten

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

[0072] Add 580mg of florfenicol and 240mg of succinic anhydride to 5ml of dichloromethane, then add 680μL of triethylamine dropwise to the above mixture, and heat the reaction in a water bath at 50℃ for 3 hours. After the reaction, the product mixture is mixed with ethyl acetate The ester is extracted 3 times, and the volume ratio of each extraction mixture to ethyl acetate is 1:2; the extract is spin-dried and purified by a silica gel column, and the light yellow powder is obtained after evaporation to dryness, which is the hapten FFD. figure 1 It is the FFD mass spectrum identification map.

Example Embodiment

[0073] Example 2 Preparation method of artificial antigen

[0074] 1. Preparation of immunogen FFD-KLH

[0075] Active fat method: Dissolve 5mg FFD hapten in 0.2mL N,N-dimethylformamide, then add 4.78mg N,N-bicyclohexylcarbodiimide and 2.65mg N-hydroxysuccinimide , The reaction was stirred overnight at 4°C, and the supernatant was taken after centrifugation and marked as solution A. Dissolve the carrier protein (14.6mg KLH) in 4mL 0.01mol/L PBS, record it as solution B, drop A solution into solution B under stirring, react for 12h at 4℃, centrifuge to take the supernatant after the reaction, and PBS solution at 4℃ After dialysis for 3 days, the artificial immunogen of florfenicol was obtained, which was divided into 1 mL centrifuge tubes at a concentration of 1 mg/mL, and stored in a refrigerator at -20°C for later use.

[0076] 2. Preparation of original FF-OVA coating

[0077] N,N-carbonyldiimidazole method: Dissolve 10.67mg FF hapten in 0.25mL N,N-dimethylformamide, then add 8.11mg of N'N-carbonyldiimidazole, stir at 37℃ for 2h, record as Solution A: Dissolve the carrier protein (13.41mg OVA) in 3mL0.01mol/L PBS, record it as solution B, slowly drip solution A into solution B under stirring, control the dropping time at about 10min, and stir the reaction at 4℃ 72h; PBS solution was dialyzed for 3 days at 4°C to obtain the artificial coating of florfenicol, which was divided into 1mL centrifuge tubes and stored in a refrigerator at -20°C for later use.

[0078] 3. Characterization of florfenicol immunogen and coating

[0079] (1) The identification of the immunogen and the encapsulating agent adopts the method of ultraviolet scanning. The ultraviolet absorption spectrum of the immunogen and the encapsulating agent is measured in the wavelength range of 200-400nm, and the scanning curves of different substances are compared to identify whether the hapten and the carrier protein are The coupling is successful. Since the carrier protein and the hapten both have the maximum absorption peak under the conditions of the ultraviolet spectrum, if the coupling is successful, the characteristic absorption peaks overlap each other, which leads to the blue shift of the maximum absorption peak.

[0080] (2) such as figure 2 , image 3 As shown, there is a characteristic absorption peak of the carrier protein at about 280 nm, and the characteristic absorption peak of the immunogen has a significant blue shift. Therefore, the scanning results of the ultraviolet spectrum can prove that the florfenicol immunogen and the coating source are successfully coupled.

Example Embodiment

[0081] Example 3 Antibody preparation and identification

[0082] When a 2.5kg female New Zealand white rabbit is immunized for the first time, the amount of immunogen injected is 0.5mL/head, 0.5mL of 1mg/mL immunogen plus an equal volume of Freund’s complete adjuvant, after sufficient emulsification, it will be more subcutaneously on the back of the rabbit. Point injection, about 200μl per point; four weeks later, the second immunization, the amount of immunogen injected is 0.5mL/mouse, emulsified with an equal volume of Freund’s incomplete adjuvant; three weeks later, the third immunization, the fourth There was also a 3 week interval between the immunization and the third, and a total of 4 immunizations. 4 One week after immunization, blood was collected from the heart to obtain antibody serum, and the antiserum was purified by caprylic acid-ammonium sulfate precipitation method to obtain polyclonal antibodies, and stored at -20°C for later use.

[0083] On the seventh day after the third booster immunization, 200 μL of blood was collected from the ear vein, incubated at 37°C for 30 min, centrifuged at 4000 r/min for 10 min, and the supernatant was taken and stored at -20°C.

[0084] The indirect ELISA method is used to determine the titer and specificity of the antiserum. The steps are as follows:

[0085] S1. Coating: Dilute 1 mg/mL of the coated antigen with coating buffer 1000 times, and make a blank control group. Add 100 μL/well to the 96-well microtiter plate and incubate overnight in a 37°C water bath.

[0086] S2. Washing: Pour out the liquid in the wells, set the parameters of the plate washer to add 300μL of washing liquid to each well, wash the plate twice, and then spin dry the washing liquid.

[0087] S3. Sealing: add 120μL of blocking solution to each hole, seal at 37°C for 3 hours, spin-dry the liquid in the hole, and place it in an oven at 37°C for 1 hour to dry.

[0088] S4. Sample addition and incubation: Add 50μL of gradiently diluted antiserum and 50μL of diluent to the titer column, shake and mix, and after reacting in a 37°C water bath for 40 minutes, wash with 300μL/well for 5 times, and then spin dry the liquid in the well.

[0089] S5. Add secondary antibody: add 100 μL of HRP-goat anti-rabbit IgG (5000 times diluted with PBS) to each well, react in a constant temperature water bath at 37°C for 30 minutes, wash with 300 μL/well for 5 times, and spin dry the liquid in the well.

[0090] S6. Color development: add 100μL of color developing solution to each well, place it in a 37℃ water bath for 10 minutes, add 50μL of stop solution (10%H 2 SO 4 ).

[0091] S7. Reading measurement: Read the absorbance (OD) with a microplate reader at a wavelength of 450nm.

[0092] (3) Choose the antiserum dilution factor whose absorbance value is in the range of 1.0 to 1.5 as the antiserum titer, and the result shows that the antiserum titer is 1:8000.

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Classification and recommendation of technical efficacy words

good accuracy
high sensitivity

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

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