CN112174851A

CN112174851A - Fluoroaminoketone hapten, fludrominoketone antigen and preparation method and application thereof

Info

Publication number: CN112174851A
Application number: CN202011240526.9A
Authority: CN
Inventors: 袁强; 王平; 梁飞敏
Original assignee: Guangzhou Wondfo Biotech Co Ltd
Current assignee: Guangzhou Wondfo Biotech Co Ltd
Priority date: 2020-11-09
Filing date: 2020-11-09
Publication date: 2021-01-05

Abstract

The invention relates to a fludrolone hapten, a fludrolone antigen, a preparation method and application thereof, wherein the structure of the fludrolone hapten is shown as a formula (I). The preparation method of the flunomide hapten comprises the following steps: the method comprises the following steps of taking the fludrolone and a compound shown as a formula (II) as raw materials, carrying out reaction, and introducing an active arm with a carboxyl group to the carbonyl group of the fludrolone to obtain the fludrolone hapten. Further, activating the flunomide hapten, and then carrying out coupling reaction with carrier protein to obtain the flunomide antigen. The invention initiates a novel artificial semi-antigen of the fluoroamine ketone and an artificial antigen of the fluoroamine ketone, applies the antigens to the immunochromatography technology, has the advantages of high sensitivity, high specificity, simplicity, rapidness, easy operation, no need of any large-scale instrument and equipment and the like, and can be used for large-scale primary screening of the fluoroamine ketone.

Description

Fluoroaminoketone hapten, fludrominoketone antigen and preparation method and application thereof

Technical Field

The invention belongs to the technical field of biology, and relates to a fludrolone hapten, a fludrolone antigen, and preparation methods and applications thereof.

Background

The flutolamine (F-Ketamine) is a phenylalamine stimulant and has the chemical name: dl-2-o-fluorophenyl-2-methylamino cyclohexanone. The fluminoketone is a derivative of phencyclidine (phencyclidine), and has certain potential of mental dependence. Fluminexone can produce a separate anesthetic state with clinical features similar to ketamine (K powder), with rigidity, light sedation, amnesia and significant analgesia, and can enter the dream, appearing hallucinations, becoming a common hallucination in wild mates and other similar activities. Currently, many countries have used flunomide, tubulofluoride in british, CLASS B, sweden and ladevian as illegal ingredients. Professor of king world jade at Beijing university reports a synthetic route of flunomide, and animal experiments prove that the flunomide has similar anesthetic effect to ketamine. The results of part of the in vitro experiments show that the half-life of flunomide (T1/2, invitro) is 69.1 + -13.1 min, while the half-life of ketamine (T1/2, invitro) is 23 + -3 min, and the clearance rate of ketamine is higher than that of flunomide. The potency of the flunomide is about 1.5 times that of the ketamine, and the duration of the potency is about 2-3 times that of the ketamine.

At the present stage, China is not listed in a national regulatory drug catalogue, the compound is not clearly specified and explained, a large number of abused people who use the fludrolone to replace K powder exist in the society, and the detection of the fludrolone is limited to High Performance Liquid Chromatography (HPLC), Gas Chromatography (GC), Thin Layer Chromatography (TLC), gas-mass spectrometry (GC-MS), liquid-mass spectrometry (LC-MS) and the like, but the methods not only need expensive instruments and equipment, but also have high requirements on detected materials, and can be carried out only by further purification treatment, so that the requirements of modern detection on rapidness, convenience and accuracy cannot be met.

The standard for detecting the flunomide cannot be achieved by using the ketamine rapid detection reagent. In order to monitor the drug and ensure the health of people, research on a fludrolone immunoassay method needs to be carried out, and an effective preparation method and a rapid detection reagent for a fludrolone artificial antigen need to be provided.

Disclosure of Invention

Aiming at the defects of the prior art, the invention aims to provide a fludrolone hapten, a fludrolone antigen, and a preparation method and application thereof. The kit specifically comprises a flunomide hapten and a preparation method thereof, a flunomide antigen and a preparation method thereof, application of the flunomide hapten or the flunomide antigen in immunodetection of flunomide in a sample, and an immunochromatographic kit for detecting the flunomide in the sample.

In order to achieve the purpose, the invention adopts the following technical scheme:

in a first aspect, the present invention provides a fluoroamidone hapten, wherein the fluoroamidone hapten has a structure represented by formula (i):

wherein x is any integer from 1 to 6, for example, x is 1, 2, 3, 4, 5 or 6; y is 2 times x.

The invention provides a novel artificial hapten and artificial antigen of fludrolone, which can be suitable for immunoassay of the fludrolone to prepare a fast detection kit of the fludrolone, and is the only product in China at the present stage. The detection kit prepared by the invention reduces cross-member interference, can be applied to a kit for hair detection materials, improves the detection sensitivity to 1ng/mg, and is the highest in sensitivity of similar products.

In a second aspect, the present invention provides a method for preparing a fluoroamine hapten as described above, comprising: reacting fludrolone with a compound shown in a formula (II) as raw materials, and introducing an active arm with carboxyl on carbonyl of the fludrolone to obtain the fludrolone hapten;

The key factor of the synthesis method of the fluoroamidone hapten related by the invention is that an active arm with carboxyl is introduced on carbonyl of a fluoroamidone molecular structure.

Preferably, the reaction is carried out under basic conditions.

Preferably, the alkaline medium comprises potassium carbonate.

Preferably, the molar ratio of the flunomide to the compound represented by the formula (II) is 1 (1-3), such as 1:1, 1:1.5, 1:2, 1:2.5 or 1:3, and other specific values within the above range can be selected, and are not described in detail herein.

Preferably, the medium of the reaction comprises aqueous methanol.

Preferably, the volume ratio of the methanol to the water is (3-4): (1-2), for example, 6:2, 6:3, 6:4, 7:2, 7:3, 7:4, 8:2, 8:3, or 8:4, and other specific values within the above range can be selected, and are not described in detail herein. Further preferably 7: 3.

Preferably, the reaction temperature is 15-65 ℃, for example, 15 ℃, 25 ℃, 30 ℃, 35 ℃, 40 ℃, 50 ℃, 60 ℃ or 65 ℃ and the like, and the time is 18-36h, for example, 18h, 20h, 22h, 25h, 30h or 36h and the like, and other specific values in the above range can be selected, and are not described in detail herein.

In a third aspect, the present invention provides a fluoroamidone antigen prepared from a fluoroamidone hapten as described above.

The flunomide hapten related to the invention is activated by coupling with a carrier protein, and the modified antigen with the carrier protein has stronger immune activity because the single small molecule does not have immune activity or has low immune activity.

In a fourth aspect, the present invention provides a method for preparing a fluoroamine antigen as described above, comprising: activating the flunomide hapten, and then carrying out coupling reaction with carrier protein to obtain the flunomide antigen.

Preferably, the activation mode of the flunomide hapten is as follows: mixing the flunixin with N-hydroxysuccinimide and cyclohexyl carbodiimide for reaction to obtain the activated flunixin.

Preferably, the molar ratio of the flunomide hapten to the N-hydroxysuccinimide to the cyclohexylcarbonyldiimine is 1 (1-1.5): (1-1.5), such as 1:1:1, 1:1:1.5, 1:1.5:1, 1:1.5:1.5, 1:1.2:1, 1:1:1.2 or 1:1.2:1.2, and others in the above range can be selected, and the description is omitted.

Preferably, the reaction medium of the reaction comprises N, N-dimethylformamide.

Preferably, the carrier protein comprises bovine serum albumin, ovalbumin, hemocyanin, bovine hemoglobin, bovine thyroid protein, human immunoglobulin IgG or human immunoglobulin IgA.

Preferably, the pH of the coupling reaction system is maintained at 6.8-7.2, for example, pH 6.8, pH 6.9, pH7.0, pH 7.1 or pH 7.2, and other specific values within the above range can be selected, which is not described in detail herein.

Preferably, the temperature of the coupling reaction is 2-8 ℃, for example, 2 ℃, 4 ℃, 6 ℃ or 8 ℃, and the time is 10-24h, for example, 10h, 15h, 18h, 20h or 24h, and other specific values within the above range can be selected, and are not described in detail herein.

Preferably, after the coupling reaction is finished, the reaction mixture is purified by dialysis, and the dialysis solution is 0.05M PBS buffer solution with pH7.0.

When the preparation method of the fluoroamidone antigen meets the requirements of temperature, time, raw material proportion, system pH environment and medium, the reaction efficiency is higher, and the yield of the fluoroamidone antigen is higher.

In a fifth aspect, the present invention provides a use of a flunomide hapten as described above or a flunomide antigen as described above for the immunoassay of flunomide in a sample.

Preferably, the sample comprises a body fluid sample or a tissue sample.

The synthesized flunomide hapten or the flunomide antigen can be used for preparing a sample to detect whether the sample contains the flunomide by using an immunization method. The sample herein refers to a sample of a mammalian or human body fluid, such as saliva, urine, sweat; alternatively, a tissue sample from a mammal or human, such as hair, liver, spleen, etc. The "immunoassay" as used herein is an immunoassay or a detection method using the principle of binding an antibody to an antigen, and includes an enzyme-linked immunosorbent assay (ELISA) and also a Lateral Flow test strip (lareal Flow) method, including a competition method.

The lateral flow test strips are made of absorbent or non-absorbent materials, and one test strip may be made of a variety of materials for fluid transfer. One material of the test strip may be superimposed on another test strip material, such as filter paper superimposed on nitrocellulose. Alternatively, one region of the test strip containing at least one material is positioned behind another region containing at least one different material. In this case, the liquid can flow between the regions, and they may or may not overlap each other. The material on the test strip may be fixed to a support or hard surface that is lined with a plastic backing to enhance the holding force of the test strip. The test strip regions may be arranged as follows: a sample addition zone, at least one reagent zone, at least one detection result zone, at least one control zone, at least one adulteration detection zone and a liquid absorption zone. If the detection zone comprises a control zone, it is preferred that the control zone is located after the analyte detection zone in the detection result zone. All of these regions or combinations thereof may be on a single test strip containing one material. In addition, the zones are made of different materials and are joined together in the direction of liquid transfer. For example, the different zones may be in direct or indirect fluid communication.

In a sixth aspect, the invention provides an immunochromatographic reagent strip for detecting fluoroamidone in a sample, which comprises a sample pad, a marking pad, a nitrocellulose membrane, a water absorption plate and a bottom plate; the nitrocellulose membrane comprises a detection line coated by the above-mentioned fluoroamidone antigen and a quality control line coated by a goat anti-mouse antibody.

Preferably, the concentration of the colloidal gold labeled fludrolone antibody in the immunochromatography reagent strip is 15-25 mug/mL; the coating concentration of the fludrolone antigen is 0.1-0.8 mg/mL; the concentration of the goat anti-mouse IgG antibody is 0.8-1.0 mg/mL; the dosage of the goat anti-mouse IgG antibody is 0.09-0.22 mu L/mm.

Preferably, the sample pad is treated by a sample pad treatment solution, wherein the sample pad treatment solution comprises a buffer solution with pH of 8-11, an aqueous rheological aid and a sealant; wherein the addition amount of the aqueous rheological additive is 0.05-10g and the addition amount of the blocking agent is 0.05-10g per 100mL of the buffer solution.

Preferably, the sample pad treatment solution further includes 0.01 to 1M Na₂CO₃Or K₂CO₃。

Preferably, the sample pad treatment solution further comprises trehalose; the addition amount of trehalose is 0.01-1g per 100mL of the buffer.

Preferably, the aqueous rheology aid is at least one of HPMC, PAA or PEO.

Preferably, the blocking agent is rabbit serum.

Preferably, the fluocinone antigen on the detection line is coated by a coating buffer solution, wherein the coating buffer solution comprises Tris-HCl buffer solution with the pH of 7-8 and the concentration of 1-50mM, S9 and urea; wherein the addition amount of the S9 is 0.01-1g and the addition amount of the urea is 2-10g per 100mL of Tris-HCl buffer solution.

The preparation method of the sample pad comprises the following steps: and soaking the glass fiber in the sample solution, and drying.

The preparation method of the marking pad comprises the following steps: (1) HAuCl₄The aqueous solution and the aqueous solution of trisodium citrate are azeotroped to prepare the colloidal gold solution, and the color is red. (2) Combining colloidal gold with protein (IgG), adding a fludrolone antibody solution into a colloidal gold solution with the pH of 9.0, adding a stabilizer, and performing centrifugal purification to obtain the immune colloidal gold marker. (3) And coating the prepared immune colloidal gold marker solution with the concentration on glass fiber, and drying to obtain the marker pad.

The preparation method of the nitrocellulose membrane comprises the following steps: a T line solution is prepared from a fluoroamidone-protein conjugate (a fluoroamidone antigen related to the invention), a C line solution is prepared from a goat anti-mouse antibody, and then the T line (detection line) and the C line (quality control line) areas on a nitrocellulose membrane are respectively coated.

The preparation method of the reagent strip comprises the following steps: the sample pad, the marking pad, the nitrocellulose membrane coated with the T line and the C line and the water absorption plate are arranged in sequence from the sample adding end. The four parts are all adhered on the bottom plate in sequence and then cut into required width to form the reagent strip.

Or can be filled according to the size of a mould (plastic shell) to form the reagent card.

In a seventh aspect, the present invention further provides a liquid reagent cup, which includes a cup body, a test paper clamping plate and a cover film, wherein the test paper clamping plate is disposed in the cup body, and is provided with a test paper slot, and the test paper slot is disposed therein with the reagent strip for detecting fluoroamine ketone in a sample and other drug detection reagent strips.

The test paper slot is internally provided with an anti-adulteration reagent strip which is selectively placed; the cover film covers the surface of the test strip through the open surface of the test strip slot.

In some of these embodiments, the length of the bottom edge of the cover film from the bottom edge of the test strip is 3-10 mm.

In some of these embodiments, the cover film is a transparent film.

Compared with the prior art, the invention has the following beneficial effects:

the invention initiates a novel artificial semi-antigen of the fludrolone and the artificial antigen of the fludrolone, the preparation method has simple and effective synthesis steps, can be completely used in immunoassay, provides a convenient way for the future research of the fludrolone for people, and can meet the domestic needs of the research.

In addition, the invention also provides an immunochromatography assay reagent strip, a reagent card and a reagent cup for detecting or determining the fludrolone, wherein the reagent strip, the reagent card and the reagent cup comprise the artificial antigen of the fludrolone. The method is applied to the immunochromatography technology, and has the advantages of high sensitivity, high specificity, simplicity, rapidness, easiness in operation, no need of any large-scale instrument and equipment and the like. Can be used for large-scale primary screening of the fludrolone.

Drawings

FIG. 1 is an ESI-MS analysis spectrum of a fluoroamidone hapten prepared in example 1;

FIG. 2 is a UV scan of the fluoroamine hapten, bovine serum albumin, and fluoroamine antigen of example 2;

FIG. 3 is a schematic view showing the structure of the immunochromatographic diagnostic reagent strip for detecting fluoroamidone prepared in example 3;

FIG. 4 is an external view of the immunochromatographic diagnostic reagent card for detecting fluoroamidone prepared in example 3;

FIG. 5 is a graph showing the results of the immunochromatographic diagnostic kit prepared in example 3 in detecting fludrolone (a is a negative result, b is a positive result, and c and d are both failure results).

FIG. 6 is a schematic diagram showing the structure of an immunochromatographic diagnostic reagent cup for detecting fluoroamidone prepared in example 6;

FIG. 7 is a schematic diagram showing the structure of the reagent cups for immunochromatography diagnosis for detecting fluoroamidone prepared in examples 12 and 16.

Detailed Description

The invention is further illustrated by the following specific embodiments. It should be understood by those skilled in the art that the examples are only for the understanding of the present invention and should not be construed as the specific limitations of the present invention.

Example 1

In this example, a fludrolone hapten was prepared as follows:

10mmol of carboxymethyl hydroxylamine hydrochloride and 10mmol of K are weighed respectively₂CO₃Adding 21mL of methanol and 9mL of purified water into a 50mL round-bottom flask, fully stirring for 20min, adding 0.1mmol of 18-crown-6 and 10mmol of flunomide, and carrying out reflux reaction at 65 ℃ for 24 h; after the reaction is finished, evaporating to dryness under reduced pressure, adding 50mL of ultrapure water and 50mL of dichloromethane into the residue, separating out a water phase, adjusting the pH value to 4 by using 2M hydrochloric acid, extracting twice by using 10mL of n-butanol, combining n-butanol layers, drying, and distilling under reduced pressure to obtain 2.3mmol of hapten for later use; ESI-MS analysis of the hapten thus obtained (295.1[ M +1 ]]) The results are shown in FIG. 1, demonstrating the successful production of the flunomide hapten.

Example 2

This example prepares a flunomide antigen by the following method:

(1) preparation of active ester of fludrolone hapten:

in a 25mL dry single neck flask, 1mmol of the hapten prepared in example 1, 1.2mmol of N-hydroxysuccinimide, 1.2mmol of cyclohexylcarbonyldiimine and 8mL of N, N-dimethylformamide are added and stirred under protection of helium at 25 ℃ for 18 h. After the reaction is finished, the reaction mixture is divided into 8 centrifuge tubes with the volume of 1.5mL, the centrifuge tube is centrifuged at 10000rpm for 30min, and the supernatant is divided into 8 glass sealed bottles with the volume of 2mL and stored for standby under the protection of helium.

(2) Preparation of a Fluoroaminoketone antigen (Fluoroaminoketone-bovine serum albumin):

100mg of BSA was taken in a 25mL single-neck flask, 8mL of 0.1M phosphate buffer solution (pH7.0) was added thereto and dissolved by stirring, the temperature of the reaction mixture was maintained at about 4 ℃ by an ice-water bath, 2.6mL of an active ester solution was slowly added dropwise to the BSA solution, and the pH of the reaction mixture was adjusted as needed by 0.1M hydrochloric acid to maintain the pH of the mixture at about 7.0. After the dropwise addition, the temperature of the reaction solution was maintained at about 4 ℃ and the reaction was stirred for 18 hours. After the reaction is finished, transferring the reaction mixture into a dialysis bag, dialyzing for many times by using 0.05M PBS buffer solution with pH7.0, monitoring by using an ultraviolet spectrophotometer until the light absorption value of the dialysate is consistent with the baseline at 200-400nm, and then freeze-drying the liquid in the dialysis bag to obtain the target antigen: fluoroaminone-bovine serum albumin.

The prepared flunomide antigen was identified as follows:

(1) and (3) coupling ratio determination: the binding ratio of the conjugate is the ratio of the number of molecules of hapten to carrier protein in the conjugate, and the binding ratio can be determined by spectrophotometry. Spectrophotometry is a principle that the absorption of light by a substance is in a proportional relationship with the concentration of the substance, and the concentrations of two coupled molecules are respectively measured. In the macromolecular and micromolecular conjugate, the two molecules have different ultraviolet scanning spectrums respectively and show the superposed properties of the spectrums. The binding ratio of the conjugate is the ratio of the number of molecules of hapten to carrier protein in the conjugate.

Respectively measuring the molar extinction coefficients () of the fludrolone hapten, the bovine serum albumin and the fludrolone antigen under a certain wavelength, and according to the additive principle of absorbance in spectral analysis and a formula: the number of moles of hapten coupled per mole of carrier protein was calculated. According to the ultraviolet spectrum scanning result, qualitative analysis can be carried out by contrasting the ultraviolet absorption spectrum characteristics of the three.

The ultraviolet scanning images of the flunomide hapten, the bovine serum albumin and the flunomide antigen are shown in figure 2, BSA has typical absorption at 279nm and 252nm, and the absorption of the flunomide hapten and the flunomide-bovine serum albumin conjugate has obvious and offset at 279nm and 252nm, which shows that the artificial antigen conjugate has different ultraviolet absorption characteristics with precursor substances thereof, and shows that the coupling of the hapten and the carrier protein is successful, and the molecular binding ratio of the hapten to the carrier protein is calculated to be 9.32 according to the formula.

(2) Measurement of protein concentration of Fluminoketone antigen (Fluminoketone-bovine serum albumin): preparing 1.5mL bovine serum albumin solution with the concentration of 0, 40, 60, 80, 100, 120, 160 and 200 mu g/mL, adding 5mL Coomassie brilliant blue staining solution, immediately mixing uniformly, standing for 20min, taking parallel samples at each concentration, measuring the light absorption value at 595nm, and drawing a relation curve between the protein concentration and the light absorption value. Diluting the antigen solution according to a certain proportion, measuring the light absorption value of the antigen solution at 595nm, and obtaining the corresponding protein concentration of the antigen solution from the curve. The protein concentration of the antigen solution was calculated to be 8.26mg/mL by the present invention.

Example 3

In this embodiment, an immunochromatographic diagnostic reagent strip and a reagent card for detecting fluoroamidone in a urine sample are prepared, and the preparation method is as follows:

(1) sample pad preparation: soaking the glass fiber in a sample pad treatment solution, and then drying, wherein the sample pad treatment solution is a 0.02M formula with the pH value of 8.4Tris and contains the following components in percentage by mass: 0.8% Triton X-100, 0.8% Tween-80, 2% rabbit serum, 1% HPMC, 1% trehalose, 1% LowCross-Buffer, 2% NaCl, 1% Na₂CO₃，0.5‰Proclin 300。

(2) Preparing a marking pad: taking HAuCl with the mass fraction of 0.01 percent₄Adding 0.75mL of trisodium citrate aqueous solution with the mass fraction of 1 percent into 100mL of the aqueous solution, and heating and boiling for 30min to turn red. Stopping heating, and cooling for later use. Mixing the colloidal gold solution with 0.1M K₂CO₃Adjusting pH to 9.0, stirring the colloidal gold solution, adding anti-fluoroamidone monoclonal antibody (provided by guangzhou Wanfu biotechnology, Inc.), stirring for 10min, centrifuging, purifying to obtain immune colloidal gold marker, coating onto glass fiber, and oven drying; wherein the concentration of the colloidal gold-labeled flunomide antibody is 20 mug/mL.

(3) Preparing a nitrocellulose membrane: a T-line solution was prepared using the fluoroamine antigen prepared in example 2, and a C-line solution was prepared using a goat anti-mouse IgG antibody, which were then coated on the T-line and C-line regions of the nitrocellulose membrane, respectively. Wherein the coating concentration of the fludrolone antigen is 0.5 mg/mL; the coating concentration of the goat anti-mouse IgG antibody is 1.0 mg/mL; the dosage of the goat anti-mouse IgG antibody is 0.22 mu L/mm. Wherein, the fludrolone antigen is coated by adopting a coating buffer solution, and the formula of the coating buffer solution is as follows: pH 8.0, 50mM Tris-HCl buffer, 0.1% S9 and 5% urea.

(4) The pasting plate mode is as follows: the nitrocellulose membrane is connected with the marking pad in the direction of a T line (detection line) and is connected with the water absorption plate in the direction of a C line (quality control line). The label pad pressed the nitrocellulose membrane 2 mm. The sample pad is connected with the marking pad, and the lower part of the sample pad is flush with the lower end of the PVC bottom plate at the position of the marking pad 1/3. The nitrocellulose membrane is pressed by the water absorption plate for 1mm, and the upper end of the nitrocellulose membrane is flush with the upper end of the PVC bottom plate. When the detection sample is dripped to the sample pad, the sample is chromatographed upwards by utilizing the capillary effect, and the sample moves along the directions of the sample pad, the marking pad, the nitrocellulose membrane and the water absorption plate, so that the reaction process is completed. Cutting to form reagent strips as shown in figure 3 (wherein 1: sample pad; 2: mark pad; 3: T line; 4: C line; 5: absorbent paper; 6: PVC base plate; 7: nitrocellulose membrane).

And filling into a mold to form a reagent card, as shown in fig. 4.

(5) And (3) judging a detection result:

60 mu L of urine sample to be detected is dripped into the sample adding area of the kit sample, after the sample is dripped on the sample pad, liquid flows to the end of the water absorption plate for chromatography due to capillary chromatography effect, if the sample liquid has no fluoroamidone, the colloidal gold particles marked with the anti-fluoroamidone antibody run to the position of a T line (detection line) on the membrane along with the sample liquid and then undergo immunological binding reaction with the fluoroamidone antigen coated on the T line, and the unknown accumulation of the colloidal gold particles on the T line leads the T line to present a macroscopic red line which is a negative result, as shown in a in figure 5. If the sample liquid contains the flunomide, the flunomide and the coated flunomide-bovine serum albumin conjugate at the T line position compete for limited antibody binding sites, and when the concentration of the flunomide in the sample liquid reaches a certain amount, all the antibody binding sites marked on the colloidal gold particles are occupied, so that the binding of the coated flunomide-bovine serum albumin conjugate at the T line position and the antibody colloidal gold of the conjugate is prevented, and a T line area has no red line and shows a positive result, as shown in b in figure 5. No matter whether the fludrolone exists in the sample liquid or not, a red line appears in a C line (quality control line) area, and the red line is used for judging whether enough products to be detected exist or not and whether the chromatographic process meets the normal standard or not and is also used as the internal control standard of the kit.

Normal results: the positive is a red line appearing on the line C (quality control line) in the display area of the kit, as shown in a in figure 5; the negativity is that a red line appears on each of a T line (detection line) and a C line (quality control line) in a display area of the kit, as shown in b in figure 5; failure results: in the kit display area, there is no red line on both lines T and C, as shown by d in FIG. 5, or only one red line appears on line T, as shown by C in FIG. 5.

Example 4

In this example, the immunochromatographic diagnostic reagent card for detecting fludrolone prepared in example 3 was used to detect a sample, which is a clinical sample obtained from guangdong plus-fu medical poison judicial expertise, according to the following operation method:

1. sample preparation

Fluoroaminone standards were added to blank urine samples, formulated to give fluoroketonic samples at concentrations of 0ng/mL, 50ng/mL, 100ng/mL, 150ng/mL, 200ng/mL, 250ng/mL, 300ng/mL, 350ng/mL, 400ng/mL, 450ng/mL, 500ng/mL, 550ng/mL, 600ng/mL, 650ng/mL, 700ng/mL, 750ng/mL, 800ng/mL, 850ng/mL, 900ng/mL, 950ng/mL, 1000ng/mL, 1050ng/mL, 1100ng/mL, 1150ng/mL, 1200ng/mL, 1250ng/mL and 1300ng/mL, respectively, according to the requirements of the test.

2. Detection and results

2.1, performing quantitative gradient test on the series of concentrations, wherein a sample is added to a sample adding area (shown in figures 3 and 4) on the reagent card during the test, and both a detection line and a quality control line on the reagent strip are negative in color during the test; the detection line does not develop color, and the quality control line develops color to be positive; the detection line is colored, the quality control line is not colored, or the detection line and the quality control line are not colored, and the test fails; the results of the sensitivity test in this example are shown in Table 1.

TABLE 1 test results of sensitivity of the test paper for immunochromatography analysis of colloidal gold

Concentration (ng/mL)	0	50	100	150	200	250	300	350	400
										Test paper results	-	-	-	-	-	-	-	-	-
Concentration (ng/mL)	450	500	550	600	650	700	750	800	850
										Test paper results	-	+	+	+	+	+	+	+	+
Concentration (ng/mL)	900	950	1000	1050	1100	1150	1200	1250	1300
										Test paper results	+	+	+	+	+	+	+	+	+

Note: "+" indicates positive; "-" indicates negative.

The test result shows that the minimum detection quantity of the test paper is 500 ng/mL.

2.2 negative samples

According to the test requirements, 1024 negative samples subjected to GC/MS analysis and test are respectively loaded on the colloidal gold immunochromatographic assay test paper for detecting the fludroxone, and the results are negative.

Example 5

Drug crossover experiment of colloidal gold immunochromatographic assay reagent card for detecting fludrolone

94 drugs and common drugs, respectively, are prepared into samples with the concentration of 100 mu l/mL by using negative urine. The color development was judged according to the colorimetric card of Guangzhou Wanfu Biotechnology Co., Ltd., the negative result was represented by "-" and the positive result was represented by "+".

TABLE 2 Cross-over test results of the colloidal gold immunochromatographic assay test paper

The colloidal gold immunochromatographic assay reagent strip for detecting fludrolone of the embodiment is prepared by a conventional preparation method and comprises a test strip and a support plate for supporting the test strip; the test strip is formed by sequentially overlapping sample filtering paper, a chromatographic material, a nitrocellulose membrane and absorbent paper, wherein the chromatographic material is pre-coated with a colloidal gold-labeled or colored-labeled fluoroamidone monoclonal antibody or polyclonal antibody. Adsorption detection lines and quality control lines are arranged on the nitrocellulose membrane; the detection line is a fluoroamidone antigen, and the area where the detection line is located is a detection area; the quality control line is a goat anti-mouse polyclonal antibody, and the area where the quality control line is located is a quality control area.

Example 6

This embodiment provides a reagent cup for immunochromatography analysis of a fluoroamine ketone colloidal gold used in a urine sample.

Referring to FIG. 6 (wherein 1: the cup cover; 2: the sealing ring; 3: the test paper clamping plate; 4: the cup body), the sample detection cup of the present embodiment comprises a cup body, a test paper clamping plate, a cover film, a cup cover and a sealing ring arranged in the cup cover; the test paper clamping plate is arranged in the cup body, a reagent strip slot is formed in the test paper clamping plate, and the fluoroamine ketone detection reagent strip and other drug detection reagent strips in embodiment 3 and anti-adulteration reagent strips selectively placed in the slot are placed in the slot; the cover film covers the surface of the reagent strip through the opening surface of the slot, and the distance between the bottom end edge of the cover film and the bottom end edge of the test strip is 3-10 mm.

Example 7

In this example, the reagent cup of example 6 was used to perform sample testing, and the sample was a clinical sample from Guangdong Fufu medical poison judicial institute, and the operation method was as follows:

1. sample preparation

Prepared in the same manner as in example 4.

2. Detection and results

2.1, performing quantitative gradient test on the series of concentrations, adding 90mL of sample into a detection cup during the test, screwing a cup cover and standing; during testing, the detection line and the quality control line on the test strip are negative in color; the detection line does not develop color, and the quality control line develops color to be positive; the detection line is colored, the quality control line is not colored, or the detection line and the quality control line are not colored, and the test fails; the results of the sensitivity test in this example are identical to those in example 4 and are not listed here.

The test result shows that the lowest detection quantity of the reagent cup is 500 ng/mL.

2.2 negative samples

According to the test requirements, 318 negative samples subjected to GC/MS analysis and test are respectively added into the reagent cups, and the results of detecting the fludrolone are all negative.

Example 8

Drug crossover experiment in reagent cups

The results of the drug crossover experiments in this example are consistent with those in example 5 and are not listed here.

Example 9

In this embodiment, an immunochromatographic diagnostic reagent strip and a reagent card for detecting fluoroamidone in a hair sample are prepared, and the preparation method is as follows:

(1) sample pad preparation: soaking the glass fiber in a sample pad treatment solution, and then drying, wherein the sample pad treatment solution is prepared by adding the following components in percentage by mass in 0.01M pH8.4 Tris: 1.2% PVP-10, 5% rabbit serum, 1% trehalose, 1% LowCross-Buffer, 0.8% Tween-80, 0.1% HPMC, 0.1% Na₂CO₃。

(2) Preparing a marking pad: taking HAuCl with the mass fraction of 0.01 percent₄Adding 0.75mL of trisodium citrate aqueous solution with the mass fraction of 1% into 100mL of the aqueous solution, and heating and boiling for 30min to turn red. Stopping heating, and cooling for later use. Mixing the colloidal gold solution with 0.1M K₂CO₃Adjusting pH to 9.0, stirring the colloidal gold solution, adding the anti-fluoroamidone monoclonal antibody (provided by guangzhou Wanfu biotechnology, Inc.), continuously stirring and stirring for 10min, performing centrifugal purification to obtain an immune colloidal gold marker, coating the immune colloidal gold marker on glass fiber, and drying for later use; wherein the concentration of the colloidal gold-labeled flunomide antibody is 20 mug/mL.

(3) Preparing a nitrocellulose membrane: a T-line solution was prepared using the fluoroamine antigen prepared in example 2, and a C-line solution was prepared using a goat anti-mouse IgG antibody, which were then coated on the T-line and C-line regions of the nitrocellulose membrane, respectively. Wherein the coating concentration of the fludrolone antigen is 0.1 mg/mL; the coating concentration of the goat anti-mouse IgG antibody is 0.8 mg/mL; the dosage of the goat anti-mouse IgG antibody is 0.09 mu L/mm. Wherein, the fludrolone antigen is coated by adopting a coating buffer solution, and the formula of the coating buffer solution is as follows: pH 8.0, 50mM Tris-HCl buffer, 0.1% S9 and 5% urea.

And filling into a mold to form a reagent card, as shown in fig. 4.

(5) Preparing a hair lysate: taking 0.02M solution with pH9.6 CB, adding the following components in percentage by mass: 5% of D-sodium erythorbate, 1% of keratinase S-221, 2% of urea, 1% of TCEP & HCl, 1% of sodium thioglycolate, 0.2% of sodium lauryl sulfate and 0.5% of Proclin 300.

(6) And (3) judging a detection result:

30mg of a hair sample to be detected is taken, 1mL of hair lysate is added into the hair, after the hair sample is broken and cracked for 2min, 60 mu L of the hair sample is dripped into a sample adding area of the kit, after the sample is dripped onto a sample pad, liquid flows to the end of a water absorption plate due to the capillary chromatography effect, if no fluoroamidone exists in the sample liquid, colloidal gold particles marked with an anti-fluoroamidone antibody run to the position of a T line (detection line) on a membrane along with the sample liquid, and then carry out immunological binding reaction with fluoroamidone antigen coated on the T line, and the unknown accumulation of the colloidal gold particles on the T line enables the T line to present a macroscopic red line, which is a negative result, as shown in a in figure 5. If the sample liquid contains the flunomide, the flunomide and the coated flunomide-bovine serum albumin conjugate at the T line position compete for limited antibody binding sites, and when the concentration of the flunomide in the sample liquid reaches a certain amount, all the antibody binding sites marked on the colloidal gold particles are occupied, so that the binding of the coated flunomide-bovine serum albumin conjugate at the T line position and the antibody colloidal gold of the conjugate is prevented, and a T line area has no red line and shows a positive result, as shown in b in figure 5. No matter whether the fludrolone exists in the sample liquid or not, a red line appears in a C line (quality control line) area, and the red line is used for judging whether enough products to be detected exist or not and whether the chromatographic process meets the normal standard or not and is also used as the internal control standard of the kit.

Example 10

In this example, the immunochromatographic diagnostic reagent card for detecting fludrolone prepared in example 9 was used to detect a sample, which is a clinical sample obtained from guangdong plus-fu medical poison judicial expertise, according to the following operation method:

1. sample preparation

According to the test requirements, a standard sample of flunomide is added to a 30mg blank hair sample, and 1mL of hair lysate is added thereto to prepare samples containing flunomide at concentrations of 0ng/mg, 0.25ng/mg, 0.5ng/mg, 0.75ng/mg, 1ng/mg, 1.25ng/mg, 1.5ng/mg, 1.75ng/mg, 2ng/mg, 2.25ng/mg, 2.5ng/mg, 2.75ng/mg, 3ng/mg, 3.25ng/mg, 3.5ng/mg, 3.75ng/mg, 4ng/mg, 4.25ng/mg, 4.5ng/mg, 4.75ng/mg and 5ng/mg, respectively.

2. Detection and results

2.1, performing quantitative gradient test on the series of concentrations, wherein a sample is added to a sample adding area (shown in figures 3 and 4) of the reagent card during the test, and both a detection line and a quality control line on the reagent strip are negative in color during the test; the detection line does not develop color, and the quality control line develops color to be positive; the detection line is colored, the quality control line is not colored, or the detection line and the quality control line are not colored, and the test fails; the results of the sensitivity test in this example are shown in Table 3.

TABLE 3 sensitivity test results

Concentration (ng/mg)	0	0.25	0.5	0.75	1	1.25	1.5
								Test paper results	-	-	-	-	+	+	+
Concentration (ng/mg)	1.75	2	2.25	2.5	2.75	3	3.25
								Test paper results	+	+	+	+	+	+	+
Concentration (ng/mg)	3.5	3.75	4	4.25	4.5	4.75	5
								Test paper results	+	+	+	+	+	+	+

Note: "+" indicates positive; "-" indicates negative.

The test result shows that the minimum detection amount of the test paper is 1 ng/mg.

2.2 negative samples

According to the test requirements, 3286 negative hair samples after GC/MS analysis test are respectively loaded on the reagent card of example 9, and the results of detecting the flutolanil are all negative.

Example 11

Drug crossover experiment of immunochromatography diagnostic kit for detecting fludrolone in hair sample

94 kinds of drugs and common drugs are respectively used for preparing samples with the concentration of 100 mu l/mL by using negative hair and hair lysate. The color development was judged according to the colorimetric card of Guangzhou Wanfu Biotechnology Co., Ltd., the negative result was represented by "-" and the positive result was represented by "+".

Example 12

This example provides a reagent cup for immunochromatography of a fluoroamine colloidal gold assay for use in a hair sample.

Referring to fig. 7 (wherein 1: the test paper clamping plate; 2: the cover connecting cup body with the sealing ring), the sample detection cup of the present embodiment includes a cover connecting cup body with a sealing ring, a test paper clamping plate and a cover film; the test paper clamping plate is arranged in the cup body, a reagent strip slot is arranged on the test paper clamping plate, and the fluoroamine ketone detection reagent strip and other drug detection reagent strips in embodiment 9 and anti-adulteration reagent strips selectively placed in the slot are placed in the slot; the cover film covers the surface of the reagent strip through the opening surface of the slot, and the distance between the bottom end edge of the cover film and the bottom end edge of the test strip is 3-10 mm.

Example 13

In this embodiment, an immunochromatographic diagnostic reagent strip and a reagent card for detecting fluoroamidone in a saliva sample are prepared, and the preparation method is as follows:

(1) sample pad preparation: soaking the glass fiber in a sample pad treatment solution, and then drying, wherein the formula of the sample pad treatment solution is 0.02M, pH8.4 Tris, and the following components are added in mass fraction: 2% rabbit serum, 1.2% PVP-10, 1% trehalose, 1% NaCl, 1% Na₂CO₃，1％HPMC，0.8％Triton X-100，0.8％Tween-80，0.5‰Proclin 300。

(2) Preparing a marking pad: 0.01% HAuCl was taken₄100mL of the aqueous solution, 0.75mL of 1% trisodium citrate aqueous solution, and boiling for 30min to turn red. Stopping heating, and cooling for later use. Mixing the colloidal gold solution with 0.1M K₂CO₃Adjusting pH to 9.0, stirring the colloidal gold solution, adding anti-fluoroamidone monoclonal antibody (provided by guangzhou Wanfu Biotechnology Co., Ltd.), and continuingStirring for 10min, centrifuging, purifying to obtain immune colloidal gold marker, coating onto glass fiber, and oven drying. Wherein the concentration of the colloidal gold-labeled flunomide antibody is 20 mug/mL.

(3) Preparing a nitrocellulose membrane: a T-line solution was prepared using the fluoroamine antigen prepared in example 2, and a C-line solution was prepared using a goat anti-mouse IgG antibody, which were then coated on the T-line and C-line regions of the nitrocellulose membrane, respectively. Wherein the coating concentration of the fludrolone antigen is 0.2 mg/mL; the coating concentration of the goat anti-mouse IgG antibody is 0.8 mg/mL; the dosage of the goat anti-mouse IgG antibody is 0.1 mu L/mm. Wherein, the fludrolone antigen is coated by adopting a coating buffer solution, and the formula of the coating buffer solution is as follows: pH 8.0, 50mM Tris-HCl buffer, 0.1% S9 and 5% urea.

And filling into a mold to form a reagent card, as shown in fig. 4.

(5) And (3) judging a detection result:

60 mu l of saliva sample to be detected is dripped into the sample adding area of the kit sample, after the sample is dripped on the sample pad, liquid flows to the end of the water absorption plate for chromatography due to capillary chromatography effect, if the sample liquid has no fluoroamidone, the colloidal gold particles marked with the anti-fluoroamidone antibody run to the position of a T line (detection line) on the membrane along with the sample liquid and then carry out immunological binding reaction with the fluoroamidone antigen coated on the T line, and the unknown accumulation of the colloidal gold particles on the T line leads the T line to present a macroscopic red line which is a negative result, as shown in a in figure 5. If the sample liquid contains the flunomide, the flunomide and the coated flunomide-bovine serum albumin conjugate at the T line position compete for limited antibody binding sites, and when the concentration of the flunomide in the sample liquid reaches a certain amount, all the antibody binding sites marked on the colloidal gold particles are occupied, so that the binding of the coated flunomide-bovine serum albumin conjugate at the T line position and the antibody colloidal gold of the conjugate is prevented, and a T line area has no red line and shows a positive result, as shown in b in figure 5. No matter whether the fludrolone exists in the sample liquid or not, a red line appears in a C line (quality control line) area, and the red line is used for judging whether enough products to be detected exist or not and whether the chromatographic process meets the normal standard or not and is also used as the internal control standard of the kit.

Example 14

In this example, the immunochromatographic diagnostic reagent card for detecting fludrolone prepared in example 13 was used to detect a sample, which is a clinical sample obtained from guangdong plus-fu medical poison judicial expertise, according to the following procedure:

1. sample preparation

According to the test requirements, a standard substance of the flunomide is added to a blank saliva sample and is respectively prepared into samples containing the flunomide, and the concentration of the samples is 0ng/mL, 10ng/mL, 20ng/mL, 30ng/mL, 40ng/mL, 50ng/mL, 60ng/mL, 70ng/mL, 80ng/mL, 90ng/mL, 100ng/mL, 110ng/mL, 120ng/mL, 130ng/mL, 140ng/mL, 150ng/mL, 160ng/mL, 170ng/mL, 180ng/mL, 190ng/mL and 200 ng/mL.

2. Detection and results

2.1, performing quantitative gradient test on the series of concentrations, wherein a sample is added to a sample adding area (shown in figures 3 and 4) of the reagent card during the test, and both a detection line and a quality control line on the reagent strip are negative in color during the test; the detection line does not develop color, and the quality control line develops color to be positive; the detection line is colored, the quality control line is not colored, or the detection line and the quality control line are not colored, and the test fails; the results of the sensitivity test in this example are shown in Table 4.

TABLE 4 sensitivity test results

Concentration (ng/mL)	0	10	20	30	40	50	60
								Test paper results	-	-	-	-	-	+	+
Concentration (ng/mL)	70	80	90	100	110	120	130
								Test paper results	+	+	+	+	+	+	+
Concentration (ng/mL)	140	150	160	170	180	190	200
								Test paper results	+	+	+	+	+	+	+

Note: "+" indicates positive; "-" indicates negative.

The test result shows that the minimum detection quantity of the test paper is 50 ng/mL.

2.2 negative samples

According to the test requirements, 1024 negative samples subjected to GC/MS analysis tests are respectively loaded on the kit in the embodiment 13, and the results are negative.

Example 15

And (3) carrying out a drug crossover experiment on the immunochromatography diagnostic reagent card for detecting the fludrolone in the saliva sample.

94 drugs and general drugs were used, and samples with a concentration of 100. mu.l/mL were prepared with negative saliva, respectively. The color development was judged according to the colorimetric card of Guangzhou Wanfu Biotechnology Co., Ltd., the negative result was represented by "-" and the positive result was represented by "+".

The results of the drug crossover experiments in this example are consistent with example 8 and are not listed here.

Example 16

This example provides a reagent cup for immunochromatography of a gold immunochromatographic assay using a fluoroamine colloid for use in a saliva test material.

Referring to fig. 7 (wherein 1: the test paper clamping plate; 2: the cover connecting cup body with the sealing ring), the sample detection cup of the present embodiment includes a cover connecting cup body with a sealing ring, a test paper clamping plate and a cover film; the test paper clamping plate is arranged in the cup body, a reagent strip slot is arranged on the test paper clamping plate, and the fluoroamine ketone detection reagent strip and other drug detection reagent strips in embodiment 13 and anti-adulteration reagent strips selectively placed in the slot are placed in the slot; the cover film covers the surface of the reagent strip through the opening surface of the slot, and the distance between the bottom end edge of the cover film and the bottom end edge of the test strip is 3-10 mm.

The applicant states that the present invention is illustrated by the above examples to describe a fluorochemical hapten, a fluorochemical antigen, and methods of preparation and use thereof, but the present invention is not limited to the above examples, i.e., it is not meant that the present invention must rely on the above examples to be practiced. It should be understood by those skilled in the art that any modification of the present invention, equivalent substitutions of the raw materials of the product of the present invention, addition of auxiliary components, selection of specific modes, etc., are within the scope and disclosure of the present invention.

The preferred embodiments of the present invention have been described in detail, however, the present invention is not limited to the specific details of the above embodiments, and various simple modifications may be made to the technical solution of the present invention within the technical idea of the present invention, and these simple modifications are within the protective scope of the present invention.

It should be noted that the various technical features described in the above embodiments can be combined in any suitable manner without contradiction, and the invention is not described in any way for the possible combinations in order to avoid unnecessary repetition.

Claims

1. A flunomide hapten, wherein the structure of the flunomide hapten is shown as formula (I):

wherein x is any integer of 1-6; y is 2 times x.

2. The method of claim 1, wherein the method comprises: reacting fludrolone with a compound shown in a formula (II) as raw materials, and introducing an active arm with carboxyl on carbonyl of the fludrolone to obtain the fludrolone hapten;

wherein x is any integer of 1-6; y is 2 times x.

3. The method for producing a fluoroamidone hapten according to claim 2 wherein said reaction is carried out under basic conditions;

preferably, the alkaline medium comprises potassium carbonate;

preferably, the molar ratio of the flunomide to the compound shown in the formula (II) is 1 (1-3);

preferably, the medium of the reaction comprises aqueous methanol;

preferably, the volume ratio of methanol to water is (3-4): (1-2), further preferably 7: 3;

preferably, the reaction temperature is 15-65 ℃ and the reaction time is 18-36 h.

4. A fluoroamidone antigen prepared from a fluoroamidone hapten as claimed in claim 1.

5. The method of producing a flunomide antigen of claim 4, wherein the method of producing comprises: activating the flunomide hapten, and then carrying out coupling reaction with carrier protein to obtain the flunomide antigen.

6. The method of claim 5, wherein the activation of the flunomide hapten is by: mixing the flunixin with N-hydroxysuccinimide and cyclohexyl carbodiimide for reaction to obtain the activated flunixin.

7. The method of claim 6, wherein the molar ratio of the fluorochemical hapten to the N-hydroxysuccinimide to the cyclohexylcarbonyldiimine is 1 (1-1.5) to (1-1.5);

preferably, the reaction medium of the reaction comprises N, N-dimethylformamide;

preferably, the carrier protein comprises bovine serum albumin, ovalbumin, hemocyanin, bovine hemoglobin, bovine thyroid protein, human immunoglobulin IgG or human immunoglobulin IgA;

preferably, the pH value of the system of the coupling reaction is maintained between 6.8 and 7.2;

preferably, the temperature of the coupling reaction is 2-8 ℃, and the time is 10-24 h;

8. Use of a flunomide hapten as defined in claim 1 or a flunomide antigen as defined in claim 4 for the immunodetection of flunomide in a sample;

preferably, the sample comprises a body fluid sample or a tissue sample.

9. An immunochromatography reagent strip for detecting fluoroamidone in a sample, which is characterized by comprising a sample pad, a marking pad, a nitrocellulose membrane, a water absorption plate and a bottom plate; the nitrocellulose membrane comprises a detection line coated with the flunomide antigen of claim 4 and a quality control line coated with goat anti-mouse antibody.

10. A liquid reagent cup, the liquid reagent cup includes cup, test paper cardboard and cover film, the said test cardboard is placed in cup, there are test paper slots on it, characterized by that, place the reagent strip including flunomide ketone in the detection sample of claim 9 and other drugs detection reagent strips in the said test paper slot.