CN111560027A

CN111560027A - Rifaximin hapten, artificial antigen, preparation method and application thereof

Info

Publication number: CN111560027A
Application number: CN202010671590.6A
Authority: CN
Inventors: 马立才; 聂靖东; 丁亚芳; 邢维维; 刘薇; 李蓉蓉
Original assignee: Beijing Wdwk Biotechnology Co ltd
Current assignee: Beijing Wdwk Biotechnology Co ltd
Priority date: 2020-07-14
Filing date: 2020-07-14
Publication date: 2020-08-21

Abstract

The invention discloses a rifaximin hapten, an artificial antigen, and a preparation method and application thereof. The rifaximin artificial antigen provided by the invention is obtained by coupling a rifaximin hapten shown in a formula I with a carrier protein. Hair brushThe rifaximin artificial antigen provided by the rifaximin has the advantages of simple synthesis method, high purity and high yield, and has great values for the preparation of rifaximin antibodies and the detection of rifaximin drug residues.

Description

Rifaximin hapten, artificial antigen, preparation method and application thereof

Technical Field

The invention belongs to the field of rapid detection of drug residues, and relates to a rifaximin artificial antigen, and a preparation method and application thereof.

Background

Rifaximin belongs to rifamycin antibacterial drugs, is a semi-synthetic derivative of rifamycin, and mainly inhibits the synthesis of bacterial RNA by irreversibly combining with beta-subunit in bacterial DNA-dependent RNA polymerase, thereby achieving the purpose of sterilization. It has good antibacterial activity against gram-positive bacteria (such as Staphylococcus aureus, Streptococcus agalactiae, Streptococcus dysgalactiae, Streptococcus uberis, Corynebacterium, etc.) and gram-negative bacteria (such as Escherichia coli, etc.).

Rifaximin is mainly used as an injection for breasts of dairy cows in the dry period in veterinary medicine, is used for preventing and treating mastitis of dairy cows in the dry period, is widely applied due to good effect, a residue marker of rifaximin in milk is specified as a prototype drug by the European Union, the Maximum Residual Limit (MRL) is 60 mug/kg, and no rifaximin residue limit is specified in veterinary medicine residue limits in China.

Currently, liquid chromatography-mass spectrometry (LC-MS) and Mass Spectrometry (MS) are used for rifaximin residue detection methods, and although the methods have strong specificity and high sensitivity, the methods are complex to operate, expensive in instruments and not suitable for screening and detecting large-scale samples. The immunochemical analysis overcomes the defects of physicochemical analysis by considering the unique advantages of qualitative and quantitative antigen-antibody and the advantages of simple and rapid operation, low cost, higher sensitivity and large analysis sample amount, and plays an increasingly important role in the residual detection of the rifaximin.

The fundamental factors influencing the quality of immunochemical analysis are the specificity and affinity of the antibody, and the properties are determined by the structure of an immune hapten molecule, so that the molecular design and synthesis of the immune hapten are the most basic and key steps for generating specific antibodies and establishing a rapid detection technology for small-molecule veterinary drug residues.

Disclosure of Invention

The invention aims to provide a rifaximin artificial antigen and a preparation method and application thereof.

The rifaximin artificial antigen provided by the invention is an antigen constructed on the basis of rifaximin hapten.

The rifaximin hapten belongs to the protection range of the invention, and the structure of the rifaximin hapten is shown as a formula I.

Formula I

The method for preparing the rifaximin hapten specifically comprises the following steps:

1. rifamycin S is dissolved in Dimethylformamide (DMF), N-bromosuccinimide (NBS) is added for reaction, and 670mg of intermediate 1 is obtained by precipitation, filtration and drying of reaction liquid. The mixture ratio of the rifamycin S, the Dimethylformamide (DMF) and the N-bromosuccinimide (NBS) is 1000 mg: 6 ml: 300 mg.

2. Dissolving the intermediate 1 in methanol, adding triethylamine and 2-aminoisonicotinic acid for reaction, and carrying out spin-drying, chromatographic purification and spin-drying on reaction liquid to obtain 386mg of product. The mixture ratio of the intermediate 1, methanol, triethylamine and 2-amino isonicotinic acid is 500 mg: 5 ml: 2 ml: 100mg, wherein the chromatography developing agent is dichloromethane and methanol, and the ratio of the dichloromethane to the methanol is 15: 1.

the rifaximin antigen constructed on the basis of the rifaximin hapten also belongs to the protection scope of the invention.

The rifaximin antigen is obtained by coupling the rifaximin hapten (shown in a formula I) with a carrier protein. In one embodiment of the invention, the carrier protein is in particular Bovine Serum Albumin (BSA) or Ovalbumin (OVA).

The preparation method of the rifaximin antigen also belongs to the protection scope of the invention.

The preparation method of the rifaximin antigen specifically comprises the following steps: coupling the rifaximin hapten (formula I) with a carrier protein through an amido bond to obtain the rifaximin antigen.

In the present invention, the rifaximin antigen is prepared according to a method comprising the following steps:

(1) dissolving the rifaximin hapten (shown in a formula I) in Dimethylformamide (DMF), adding 1- (3-dimethylaminopropyl) -3-ethylcarbodiimide hydrochloride (EDC) and N-hydroxysuccinimide (NHS), and reacting for 2-3h at 20-25 ℃ by magnetic stirring to obtain a solution I;

wherein the ratio of the rifaximin hapten (formula I), the Dimethylformamide (DMF), the 1- (3-dimethylaminopropyl) -3-ethylcarbodiimide hydrochloride (EDC) and the N-hydroxysuccinimide (NHS) is 30.4 mg: 1.5 ml: 25.75 mg: 15.46 mg.

(2) Putting the carrier protein into 0.1M sodium bicarbonate buffer solution, stirring at 200rpm for 10min, and fully dissolving to obtain solution II; the ratio of the carrier protein to the 0.1M sodium bicarbonate buffer solution is 33.6-50 mg: 3.5 ml;

wherein, if the carrier protein is Bovine Serum Albumin (BSA), the ratio of the Bovine Serum Albumin (BSA) to the 0.1M sodium bicarbonate buffer solution is 50 mg: 3.5 ml; if the carrier protein is Ovalbumin (OVA), the ratio of the Ovalbumin (OVA) to the 0.1M carbonic acid buffer solution is 33.6 mg: 3.5 ml;

(3) mixing the solution I and the solution II, specifically, dropwise adding the solution I into the solution II under the condition of 0-4 ℃ and stirring at 1000rpm, and stirring at 500rpm for 24 hours to obtain a solution III;

(4) the rifaximin antigen was obtained by dialysis of the solution III with phosphate buffer (0.01M PBS, pH 7.2) at 4 ℃ for 3 days with stirring.

The rifaximin hapten (formula I) or the application of the rifaximin antigen in qualitative or quantitative detection of rifaximin also belongs to the protection scope of the invention.

Antibodies produced using the rifaximin antigens are also within the scope of the invention. The antibody may be a polyclonal antibody, a monoclonal antibody or an antiserum.

The rifaximin hapten and the rifaximin antigen provided by the invention have the advantages of simple synthesis method, high purity and high yield, and have great values for the preparation of rifaximin antibodies and the detection of rifaximin drug residues.

Drawings

Figure 1 is a rifaximin hapten mass spectrum.

Figure 2 is a standard curve for rifaximin.

FIG. 3 shows the consistency of the results of ELISA detection and LC-MS detection of two samples (beef and milk).

Detailed Description

The experimental procedures used in the following examples are all conventional procedures unless otherwise specified.

Materials, reagents and the like used in the following examples are commercially available unless otherwise specified.

Example 1 preparation and characterization of rifaximin haptens

1. Preparation of rifaximin haptens

A50 ml round bottom flask was rinsed clean, blown dry with ethanol, mounted on a stirrer and a stir bar was added. Weighing 1g rifamycin S, dissolving with 6ml DMF, stirring, keeping sample after dissolving completely, weighing 300mg NBS when the solution is purple, adding into the solution, stirring, treating when TLC finds that the reaction is complete, adding into ice water, precipitating, filtering to obtain solid, and drying to obtain 670mg intermediate 1.

Weighing 500mg of the intermediate 1, dissolving the intermediate 1 in 5ml of methanol, stirring, adding 2ml of triethylamine, adding 100mg of 2-aminoisonicotinic acid, stirring, monitoring by a dot plate, treating the reaction when the raw materials are unchanged, spin-drying the reaction solution, purifying by a residue column chromatography, and preparing a developing agent according to the following proportion: (dichloromethane: methanol =15: 1) the desired solution was collected and spin-dried to yield 386mg of product.

The reaction equation is as follows:

2. structural identification of rifaximin haptens

Mass spectrometry detection (figure 1) was carried out on 386mg of the obtained product, and the result shows that the chemical structural formula is shown as formula I (MW = 815.87), namely the rifaximin hapten.

Formula I

Example 2 preparation of rifaximin Artificial antigen

1. Synthesis of immunogens

(1) 30.4mg of rifaximin hapten is dissolved in 1.5ml of DMF, stirred at 200rpm for 10min, added with 25.75mg of EDC and 15.46mg of NHS for dissolution, and stirred at room temperature (500 rpm) for activation for 2-3 h.

(2) 50mg of BSA was weighed and dissolved in 3.5ml of 0.1M sodium bicarbonate solution, stirred at 200rpm for 10min to dissolve it sufficiently, the temperature was reduced to 0-4 ℃ in an ice bath, and the reaction solution in step 1 was added dropwise (1 ml/min) under stirring at 1000rpm, and stirred at 500rpm for 24 h.

(3) The reaction product was placed in a dialysis bag (10 cm) rinsed with distilled water, dialyzed at 1L of 0.01M PBS (1X, pH 7.2) with stirring (100 rpm) at 4 ℃ for 3d, the solution was changed 3 times a day (once in the morning, at night), the total of the solutions was changed 9 times, the dialyzed product was centrifuged at 5000rpm for 6min, and 1.5 ml/tube was dispensed, and the antigen was numbered and stored at-20 ℃ for further use.

2. Synthesis of coatingen

(1) 30.4mg of rifaximin hapten 4 was dissolved in 1.5ml of DMF, stirred at 200rpm for 10min, dissolved by adding 25.75mg of EDC and 15.46mg of NHS, and activated at room temperature for 2-3h with stirring (500 rpm).

(2) Weighing 33.6mg OVA, dissolving in 3.5ml 0.1M sodium bicarbonate solution, stirring at 200rpm for 10min to fully dissolve, cooling at 0-4 deg.C in ice bath, stirring at 1000rpm, dropwise adding the reaction solution of step 1 (1 ml/min), and stirring at 500rpm for 24 h.

EXAMPLE 3 preparation of monoclonal antibodies by immunization of animals with rifaximin Artificial antigen

First, animal immunization

Dissolving 100 mu g of immunogen (rifaximin-BSA) prepared in example 2 by using physiological saline, uniformly mixing the dissolved immunogen with Freund complete adjuvant in equal volume, injecting and immunizing Balb/c female mice with 6-8 weeks old by subcutaneous injection at the neck and back, uniformly mixing the immunogen with Freund incomplete adjuvant in equal volume on 7, 14 and 28 days after primary immunization, performing additional immunization once respectively, and performing additional immunization once by using 100 mu g of immune complex in 3 days before fusion without adding Freund adjuvant.

Second, cell fusion and cloning

Mixing splenocytes of immunized mice with myeloma cells of mice (SP 2/0) in logarithmic growth phase, slowly adding preheated fusion agent (PEG 4000) within 45s for fusion, suspending with HAT medium, adding appropriate amount of feeder cells, culturing in 96-well culture plate at 37 deg.C and 5% CO₂Culturing in an incubator, half-changing the culture medium with HT after 5 days, and completely changing the culture medium after 9 days.

3. After cell fusion, when the cells grow to 1/4 of the culture hole area, hybridoma cells are screened by a step screening method. The primary selection adopts an indirect ELISA method, an enzyme label plate is coated with coating antigen (the optimal coating concentration and the positive serum dilution are titrated conventionally by a square matrix method in advance), culture supernatant of a detected hole is added, incubation and washing are carried out, and then goat anti-mouse IgG-HRP, IgM-HRP and OPD are added for color reaction. The screened positive hole is screened by an indirect competitive ELISA method, the cell supernatant is mixed with rifaximin with the same volume of 100 mu g/ml, the mixture is treated by water bath at 37 ℃ for 30min, and then the mixture is added into the coated enzyme label plate. The rifaximin was also replaced with PBS as a control and the rest of the procedure was as above. OD if blocked by rifaximin_450nmIf the value is reduced to 50% or less of the control well, the wells are judged to be positive, and the wells which are positive after 2-3 detections are subcloned immediately by a limiting dilution method.

Preparation and purification of monoclonal antibody

Carrying out amplification culture on the hybridoma cells after 2-3 times of subcloning and strain establishment, collecting supernatant, measuring titer by using indirect ELISA, freezing, taking 0.5 ml/mouse of liquid paraffin for intraperitoneal injection of Balb/c mice with the age of 8-10 weeks, and carrying out intraperitoneal injection on the hybridoma cells 1-2 × 10 after 7-10 days⁵Ascites was extracted 7 to 10 days later. Collecting cell supernatant or ascites, and measuring titer by indirect ELISA (P/N for measuring titer)>2.1 of the cell supernatant or ascites, expressed as the maximum dilution factor), the results showed that the titer of the cell supernatant was 1: 10000, ascites titer 1: 50000. then, the mixture is purified by an octanoic acid-saturated ammonium sulfate method and is placed into a-20 ℃ environment for storage after purification.

Through detection, the amino acid sequence of the variable region of the heavy chain of the rifaximin monoclonal antibody is shown as the sequence 1 in the sequence table, and the amino acid sequence of the variable region of the light chain of the rifaximin monoclonal antibody is shown as the sequence 2 in the sequence table.

Example 4 rifaximin enzyme linked immunosorbent assay kit for rifaximin detection

Assembly of rifaximin enzyme-linked immunoassay kit

1. The rifaximin enzyme linked immunosorbent assay kit comprises the following components:

(1) rifaximin standard working solution: 6 bottles, 1.5 ml/bottle, the concentration is 0 mug/l, 0.3 mug/l, 0.9 mug/l, 2.7 mug/l, 8.1 mug/l, 24.3 mug/l;

(2) rifaximin enzyme label plate: 1 piece (8 wells × 12 strips) of an ELISA plate coated with "rifaximin-OVA" prepared in example 2;

(3) rifaximin antibody working solution: 1 vial (7 ml) of antibody dilution 1:8000, containing 6% (volume fraction) goat serum in 0.2M PBS, the rifaximin antibody being purified from the antiserum prepared in example 3;

(4) enzyme label concentrate: 1 bottle (1 ml), wherein the enzyme label is goat anti-mouse antibody labeled by horseradish peroxidase;

(5) sample diluent: 1 vial (10X, 15 ml) of 0.01M PBS pH 7.4;

(6) washing liquid: 1 vial (20X, 25 ml) of 0.01M PBST solution, pH 7.4;

(7) each of the substrate A solution and the substrate B solution was 1 bottle (7 ml). Wherein the substrate A is 2% carbamide peroxide aqueous solution. The substrate B is 1% tetramethyl benzidine aqueous solution;

(8) stopping liquid: 1 bottle (7 ml) of 2M H₂SO₄A solution;

(9) a cover plate film;

(10) a valve bag.

2. Equipment and materials not provided for

(1) Device

An enzyme-labeling instrument (detection wavelength 450nm and reference wavelength 630 nm), a balance (precision: 0.01 g), a vortex oscillator, a centrifuge (4000 g), a shaker (300 rpm), a nitrogen blowing instrument, a micropipette and a timer.

(2) Reagent

Sample extracting solution: weighing 20g NaCl, 26g K₂HPO₄•3H₂O in a clean beaker, add 200mL of deionized water to dissolve.

Ethyl acetate and n-hexane.

3. Storage of

The kit is stored at 2-8 deg.C, and has expiration date of 1 year.

The unused enzyme label plate should be sealed and stored at 2-8 deg.C.

4. Principle of detection of kit

And (3) competing antibodies of the rifaximin in the sample and the antigen specificity fixed on the enzyme label plate, adding an enzyme label, catalyzing a substrate to develop color, and judging the content of the rifaximin in the sample according to the color depth of the color development. Dark color, low content, light color and high content.

Application method of rifaximin enzyme-linked immunoassay kit

1. Sample pretreatment

(1) Animal meat and tissue (dilution factor: 1)

a) Taking 2 +/-0.02 g of tissue sample into a 50ml centrifuge tube, adding 2ml of sample extracting solution, and fully whirling for 30 s; b) adding 8ml ethyl acetate, and vortexing for 1 min; c) shaking at 300rpm for 10 min; d) centrifuging for 10min at a speed of more than 4000 g; e) taking 4ml of supernatant fluid to a new 4ml centrifuge tube; f) drying in 50-60 deg.C water bath with nitrogen; g) adding 1ml of sample diluent, adding 2ml of n-hexane, and fully whirling for 1 min; h) centrifuging for 5min at a speed of more than 4000g, and completely removing upper-layer n-hexane and middle-layer impurities; i) 50. mu.l of the solution was taken for detection.

(2) Liquid milk (dilution factor: 2)

a) Taking 2ml of liquid milk, putting into a 50ml centrifuge tube, adding 8ml of ethyl acetate, and violently whirling for 1 min; b) centrifuging for 10min at a speed of more than 4000 g; c) taking 2ml of supernatant liquid to a new 4ml centrifuge tube; d) drying in 50-60 deg.C water bath with nitrogen; e) adding 1ml of sample diluent, adding 2ml of n-hexane, and fully whirling for 1 min; f) centrifuging for 5min at a speed of more than 4000g, and completely removing upper-layer n-hexane and middle-layer impurities; g) 50. mu.l of the solution was taken for detection.

2. Detection step

(1) Inserting the lath into the ELISA plate frame, recording the positions of each standard product and each sample, suggesting that the two holes are parallel, sealing the unused lath by a self-sealing bag, and immediately storing in an environment of 2-8 ℃;

(2) respectively adding 50 mu l of rifaximin standard substance working solution (or sample solution to be detected) with each concentration into the corresponding standard substance (or sample hole to be detected);

(3) adding 50 mul rifaximin antibody working solution into each hole;

(4) covering a cover plate film, slightly oscillating the ELISA plate for 10s, fully and uniformly mixing, and reacting for 20min at room temperature (25 +/-2 ℃) in a dark place;

(5) uncovering the cover plate film;

(6) pouring out the liquid in the plate holes, adding 260 mul of washing working solution into each hole, and fully washing for 4 times, wherein each time of soaking is 15-30 s;

(7) pouring out liquid in the plate hole, pouring the enzyme label plate on absorbent paper, and patting dry;

(8) immediately adding 100 mul of enzyme marker working solution into each hole;

(9) covering a cover plate film, slightly oscillating the ELISA plate for 10s, fully and uniformly mixing, and reacting for 20min at room temperature (25 +/-2 ℃) in a dark place;

(10) repeating steps (5) - (7);

(11) immediately adding 100 mu L of mixed solution of the substrate A solution and the substrate B solution into each hole (the substrate A solution and the substrate B solution are mixed according to the volume of 1:1 and must be fully and uniformly mixed, and the mixed solution is used within 5min, so that the use of metal holding and stirring reagents is avoided);

(12) covering a cover plate film, slightly oscillating the ELISA plate for 10s, fully and uniformly mixing, and reacting for 15-20min at room temperature (25 +/-2 ℃) in a dark place;

(13) uncovering the cover plate membrane, adding 50 mu l of stop solution into each hole, slightly oscillating the ELISA plate for 10s, and fully and uniformly mixing;

(14) and reading the absorbance value of the ELISA plate by using an ELISA reader at the dual wavelength of 450nm and 630nm within 5min after termination.

3. Result calculation or determination

(1) The average absorbance value of each standard (or sample to be measured) is divided by the absorbance value of the zero standard (standard with the concentration of 0 mug/l) and multiplied by 100 to obtain the corresponding absorbance percentage of each standard, namely the percent absorbance value.

(2) And drawing a standard curve by taking the percent absorbance value of each standard as a vertical coordinate and the corresponding rifaximin concentration as a horizontal coordinate.

(3) Substituting the percent absorbance value of the sample to be detected into a standard curve equation to obtain the corresponding concentration of the sample to be detected, and multiplying the dilution times of the corresponding samples to obtain the actual content of the rifaximin in the original sample to be detected.

Three, rifaximin enzyme linked immunosorbent assay kit is used for detecting rifaximin

The sensitivity (IC) of the kit developed according to the invention was determined according to the method of using the two kits of this example₅₀) The detection limit,

1. Sensitivity of kit (IC)₅₀) Measurement of

The rifaximin standard substance with the detection concentration of 0.0, 0.3, 0.9, 2.7, 8.1, 24.3 and 72.9 mug/l respectively is detected. In pairs of standard substance concentrationsThe numerical values are abscissa and absorbance B/B₀Is the ordinate (B is the OD value of each standard concentration, B₀OD value of 0. mu.g/l) was plotted as a standard curve.

The rifaximin standard curve is shown in figure 2.

The rifaximin standard curve equation is as follows: y =0.0163+1.057/(1+ (x/0.8054) ^1.0858)

Coefficient of correlation (R)²) Is 0.9993, IC₅₀0.8054. mu.g/l, the sensitivity of the rifaximin enzyme linked immunosorbent assay kit is 0.81. mu.g/l.

2. Determination of minimum detection limit

And (3) taking 20 blank samples for detection, calculating a measured value according to a standard curve, calculating an average value, and adding 3 times of standard deviation to obtain a minimum detection limit, wherein the result is shown in table 1, and the minimum detection limit of the rifaximin in the beef is 10 mug/kg, and the minimum detection limit of the rifaximin in the milk is 8 mug/l.

TABLE 1 statistical table of blank sample measurement results (mug/kg)

3. Determination of method accuracy and precision

20 blank samples are respectively measured, pretreatment is carried out according to the method provided by the invention, the measured values are obtained according to the standard curve, the average value of the measured values is calculated, and 3 times of standard deviation is added to obtain the lowest detection limit. The results are shown in tables 2-3, and the results show that the recovery rate of each additive concentration of the beef and milk samples is 92.26-107.78%; the variation coefficient between batches is less than 10%.

TABLE 2 accuracy and precision of the kit (beef)

TABLE 3 accuracy and precision of the kit (milk)

4. Specificity detection

The specificity of the rifaximin enzyme linked immunosorbent assay kit is determined by carrying out a cross reaction test with corresponding substances. The smaller the cross-reaction, the better the specificity.

Respectively performing serial dilution on rifaximin and other analogs (rifamycin S, rifamycin SV, rifamycin B, rifampicin, rifapentine and rifabutin), respectively operating according to the step two 2, replacing the 'rifaximin standard work solution' with the serial dilution of rifaximin and other analogs, making a standard curve, and finding out respective 50% Inhibition Concentrations (IC) on the curve₅₀) The specific method comprises the following steps: obtaining a corresponding rifaximin concentration (mug/l) with a value on the ordinate equal to 50%, i.e. IC₅₀The value is obtained. The cross-reactivity of the kit to rifaximin and various analogs was calculated using the following formula.

Cross-reactivity (%). ratio (rifaximin concentration causing 50% inhibition/rifaximin analogue concentration causing 50% inhibition) × 100%

The results are shown in table 4, and it can be seen from table 4 that the cross-reactivity rate of the rifaximin enzyme linked immunosorbent assay kit to various analogues is less than 0.1%. The result shows that the rifaximin enzyme linked immunosorbent assay kit has extremely high specificity on rifaximin, can effectively eliminate the interference of other analogues, and can be specially used for detecting the rifaximin.

TABLE 4 specificity of rifaximin enzyme linked immunosorbent assay kit

Name of drug	Cross reaction Rate (%)
		Rifaximin	100
Rifamycin S	＜0.1
		Rifamycin SV	＜0.1
Rifamycin B	＜0.1
		Rifampicin	＜0.1
Rifapentine	＜0.1
		Rifabutin	＜0.1

5. Rifaximin enzyme-linked immunoassay kit for detecting recovery rate determination of rifaximin

And (3) determining the recovery rate of the rifaximin by using a rifaximin enzyme-linked immunoassay kit. The specific method is as step two.

The result shows that the recovery rate of rifaximin detected by the rifaximin enzyme-linked immunoassay kit is 80-120%.

6. Rifaximin enzyme-linked immunoassay kit and instrument method detection result comparison

Two samples (beef and milk, 60 parts in total) are detected by an ELISA method, and are respectively compared with the detection result of LC-MS for confirmation. Of these, 52 samples were obtained, and the results were negative in both methods. The detection accuracy of the remaining 8 positive samples is shown in fig. 3, and the ELISA results show that the positive samples are significantly related to LC-MS and are both accurate and reliable. Linearly fitted R²At 0.9935, the slope (slop) was 0.9069. The consistency of the detection results of the ELISA method and the LC-MS is good.

The consistency of the ELISA method for detecting two samples (beef and milk) and the LC-MS detection result is shown in figure 3.

Example 5 rifaximin detection by rifaximin colloidal gold test strip

Composition of rifaximin colloidal gold test strip

The rifaximin colloidal gold test strip consists of a sample absorption pad, a colloidal gold pad, a reaction membrane and a water absorption pad;

along the axial direction of the test strip, a sample absorption pad, a colloidal gold pad, a reaction membrane and a water absorption pad are sequentially connected, the tail end of the sample absorption pad is connected with the initial end of the colloidal gold pad, the tail end of the colloidal gold pad is connected with the initial end of the reaction membrane, and the tail end of the reaction membrane is connected with the initial end of the water absorption pad;

the colloidal gold pad is coated with the rifaximin monoclonal antibody obtained in the example 3 and marked by the colloidal gold;

the reaction membrane is provided with a detection area and a quality control area, and the detection area (T line) and the quality control area (C line) are both in a strip shape which is vertical to the axial direction of the test strip; the detection area is positioned at one side close to the tail end of the colloidal gold pad; the quality control area is positioned on one side far away from the tail end of the colloidal gold pad; the detection zone was coated with rifaximin-OVA prepared in example 2, and the quality control zone was coated with goat anti-mouse secondary antibody.

The sample hole is positioned at one end of the sample absorption pad far away from the tail end of the colloidal gold pad.

Secondly, preparation of the test strip

1. Colloidal gold-labeled antibody

(1) Preparation of colloidal gold solution

Heating 100ml of 0.01% chloroauric acid aqueous solution to boiling with a constant temperature electromagnetic stirrer, adding 2.5ml of 1% trisodium citrate aqueous solution under the condition of continuous stirring, and continuously stirring and heating for 20min to obtain a bright red solution. Cooling at room temperature, restoring the original volume with deionized water, and storing at 2-8 deg.C.

(2) Preparation of gold-labeled antibody solution

With 0.1mol/l K₂CO₃Adjusting the pH of the colloidal gold solution to 8.2 with the aqueous solution, adding 10ml into a 50ml beaker, stirring with an electromagnetic stirrer at 250r/min, dropwise adding the monoclonal antibody solution, and dropwise adding 3ml5g/100 ml BSA in water, stirring was continued for 10 min.

(3) Centrifuging the gold-labeled antibody solution at low speed (1500 r/min) at 20-24 deg.C for 20min, discarding precipitate formed by coagulated gold particles, and collecting red supernatant solution.

(4) And (3) centrifuging the solution in the step (3) at 4 ℃ and 11000r/min for 40min, dividing the solution into three layers (transparent supernatant, a flowing dark red precipitate at the bottom of a tube and a black compact gold particle layer on the bottom wall of the tube), transferring the flowing dark red precipitate into another centrifuge tube, suspending the flowing dark red precipitate to the volume of the original gold-labeled antibody solution by using 0.01mol/l phosphate buffer solution containing 1g/100ml BSA, centrifuging at 4 ℃ and 11000r/min for 40min overnight, and collecting the precipitate.

(5) Using a mixture of 1g/100ml BSA and 0.02g/100ml NaN₃0.01mol/l phosphate buffer solution the pellet of step (4) was suspended to 1/40 of the volume of the original gold-labeled antibody solution and stored at 2-8 ℃.

2. Spraying gold: and (3) spraying the suspension obtained in the step (1) onto a glass fiber membrane to prepare a colloidal gold pad.

3. Film spraying: rifaximin-OVA prepared in example 2 is sprayed on the reaction membrane at the line T and goat anti-mouse antibody is sprayed on the reaction membrane at the line C.

4. Assembling: assembling a sample absorption pad (cellulose filter membrane), a colloidal gold pad, a nitrocellulose membrane and a water absorption pad according to a conventional method, and then cutting into strips to obtain the detection. The test strip can also be put into a plastic card to form a test card for detection.

Thirdly, detecting by using a test paper card

1. Sample pretreatment and detection

The sample pretreatment method was as in step two 1 of example four.

Taking out the test paper card, unsealing, horizontally placing on a table, sucking the sample solution to be tested, and dropwise adding 3-5 drops of the sample solution into the sample hole; judging result in 5-10min, and judging result after 15min is invalid.

And (4) judging a result standard:

negative: c line is developed, T line is visible with naked eyes, and the color is judged to be negative no matter the color is dark or light;

positive: c line is colored, T line is not colored, and the result is positive;

and (4) invalidation: the C line is not colored, and the test paper card is judged to be invalid no matter whether the T line is colored or not.

Sequence listing

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<120> rifaximin hapten, artificial antigen, preparation method and application thereof

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<210>2

<211>105

<212>PRT

<213> Artificial Sequence (Artificial Sequence)

<400>2

Val Tyr Ser Met Thr Gly Asp Gln Ile Ser Phe Pro Ser Thr Val Val

1 5 10 15

Asp Ser Thr Cys Ala Lys Ile Ser Gln Asp Arg Thr Ala Val Ala Tyr

20 25 30

Gln Gln Lys Pro Gln Lys Ser Pro Gly Ile Trp Leu Val Tyr Trp Ala

35 40 45

Ser Thr Val Pro Arg Arg His Gly Ser Glu Phe Thr Gly Ser Gly Thr

50 55 60

Gly Leu Thr Ile Ser Asn Phe Thr Val Gln Ser Thr Asp Glu Asp Leu

65 70 75 80

Ala Asp Tyr Tyr Ile Ser Phe Cys Tyr Pro Tyr Thr Phe Gly Gly Gln

85 90 95

Gln Gly Thr Lys Gly Ile Arg Lys Val

100 105

Claims

1. A compound having the structure of formula I:

formula I.

2. A process for preparing a compound of claim 1, comprising the steps of:

dissolving rifamycin S in dimethylformamide, adding N-bromosuccinimide for reaction, precipitating, filtering and drying reaction liquid to obtain 670mg of an intermediate 1, wherein the ratio of the rifamycin S to the dimethylformamide to the N-bromosuccinimide is 1000 mg: 6 ml: 300 mg;

dissolving the intermediate 1 in methanol, adding triethylamine and 2-aminoisonicotinic acid for reaction, carrying out spin-drying, chromatographic purification and spin-drying on reaction liquid to obtain 386mg of product, wherein the ratio of the intermediate 1 to the methanol to the triethylamine to the 2-aminoisonicotinic acid is 500 mg: 5 ml: 2 ml: 100mg, wherein the chromatography developing agent is dichloromethane and methanol, and the ratio of the dichloromethane to the methanol is 15: 1.

3. a rifaximin antigen, which is an antigen obtained by coupling a compound of claim 1 to a carrier protein.

4. The rifaximin antigen of claim 3, wherein: the carrier protein can be bovine serum albumin, ovalbumin, human serum albumin, hemocyanin, murine serum protein, thyroid protein or rabbit serum protein.

5. A process for the preparation of a rifaximin antigen according to claim 3 or 4, comprising the steps of: coupling a compound of claim 1 to a carrier protein via an amide bond to obtain the rifaximin antigen.

6. The process for the preparation of rifaximin antigen according to claim 5, characterized in that: the method comprises the following steps:

(1) dissolving the compound in dimethylformamide, adding 1- (3-dimethylaminopropyl) -3-ethylcarbodiimide hydrochloride and N-hydroxysuccinimide, and reacting for 2-3h at 20-25 ℃ by magnetic stirring to obtain a solution I;

wherein the mixture ratio of the compound, the dimethylformamide, the 1- (3-dimethylaminopropyl) -3-ethylcarbodiimide hydrochloride and the N-hydroxysuccinimide is 30.4 mg: 1.5 ml: 25.75 mg: 15.46 mg;

wherein, if the carrier protein is bovine serum albumin, the ratio of the bovine serum albumin to the 0.1M sodium bicarbonate buffer solution is 50 mg: 3.5 ml; if the carrier protein is egg white protein, the ratio of the egg white protein to the 0.1M carbonic acid buffer solution is 33.6 mg: 3.5 ml;

(4) the rifaximin antigen was obtained by dialysis of the solution III with 0.01M phosphate buffer ph7.2 for 3 days at 4 ℃ with stirring.

7. Use of a compound according to claim 1 or a rifaximin antigen according to claim 3, characterized in that the rifaximin is detected qualitatively or quantitatively; rifaximin antibodies were prepared.

8. Antibodies produced using the rifaximin antigen of claim 3, characterized in that the antibodies are anti-rifaximin monoclonal antibodies; the amino acid sequence of the heavy chain variable region of the anti-rifaximin monoclonal antibody is shown as sequence 1 in the sequence table; the amino acid sequence of the variable region of the light chain of the anti-rifaximin monoclonal antibody is shown as the sequence 2 in the sequence table.

9. Rifaximin antigen according to claim 3, characterized by the possibility of being used for the preparation of enzyme linked immunosorbent kits and colloidal gold test cards.

10. The rifaximin antigen of claim 9, wherein the samples detected by the enzyme linked immunosorbent assay kit and the colloidal gold detection card can be animal meat or tissue, liquid milk, the detection limits of the rifaximin in the samples are 10 μ g/kg and 8 μ g/l respectively, and the sensitivity is 0.81 μ g/l.