CN111533705A

CN111533705A - Diazepam hapten and artificial antigen as well as preparation methods and application thereof

Info

Publication number: CN111533705A
Application number: CN202010654112.4A
Authority: CN
Inventors: 马立才; 聂靖东; 丁亚芳; 邢维维; 李蓉蓉; 刘薇
Original assignee: Beijing Wdwk Biotechnology Co ltd
Current assignee: Beijing Wdwk Biotechnology Co ltd
Priority date: 2020-07-09
Filing date: 2020-07-09
Publication date: 2020-08-14

Abstract

The invention discloses a diazepam hapten, an artificial antigen, and preparation methods and applications thereof. The diazepam artificial antigen provided by the invention is represented by the formula I

Description

Diazepam hapten and artificial antigen as well as preparation methods and application thereof

Technical Field

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

Background

Diazepam, also known as diazepam, is a sedative drug, is mainly used for anxiety, sedation and hypnosis, can also be used for resisting epilepsy and convulsion, and plays an important role in improving the sleep of patients, resisting anxiety and relieving dysphoria. But the traditional Chinese medicine composition also shows various adverse reactions such as lethargy, headache, hypodynamia and the like while playing a therapeutic role, and is easy to addict particularly after being continuously used. With the wide clinical application of sedative drugs including diazepam, the indirect toxic and side effects of the sedative drugs are more and more emphasized. The accessory "highest residual quantity of veterinary drug in animal food" announced by 235 of Ministry of agriculture clearly stipulates that sedative drugs such as diazepam can only be used for treatment, and cannot be detected in animal food. After the "clenbuterol event" in 2011, the Ministry of health released a list of inedible substances that could be illegally added to food in the current 4 months, including neuroleptic (i.e., diazepam) drugs. Therefore, the establishment of the method for rapidly and efficiently detecting the residual amount of diazepam in the food is of great significance.

At present, the detection methods of diazepam mainly comprise gas chromatography-mass spectrometry (GC-MS), High Performance Liquid Chromatography (HPLC), liquid chromatography-tandem mass spectrometry (LC-MS/MS) and the like, and although the methods have strong specificity and high sensitivity, the methods are complex to operate and expensive in instruments, and are not suitable for screening and detecting large-scale samples. The immunochemical analysis method has unique advantages in the qualitative and quantitative aspects of antigen and antibody, is simple, convenient and quick to operate, low in cost, high in sensitivity and large in analysis sample amount, and makes up for the defects of physicochemical analysis.

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 diazepam artificial antigen and a preparation method and application thereof.

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

The diazepam hapten belongs to the protection range of the invention, and has a structure shown in a formula I.

Formula I

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

1. adding 2-amino-5-chlorobenzophenone into Tetrahydrofuran (THF), stirring, adding bromoacetyl bromide for reaction, and spin-drying, extracting and spin-drying the reaction liquid to obtain a dry yellow solid 1. The proportion of the 2-amino-5-chlorobenzophenone, Tetrahydrofuran (THF) and bromoacetyl bromide is 200 mg: 5 ml: 261mg, the reaction condition of bromoacetyl bromide is ice bath, the extractant is water and ethyl acetate, the ratio of water to ethyl acetate is 5 ml: 15 ml.

2. Dissolving the solid 1 in methanol, adding hexamethylenetetramine for reaction, adjusting the pH of the reaction solution, heating, spin-drying, extracting, and spin-drying to obtain a solid 2. The proportion of the solid 1, the methanol and the hexamethylene tetramine is 150 mg: 3 ml: 120mg, the pH is adjusted by hydrochloric acid to be pH =5, the extracting agent is water and ethyl acetate, and the ratio of water to ethyl acetate is 3 ml: 9 ml.

3. Dissolving the solid 2 in Dimethylformamide (DMF), adding sodium hydride and methyl 4-bromobutyrate for reaction, adding water, precipitating, and filtering to obtain a solid 3. The proportion of the solid 2, Dimethylformamide (DMF), sodium hydride, methyl 4-bromobutyrate and water is 50 mg: 1 ml: 6 mg: 63 mg: 5 ml.

4. Dissolving the solid 3 in methanol, adding 2N sodium hydroxide solution for reaction, adjusting the pH of the reaction solution, spin-drying, washing with water, and filtering to obtain the hapten 4. The proportion of the solid 3, the methanol and the 2N sodium hydroxide solution is 100 mg: 5 ml: 15 ml.

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

The diazepam antigen is obtained by coupling the diazepam hapten (shown in a formula I) with 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 diazepam antigen also belongs to the protection scope of the invention.

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

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

(1) dissolving the diazepam 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 diazepam hapten (formula I), the Dimethylformamide (DMF), the 1- (3-dimethylaminopropyl) -3-ethylcarbodiimide hydrochloride (EDC) and the N-hydroxysuccinimide (NHS) is 15.07 mg: 1.5 ml: 21.45 mg: 12.88 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 33.57-50 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 33.57 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 diazepam antigen was obtained by dialyzing the solution III with phosphate buffer (0.01M PBS, pH 7.2) at 4 ℃ for 3 days under stirring.

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

Antibodies prepared by using the diazepam antigen also belong to the protection scope of the invention. The antibody may be a polyclonal antibody, a monoclonal antibody or an antiserum.

The diazepam hapten and the diazepam antigen provided by the invention have the advantages of simple synthesis method, high purity and high yield, and have great values for preparation of a diazepam antibody and detection of diazepam drug residue.

Drawings

FIG. 1 is a diazepam hapten mass spectrum.

Figure 2 is a standard curve for diazepam.

FIG. 3 shows the consistency of the results of the detection of three samples (pork, pig urine, pig feed) by ELISA and GC-MS.

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 identification of diazepam haptens

1. Preparation of diazepam haptens

(1) Washing a 50ml round-bottom flask, drying by using ethanol, fixing the flask on a stirrer, adding a stirrer, weighing 200mg of 2-amino-5-chlorobenzophenone, dissolving the benzophenone with 5ml of THF, stirring, keeping a sample after the benzophenone is completely dissolved, adding 261mg of bromoacetyl bromide in an ice bath, stirring, performing spot plate monitoring reaction, after the reaction is completely performed, spin-drying a solvent, adding 5ml of water and 15ml of ethyl acetate, performing extraction and liquid separation, and spin-drying the ethyl acetate to obtain a dry yellow solid 1.

(2) 150mg of the yellow solid 1 described above are dissolved in 3ml of methanol, 120mg of hexamethylenetetramine are added, the pH is adjusted to 5 with hydrochloric acid, the reaction is heated under reflux, the reaction is monitored by dot-on-plate and is completed, the solvent is evaporated, 3ml of water and 9ml of ethyl acetate are added for extraction, and the organic phase is evaporated to give a solid 2.

(3) 50mg of the solid 2 is dissolved by 1ml of DMF, 6mg of sodium hydride is added under ice bath, 63mg of methyl 4-bromobutyrate is added, the reaction is monitored by a dot plate, 5ml of water is added into the system after the reaction is completed, a precipitate is separated out, and the solid 3 is obtained by filtration.

(4) Dissolving 100mg of the solid 3 in 5ml of methanol, stirring, adding 1.5ml of 2N sodium hydroxide solution, spotting the solution on a plate to monitor the reaction progress, adjusting the pH value to 6 by using acetic acid after complete reaction, spin-drying, washing with water, and filtering to obtain hapten 4.

The reaction equation is as follows:

2. structural identification of diazepam haptens

Mass spectrometry detection is carried out on the obtained hapten 4 (figure 1), and the result shows that the chemical structural formula is shown as formula I (MW = 356.81), namely the diazepam hapten.

Formula I

Example 2 preparation of diazepam Artificial antigen

1. Synthesis of immunogens

(1) 15.07mg of diazepam hapten 4 was dissolved in 1.5ml of DMF and stirred at 200rpm for 10min, 21.45mg of EDC and 12.88mg of NHS were added and dissolved 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

(2) 33.57mg of OVA is weighed and dissolved in 3.5ml of 0.1M sodium bicarbonate solution, the mixture is stirred for 10min at 200rpm to be fully dissolved, the temperature is reduced to 0-4 ℃ in ice bath, the reaction liquid in the step 1 is dropwise added (1 ml/min) under the stirring of 1000rpm, and the mixture is stirred and reacted for 24h at 500 rpm.

EXAMPLE 3 preparation of monoclonal antibodies by immunizing animals with diazepam Artificial antigen

First, animal immunization

Dissolving the immunogen (diazepam-BSA) prepared in the example 2 by using normal saline according to 100 mu g/mouse, uniformly mixing the dissolved immunogen and Freund's complete adjuvant in equal volume, injecting and immunizing Balb/c female mice with 6-8 weeks old by subcutaneous injection at the back of the neck, uniformly mixing the immunogen and Freund's incomplete adjuvant in equal volume at 7, 14 and 28 days after primary immunization, performing additional immunization once respectively, and performing additional immunization once by using an immune compound 100 mu g/mouse 3 days before fusion and without adding Freund's 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. And screening the screened positive holes by using an indirect competitive ELISA method, mixing cell supernatant with 100 mu g/ml diazepam in equal volume, performing water bath at 37 ℃ for 30min, and adding the mixture into the coated ELISA plate. Meanwhile, PBS was used as a control instead of diazepam, and the rest steps were as above. OD if blocked by diazepam_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 diazepam monoclonal antibody is shown as sequence 1 in a sequence table, and the amino acid sequence of the variable region of the light chain of the diazepam monoclonal antibody is shown as sequence 2 in the sequence table.

Example 4 detection of diazepam by diazepam ELISA kit

Assembling of diazepam enzyme-linked immunoassay kit

1. The diazepam enzyme-linked immunoassay kit comprises the following components:

(1) diazepam standard working solution: 6 bottles, 1.5 ml/bottle, the concentration is 0 mug/l, 0.03 mug/l, 0.09 mug/l, 0.27 mug/l, 0.81 mug/l, 2.43 mug/l;

(2) diazepam microplate: 1 (8 wells × 12 strips) of an ELISA plate coated with the diazepam-OVA prepared in example 2;

(3) diazepam antibody working solution: 1 bottle (7 ml), diluting the antibody at a ratio of 1:40000 for an antibody diluent of 0.2M PBS containing 6% (volume fraction) goat serum, wherein the diazepam antibody is 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) compounding the solution: 1 vial (2X, 50 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

A microplate reader (detection wavelength 450nm, reference wavelength 630 nm), a homogenizer, a balance (precision: 0.01 g), a vortex oscillator, a centrifuge (4000 g), a nitrogen blower, a micropipette and a timer.

(2) Reagent

1M sodium hydroxide solution: 20g of sodium hydroxide was weighed into a clean beaker and dissolved by adding 500ml of deionized water.

Redissolving the working solution: diluted with deionized water at a 1:1 volume ratio (1 part concentrated complex solution +1 part deionized water).

Washing the working solution: diluted with deionized water at a 1:19 volume ratio (1 part concentrated wash +19 parts deionized water).

Acetonitrile, 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 diazepam 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 diazepam in the sample according to the color depth of the color development. Dark color, low content, light color and high content.

Use method of diazepam enzyme-linked immunoassay kit

1. Sample pretreatment

(1) Tissue (muscle, liver, etc.) (dilution factor: 10)

a) Weighing 2.0 +/-0.05 g of homogenized tissue sample in a 50ml centrifuge tube; b) adding 7.2ml deionized water and 0.8ml 1M sodium hydroxide solution in sequence, and oscillating for 5 min; c) centrifuging for 10min at a speed of more than 4000 g; d) taking 1ml of supernatant fluid to a 50ml centrifuge tube, adding 10ml of n-hexane, and oscillating for 5 min; e) centrifuging for 10min at a speed of more than 4000 g; f) taking 5ml of the upper organic phase in a 10ml new centrifugal tube, and drying by nitrogen in a water bath at 50-60 ℃; g) adding 1ml of redissolution working solution, and whirling for 2 min; h) 50. mu.l of the solution was taken for detection.

(2) Urine (dilution factor: 10)

a) Taking 1ml of clear urine sample in a 50ml centrifuge tube; b) sequentially adding 3.6ml of deionized water and 0.4ml of 1M sodium hydroxide solution, and oscillating for 2-5 min; c) taking 1ml of mixed solution to a 50ml centrifuge tube; d) adding 10ml n-hexane, and shaking with oscillator for 5 min; e) centrifuging for 10min at a speed of more than 4000 g; f) taking 5ml of the upper organic phase in a 10ml new centrifugal tube, and drying by nitrogen in a water bath at 50-60 ℃; g) adding 1ml of redissolution working solution, and whirling for 2 min; h) 50. mu.l of the solution was taken for detection.

(3) Feed (dilution factor: 20)

a) Weighing 1.0 +/-0.05 g of feed sample in a 50ml centrifuge tube; b) sequentially adding 3.7ml of deionized water and 0.3ml of 1M sodium hydroxide solution, and whirling for 1 min; c) then adding 10ml of n-hexane, and oscillating for 10 min; d) centrifuging for 10min at a speed of more than 4000 g; e) transferring 1ml of the upper organic phase into a 10ml new centrifugal tube, and drying by nitrogen in a water bath at 50-60 ℃; f) adding 1ml of redissolution working solution, and whirling for 2 min; g) diluting with redissolving working solution at a volume ratio of 1:1 (1 part of sample solution +1 part of redissolving working solution), and vortexing for 1 min; h) 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 diazepam 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) preparing an enzyme marker working solution: diluting with antibody working solution at a volume ratio of 1:10 (1 part of enzyme marker concentrated solution +10 parts of antibody working solution), and gently mixing. The solution is mixed evenly and then used immediately, and can not be stored;

(4) adding 50 mul of enzyme marker working solution into each hole;

(5) covering a cover plate film, slightly oscillating the ELISA plate for 10s, fully and uniformly mixing, and reacting for 30min at room temperature (25 +/-2 ℃) in a dark place;

(6) uncovering the cover plate film;

(7) 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;

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

(9) immediately adding 100 mu l A and B mixed solution into each hole;

(10) 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;

(11) 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;

(12) 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 (3) drawing a standard curve by taking the percent absorbance value of each standard as a vertical coordinate and the corresponding diazepam concentration as a horizontal coordinate.

(3) And 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 concentration by the dilution multiple of the corresponding sample to obtain the actual content of the diazepam in the original sample to be detected.

Third, diazepam enzyme linked immunosorbent assay kit detects diazepam

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

And detecting diazepam standard products with the concentrations of 0.00, 0.03, 0.09, 0.27, 0.81, 2.43 and 7.29 mu g/l respectively. Taking the logarithm value of the concentration of the standard substance as the abscissa and the 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 diazepam standard curve is shown in figure 2.

The diazepam standard curve equation is: y =0.0154+0.9868/(1+ (x/0.1705) ^1.772)

Coefficient of correlation (R)²) Is 0.9996, IC₅₀0.1705 mug/l, the sensitivity of the diazepam enzyme linked immunosorbent assay kit is 0.17 mug/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, the minimum detection limit of the diazepam in pork and pig urine is 5 mug/kg, and the minimum detection limit of the diazepam in pig feed is 20 mug/kg.

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 2-4, and the results show that the recovery rate of each additive concentration of pork, pig urine and pig feed samples is 92.35-108.90%; the variation coefficient between batches is less than 10%.

TABLE 2 kit accuracy and precision (pork)

TABLE 3 kit accuracy and precision (pig urine)

TABLE 4 accuracy and precision of the kit (pig feed)

4. Specificity detection

The specificity of the diazepam enzyme-linked immunoassay kit is determined by performing a cross-reaction test with a corresponding substance. The smaller the cross-reaction, the better the specificity.

Diazepam and other analogs (nitrazepam, clonitro)Respectively carrying out serial dilution on diazepam, flurazepam and flurazepam), respectively operating according to the second step 2, replacing 'diazepam standard substance working solution' with serial dilution of diazepam and other analogues to prepare a standard curve, and finding out respective 50% Inhibition Concentration (IC) on the curve₅₀) The specific method comprises the following steps: the corresponding diazepam concentration (. mu.g/l) with a value on the ordinate equal to 50%, i.e.IC, was obtained₅₀The value is obtained. The cross-reactivity of the kit to diazepam and various analogues was calculated using the following formula.

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

The results are shown in Table 5, and it can be seen from Table 5 that the cross-reactivity of the diazepam ELISA kit to various analogues is less than 0.1%. The diazepam enzyme linked immunosorbent assay kit has extremely high specificity to diazepam, can effectively eliminate the interference of other analogues, and can be specially used for detecting the diazepam.

TABLE 5 specificity of diazepam enzyme-linked immunosorbent assay kit

5. Recovery rate determination of diazepam detected by diazepam enzyme-linked immunosorbent assay kit

The recovery rate of diazepam is detected by a diazepam enzyme linked immunosorbent assay kit. The specific method is as step two.

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

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

Three samples (60 parts of pork, pig urine and pig feed in total) are detected by an ELISA method, and are respectively compared with the detection result of GC-MS for confirmation. Of these, 49 samples were tested, and the results were negative for both methods. The detection accuracy of the remaining 11 positive samples is shown in fig. 3, and the ELISA results show significant correlation with GC-MS, which are both accurate and reliable. Linearly fitted R²In the order of 0.9829, is,the slope (slop) is 0.8003. The consistency of the detection results of the ELISA method and the GC-MS is good.

The consistency of the results of ELISA detection of three samples (pork, pig urine and pig feed) and GC-MS detection is shown in FIG. 3.

Example 5 detection of diazepam by a diazepam colloidal gold test strip

Composition of diazepam colloidal gold test strip

The diazepam 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 diazepam 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 diazepam-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 K₂CO₃Of colloidal gold solutions adjusted with aqueous solutionThe pH value is 8.2, 10ml of the mixture is added into a 50ml beaker, the mixture is stirred by an electromagnetic stirrer for 250r/min, the monoclonal antibody solution is dropwise added, 3ml of a 5g/100 ml BSA aqueous solution is dropwise added, and the stirring is 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: the diazepam-OVA prepared in example 2 and the goat anti-mouse antibody are sprayed on the T line and the C line of the reaction membrane.

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

<110> Beijing Weideweikang Biotech Ltd

<120> diazepam hapten and artificial antigen as well as preparation methods and application thereof

<160>2

<170>SIPOSequenceListing 1.0

<210>1

<211>119

<212>PRT

<213> Artificial Sequence (Artificial Sequence)

<400>1

Gln Leu Gln Gln Ser Glu Val Gly Ala Glu Leu Ala Arg Pro Gly Gly

1 5 10 15

Ser Lys Leu Ser Ser Cys Ala Ser Gly Lys Tyr Phe Val Thr Ala Tyr

20 25 30

Trp Met Trp Leu Gln Leu Glu Arg Gln Pro Gly Arg Gly Leu Glu Trp

35 40 45

Ile Gly Val Pro Gly Asp Ile Tyr Gly Asp Ala Arg Tyr Thr Gln Lys

50 55 60

Phe Gln Gly Lys Ala Thr Tyr Thr Ala Asp Lys Ser Leu Thr Ala Ser

65 70 75 80

Ser Met Gln Leu Ser Ser Leu Ala Ser Glu Val Tyr Tyr Cys Ser Ala

85 90 95

Asp Ala Arg Trp Phe His His Asp Tyr Val Met Asp Tyr Trp Gly Gln

100 105 110

Gly Thr Thr Val Ser Thr Val

115

<210>2

<211>106

<212>PRT

<213> Artificial Sequence (Artificial Sequence)

<400>2

Ile Gln Leu Thr Gln Ser Asp Pro Lys Ser Met Val Ser Met Ser Glu

1 5 10 15

Glu Lys Ala Leu Ser Thr Arg Val Cys Ser Glu Gly Asn Val Tyr Val

20 25 30

Ser Trp Tyr Thr Lys Gln Gln Pro Gln Ser Pro Glu Arg Asn Ser Gly

35 40 45

Val Ile Leu Tyr Arg Cys Thr Leu Gly Asp Val Pro Phe Thr Gly Ser

50 55 60

Arg Gly Ser Gly Thr Asp Phe Thr Ile Leu Thr Ser Ser Val Gln Ala

65 70 75 80

Leu Ala Glu Asp Asp Tyr Cys Gly His Gln Ile Tyr Asn Tyr Pro Leu

85 90 95

Thr Phe Gly Ala Gly Thr Lys Leu Glu Ile

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:

adding tetrahydrofuran into 2-amino-5-chlorobenzophenone, stirring, adding bromoacetyl bromide for reaction, and carrying out spin-drying, extraction and spin-drying on reaction liquid to obtain a dry yellow solid 1, wherein the ratio of the 2-amino-5-chlorobenzophenone to the tetrahydrofuran to the bromoacetyl bromide is 200 mg: 5 ml: 261mg, the reaction condition of bromoacetyl bromide is ice bath, the extractant is water and ethyl acetate, the ratio of water to ethyl acetate is 5 ml: 15 ml;

dissolving the solid 1 in methanol, adding hexamethylenetetramine for reaction, adjusting the pH of a reaction solution, heating, spin-drying, extracting, and spin-drying to obtain a solid 2, wherein the proportion of the solid 1, the methanol and the hexamethylenetetramine is 150 mg: 3 ml: 120mg, the pH is adjusted by hydrochloric acid to be pH =5, the extracting agent is water and ethyl acetate, and the ratio of water to ethyl acetate is 3 ml: 9 ml;

dissolving the solid 2 in dimethylformamide, adding sodium hydride and methyl 4-bromobutyrate for reaction, adding water, precipitating and filtering to obtain a solid 3, wherein the proportion of the solid 2, the dimethylformamide, the sodium hydride, the methyl 4-bromobutyrate and the water is 50 mg: 1 ml: 6 mg: 63 mg: 5ml of the solution;

dissolving the solid 3 in methanol, adding a 2N sodium hydroxide solution for reaction, adjusting the pH of a reaction solution, spin-drying, washing with water, and filtering to obtain the hapten 4, wherein the ratio of the solid 3 to the methanol to the 2N sodium hydroxide solution is 100 mg: 5 ml: 15 ml.

3. A diazepam antigen obtained by coupling a compound of claim 1 to a carrier protein.

4. The diazepam 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 method of preparing a diazepam 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 diazepam antigen.

6. The method of preparing a diazepam antigen according to claim 5, wherein: 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 15.07 mg: 1.5 ml: 21.45 mg: 12.88 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 33.57 mg: 3.5 ml;

(4) and (3) dialyzing the solution III with 0.01M phosphate buffer solution with pH7.2 at 4 ℃ for 3 days under stirring to obtain the diazepam antigen.

7. Use of a compound according to claim 1 or a diazepam antigen according to claim 3, characterized in that the diazepam is detected qualitatively or quantitatively; preparing diazepam antibody.

8. An antibody produced using the diazepam antigen of claim 3, wherein said antibody is an anti-diazepam monoclonal antibody; the amino acid sequence of the heavy chain variable region of the diazepam-resistant monoclonal antibody is shown as sequence 1 in a sequence table; the amino acid sequence of the light chain variable region of the diazepam-resistant monoclonal antibody is shown as a sequence 2 in a sequence table.

9. The diazepam antigen of claim 3, which can be used to prepare ELISA kits and colloidal gold test cards.

10. The diazepam antigen of claim 9, wherein the detection samples of the ELISA and colloidal gold detection card can be tissues, urine and feeds, the detection limits for detecting diazepam in the above samples are 5 μ g/kg, 5 μ g/l and 20 μ g/kg respectively, and the sensitivity is 0.17 μ g/l.