A toluimidazole hapten, artificial antigen and toluimidazole monoclonal antibody and preparation and application thereof

Abstract

The application discloses a toluidine imidazole hapten, artificial antigen and toluidine imidazole monoclonal antibody and a preparation and application thereof. The structure formula of the toluidine imidazole hapten is shown as formula I. The toluidine imidazole artificial antigen is obtained by coupling the toluidine imidazole hapten with a carrier protein, and the toluidine imidazole monoclonal antibody is prepared by using the toluidine imidazole artificial antigen. The monoclonal antibody has the characteristics of high specificity, high sensitivity, high accuracy and high accuracy. The fluorescent immunochromatographic test strip developed by using the toluidine imidazole artificial antigen and the toluidine imidazole monoclonal antibody is simple and efficient in operation method, guarantees high accuracy and sensitivity, and is very suitable for detecting the residual amount of toluidine imidazole in livestock and aquatic products on the spot.

Description

TECHNICAL FIELD

[0001] The present application relates to the technical field of biological detection, more particularly to a mebendazole hapten, artificial antigen and mebendazole monoclonal antibody, and preparation and application thereof. BACKGROUND

[0002] Mebendazole (MBZ) is an organic compound with the chemical formula C 16 H 13 N3O3, which is a broad-spectrum anthelmintic drug with significant larvicidal and ovicidal effects. It has good curative effect on animal and fish digestive tract parasitic diseases. Mebendazole is widely used in livestock and aquaculture due to its excellent parasiticidal effect. However, long-term and repeated use of mebendazole anthelmintic drugs can cause drug resistance in parasites. In addition, the use of large doses of mebendazole can cause drug residues in livestock and aquaculture products, which can cause abdominal cramps, nausea, vomiting and diarrhea after consumption, seriously endangering human health. At the same time, the undegraded chemical drugs excreted by animals will enter the ecological cycle and further pollute the environment, forming a vicious cycle.

[0003] The current detection method for mebendazole residues in livestock and aquaculture meat is mainly instrument method. For example, the national standard “GB31656.1-2021 Determination of mebendazole and metabolites in aquatic products by high performance liquid chromatography” specifies the sample preparation and liquid chromatography method for detecting mebendazole and its metabolites aminomethylbenzimidazole and hydroxymethylbenzimidazole residues in aquatic products. It is suitable for detecting mebendazole and its metabolites aminomethylbenzimidazole and hydroxymethylbenzimidazole residues in fish, shrimp and crab edible tissues.

[0004] Although the instrument method is accurate, the test instrument used is generally expensive, the detection process is precise and complicated, and it takes a long time. It requires special testing environment and operating personnel. However, livestock breeding and aquaculture are mostly in remote areas, and the instrument method cannot meet the requirements of on-site sample detection, rapidness and simplicity.

[0005] Therefore, it is necessary to develop a simple and efficient method for detecting mebendazole residues suitable for on-site personnel. SUMMARY

[0006] Therefore, it is necessary to develop a simple and efficient method for detecting mebendazole residues suitable for on-site personnel.

[0007] The present application relates to the technical field of biological detection, more particularly to a mebendazole hapten, artificial antigen and mebendazole monoclonal antibody, and preparation and application thereof.

[0008] The present application relates to the technical field of biological detection, more particularly to a mebendazole hapten, artificial antigen and mebendazole monoclonal antibody, and preparation and application thereof.

[0009] The application also aims to provide a tolyimidazole artificial antigen.

[0010] The application also aims to provide a tolyimidazole monoclonal antibody.

[0011] The application also aims to provide a method for preparing a tolyimidazole test strip by using the tolyimidazole artificial antigen and the tolyimidazole monoclonal antibody.

[0012] The above-mentioned objects of the application are achieved by the following technical solutions.

[0013] The first aspect of the application provides a tolyimidazole hapten, the structural formula of which is shown as formula I.

[0014]

[0015] The second aspect of the application provides a preparation method of a tolyimidazole hapten, which comprises the following steps: S1, tolyimidazole, potassium tert-butoxide and tert-butyl 4-bromobutyrate are completely reacted with DMSO as a solvent, and the organic phase is extracted and combined to obtain a tolyimidazole hapten intermediate product;

[0016] S2, the intermediate product obtained in step S1 is dissolved in a mixed solution of trifluoroacetic acid and dichloromethane, hydrolysis is performed, and the solvent is removed by rotary evaporation, and then the tolyimidazole hapten is obtained by purification and drying.

[0017] Further, the molar ratio of tolyimidazole, potassium tert-butoxide and tert-butyl 4-bromobutyrate is 34:41:34; the volume ratio of trifluoroacetic acid and dichloromethane in the mixed solution of trifluoroacetic acid and dichloromethane is 1:2.

[0018] The third aspect of the application provides a tolyimidazole artificial antigen, which is obtained by coupling the tolyimidazole hapten with a carrier protein at a molar ratio of 1:100; the structural formula of the tolyimidazole artificial antigen is shown as formula (II),

[0019]

[0020] Further, the carrier protein is chicken egg white protein or lactoferrin.

[0021] The fourth aspect of the application provides a tolyimidazole monoclonal antibody prepared from the tolyimidazole artificial antigen, and the carrier protein is lactoferrin.

[0022] The fifth aspect of the present application provides a method for preparing a test strip for detecting tolmetinazole by using the tolmetinazole artificial antigen and the tolmetinazole monoclonal antibody, comprising the following steps: S1, washing and activating the time-resolved fluorescent microspheres, then reconstituting with a 50 mM MES buffer solution with pH 8.0, and then coupling with the tolmetinazole monoclonal antibody to prepare a fluorescent probe; S2, spraying the tolmetinazole artificial antigen with chicken egg white protein as a carrier protein and the goat anti-mouse IgG secondary antibody on the test line T and the control line C on the nitrocellulose membrane respectively; S3, spraying the fluorescent probe on the conjugate pad after soaking the conjugate pad in the conjugate pad treatment solution prepared by the present application; S4, sequentially laminating the conjugate pad, the nitrocellulose membrane and the absorbent paper on the bottom plate to combine into a test strip; and S5, preparing a sample diluent by adding 1% (w / w) PVP and 10% (w / w) methanol to the 0.01 mol / L PBS buffer solution.

[0023] Further, in step S1, 40 μg of the tolmetinazole monoclonal antibody is coupled with every 50 μL of the time-resolved fluorescent microspheres.

[0024] Further, in step S2, the 1.4 mg / mL tolmetinazole artificial antigen and the 0.05 mg / mL goat anti-mouse IgG secondary antibody are sprayed on the test line T and the control line C at a spraying speed of 1 μL / cm respectively; and in step S3, 5 μL of the fluorescent probe is added to 60 μL of the fluorescent diluent and then sprayed on the conjugate pad at a speed of 2 μL / cm.

[0025] Further, in step S5, the conjugate pad treatment solution is prepared by adding 0% to 10% (w / w) trehalose, 0% to 2% (w / w) casein and 0% to 0.4% (w / w) Tween-20 to the 20 mM sodium citrate buffer solution.

[0026] Compared with the prior art, the present application has the following beneficial effects:

[0027] The tolmetinazole hapten prepared by the present application has a simple preparation method, and the tolmetinazole artificial antigen prepared by using the hapten and the monoclonal antibody prepared by coupling with the carrier protein has high sensitivity, strong recognition and high specificity to tolmetinazole, and the cross-reactivity rate to common benzimidazole drugs is less than 1%, the half-inhibitory concentration is 0.13 ng / mL, and the detection limit is 0.01 ng / mL, so that the sensitive detection of tolmetinazole can be realized; and the fluorescent immunochromatographic test strip prepared by using the tolmetinazole artificial antigen and the tolmetinazole monoclonal antibody has high accuracy and sensitivity under the premise of simple and efficient operation method, and is very suitable for detecting the residual amount of tolmetinazole in livestock and aquatic products in the field. BRIEF DESCRIPTION OF DRAWINGS

[0028] In order to more clearly illustrate the scheme in the present application or the prior art, the drawings required to be used in the following embodiment or prior art description will be briefly introduced. Obviously, the drawings in the following description are some embodiments of the present application, and other drawings can also be obtained by those skilled in the art without creative labor.

[0029] Figure 1 Synthetic route map of the tolylimidazole hapten of embodiment 1 of the present application;

[0030] Figure 2 ESI-MS mass spectrum identification map of the tolylimidazole hapten of embodiment 1 of the present application;

[0031] Figure 3a UV scanning map of the tolylimidazole hapten, artificial antigen of the lactoferrin carrier and lactoferrin carrier protein of embodiment 2 of the present application;

[0032] Figure 3b UV scanning map of the tolylimidazole hapten, artificial antigen of the chicken egg white protein carrier and chicken egg white protein carrier protein of embodiment 2 of the present application;

[0033] Figure 4 Inhibition curve of the antibody prepared from the tolylimidazole artificial antigen of embodiment 3 of the present application on tolylimidazole;

[0034] Figure 5 Detection principle map of the fluorescent immunochromatographic test strip for detecting tolylimidazole;

[0035] Figure 6 Effect of different pH values of the MES solution used for resolubilizing the activated fluorescent microspheres on the test strip in the coupling process of the antibody in embodiment 7;

[0036] Figure 7 Effect of adding different tolylimidazole monoclonal antibodies and coupling with fluorescent microspheres on the detection results of the test strip in embodiment 7;

[0037] Figure 8 Effect of different amounts of fluorescent probes used in the preparation of the conjugate pad on the negative and positive detection results of the test strip in embodiment 7;

[0038] Figure 9a Effect of the ion components in the treatment solution of the conjugate pad on the test strip in embodiment 9;

[0039] Figure 9b Effect of the mass content of trehalose in the treatment solution of the conjugate pad on the test strip in embodiment 9;

[0040] Figure 9c Effect of the mass content of casein in the treatment solution of the conjugate pad on the test strip in embodiment 9;

[0041] Figure 9d The effect of the mass content of Tween-20 in the conjugate pad treatment solution on the test strip in Example 9;

[0042] Figure 10a This illustrates the effect of the ion concentration of the PBS buffer in the sample dilution solution on the detection of the test strip in Example 10.

[0043] Figure 10b This illustrates the effect of the PVP content in the sample diluent on the test strip detection in Example 10.

[0044] Figure 10c This illustrates the effect of the methanol content in the sample diluent on the test strip detection in Example 10.

[0045] Figure 11 This is a schematic diagram of the standard curve and corresponding fluorescence results for a mebendazole fluorescent immunochromatographic test strip. Detailed Implementation

[0046] To enable those skilled in the art to better understand the present invention, the technical solutions of the present invention will be clearly and completely described below with reference to the accompanying drawings of the embodiments of the present invention. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort should fall within the scope of protection of the present invention.

[0047] To enable those skilled in the art to better understand the present application, the technical solutions in the embodiments of the present application will be clearly and completely described below with reference to the accompanying drawings.

[0048] Example 1: Synthesis and Identification of Mebendazole Hapten

[0049] S1, according to Figure 1 The synthetic route for the mebendazole hapten shown is as follows: Mebendazole (100 mg, 0.34 mmol) and potassium tert-butoxide (45.8 mg, 0.41 mmol) were dissolved in DMSO. The solution was sonicated until completely dissolved. Tert-butyl 4-bromobutyrate (0.34 mmol, 60 μL) was added, and the mixture was stirred at room temperature for 2 h. After the reaction was complete, the mixture was extracted three times with ethyl acetate. The combined ethyl acetate layers were washed twice with saturated brine. An appropriate amount of anhydrous sodium sulfate was added to remove water. The ethyl acetate was removed by rotary evaporation under reduced pressure, and the intermediate product was purified by silica gel column chromatography.

[0050] S2, the intermediate product obtained in step S1 was dissolved in a solution of trifluoroacetic acid: dichloromethane = 1:2 (volume ratio) and hydrolyzed at 0°C for 3 h. The solvent was removed by rotary evaporation, and the brown oil obtained was dried by rotary evaporation under reduced pressure and purified by silica gel column chromatography to obtain the imidazole hapten, which has the structural formula shown in formula (I) and a molecular weight of 381.4,

[0051]

[0052] S3, the ESI-MS identification result of the imidazole hapten is shown in Figure 2 which is consistent with the molecular weight of the imidazole hapten (381.4), proving that the synthesis of the imidazole hapten is successful.

[0053] Example 2: Synthesis and identification of imidazole artificial antigen

[0054] 1. Synthesis of artificial antigen

[0055] The structural formula of the imidazole artificial antigen is shown in formula (II). The imidazole hapten and the carrier protein were coupled by the active ester method. The imidazole hapten and lactoferrin coupled artificial antigen were used as the immunogen, and the imidazole hapten and chicken ovalbumin coupled artificial antigen were used as the coating antigen. The molar ratio of the imidazole hapten to lactoferrin and chicken ovalbumin was 1:100. Two portions of 19.1 mg of the imidazole hapten were dissolved in 500 μL of DMF, and 14.4 mg of EDC and 8.6 mg of NHS were added while stirring. The reaction was carried out at room temperature in the dark for 4 h to obtain the activated solution. 40 mg of lactoferrin and chicken ovalbumin were dissolved in 3 mL of CB (0.1 M, pH 9.6) buffer, and the activated solution was added while stirring in an ice bath. The pH was measured after the addition was completed, and if the alkalinity was insufficient, NaOH solution was added to adjust the pH to about 9.6. The coupling was carried out at room temperature in the dark overnight, and the coupling mixture was obtained the next day. The coupling mixture was transferred to a dialysis bag and dialyzed in 0.01 M PBS at 4°C for 3 days, and the dialysis solution was replaced every 12 h. The final concentration of the artificial antigen was 5 mg / mL, and the antigen was aliquoted and stored in a -20°C freezer,

[0056]

[0057] 2. Identification of artificial antigen

[0058] The UV scanning determination (190-400 nm) was carried out on the imidazole hapten (MBZ-H1), the lactoferrin carrier artificial antigen (MBZ-H1-LF), the chicken ovalbumin carrier artificial antigen (MBZ-H1-OVA), the lactoferrin carrier protein (LF), and the chicken ovalbumin carrier protein (OVA), as shown in Figure 3aAs shown, the ultraviolet absorption peak of the artificial antigen of the lactoferrin carrier is obviously offset compared with the ultraviolet absorption peak of the toluidine imidazole hapten, and the artificial antigen has the characteristic absorption peaks of the toluidine imidazole hapten and the lactoferrin, indicating that the artificial antigen is coupled successfully. Figure 3b As shown, the ultraviolet absorption peak of the artificial antigen of the chicken egg white protein carrier is obviously offset compared with the ultraviolet absorption peak of the toluidine imidazole hapten, and the artificial antigen has the characteristic absorption peaks of the toluidine imidazole hapten and the chicken egg white protein, indicating that the artificial antigen is coupled successfully.

[0059] Example 3 Preparation and identification of toluidine imidazole monoclonal antibody

[0060] 1. Preparation of toluidine imidazole monoclonal antibody

[0061] The artificial antigen with the carrier protein prepared in Example 2 being lactoferrin is emulsified with an equal amount of immunoadjuvant (Freund's complete adjuvant for the first immunization, and Freund's incomplete adjuvant for subsequent booster immunization) to immunize mice. 6-7 week old Balb / c mice are immunized by subcutaneous injection on the back and intraperitoneal injection, 2 weeks later, the second immunization is performed, and then the booster immunization is performed every 2 weeks. One week after the fourth booster immunization, the mice are bled from the tail, and the serum titer is determined by indirect competitive ELISA. When the titer no longer increases, the intraperitoneal injection is used for booster immunization. 3 days later, the heart is bled, and the supernatant is obtained by centrifugation at 10000 r / min for 15 min at 4°C after water bath for 0.5-1 h, which is the antiserum, i.e. polyclonal antibody.

[0062] PEG is used to mix the spleen cells with SP2 / 0 myeloma cells for cell fusion, and after subcloning and three times of limiting dilution, part of the positive hybridoma is preserved by freezing, and the other part is injected into the abdominal cavity of mice to produce ascites. The ascites is collected after 7 days, and the ascites is purified by protein G immunoaffinity column to obtain the monoclonal antibody.

[0063] 2. Performance identification of monoclonal antibody

[0064] The performance of the toluidine imidazole monoclonal antibody is identified by indirect competitive ELISA, and the detection steps are as follows:

[0065] (1) Coating: dilute the coating liquid to 500 ng / mL, 100 μL / well, and coat overnight in a 37°C water bath;

[0066] (2) Blocking: discard the coating liquid, wash the plate twice, pat dry with absorbent paper, add 120 μL of blocking solution to each well, and incubate at 37°C in a water bath for 3 h. Shake dry, and dry in a 37°C oven for 1 h;

[0067] (3) Add antibody and drug: the monoclonal antibody of metronidazole was diluted to 62.5 ng / mL, and the standard sample of metronidazole was diluted to a series of concentrations with PBS starting from 100 ng / mL. The value column: 50 μL of PBS buffer was added to each well, and then the diluted monoclonal antibody was added to the wells in turn at 50 μL / well, and PBS was added to the last well as a blank control; the inhibition column: 50 μL of the series of drugs diluted with PBS buffer was added to each well, and then the diluted antibody was added to the wells in turn at 50 μL / well, and PBS was added to the last well as a blank control; incubation at 37°C for 40 min, and washing of the plate 5 times;

[0068] (4) Add secondary antibody: add goat anti-mouse secondary antibody (100 μL / well) diluted 5000 times with PBST buffer, incubate at 37°C for 30 min, and wash the plate 5 times;

[0069] (5) Color development: mix equal volumes of TMB substrate buffer A and B to obtain a substrate solution, add the substrate solution (100 μL / well), and incubate at 37°C for 10 min;

[0070] (6) Termination: terminate the reaction by adding 10% H2SO4 termination solution (50 μL / well) to the enzyme-labeled plate;

[0071] (7) Reading: read the absorbance value (OD) at a wavelength of 450 nm using an enzyme-labeled instrument.

[0072] Take the OD value as the vertical coordinate, and the corresponding standard sample concentration logarithmic value as the horizontal coordinate. Apply Origin software to perform curve fitting on the four-parameter function, take IC 10 as the detection limit, take IC 20 ~ IC 80 as the detection range; establish the standard curve of ELISA with the metronidazole standard sample, and the results are shown in Figure 4 , and the related standard curve parameters are shown in Table 1.

[0073] Table 1: Detection parameters of metronidazole monoclonal antibody for metronidazole

[0074]

[0075]

[0076] 3. Specificity of antibody

[0077] The monoclonal antibody prepared in step 1 was used for enzyme-linked immunoassay (ELISA) of metronidazole structural analogs and functional analogs, and the corresponding IC 50 was obtained by fitting, and the cross-reactivity was calculated, and the results are shown in Table 2.

[0078] Table 2: Cross-reactivity of metronidazole monoclonal antibody for its structural and functional analogs

[0079]

[0080]

[0081] As shown in Table 2, the cross-reactivity of the antibody to methimazole is 100%, and the cross-reactivity to other benzimidazole drugs is less than 1%, indicating that the monoclonal antibody of the application can specifically recognize methimazole, and further detect the content of methimazole.

[0082] Preparation method of the fluorescent probe

[0083] The time-resolved fluorescent microspheres used in the application are Eu-time-resolved fluorescent nanomicrospheres (labeled with Eu 3+ The fluorescent microspheres are labeled and carboxyl-functionalized, with a particle size of 210 nm and a solid content of 1%

[0084] 1. Washing of the fluorescent microspheres: 500 μL of 50 mM MES (pH = 6.5) solution was taken in a 1.5 mL centrifuge tube, 50 μL of the fluorescent microspheres were added, and the mixture was uniformly shaken and ultrasonically treated for 30-60 s. Centrifugation was performed at 15000 r / min for 20 min at 10℃, the supernatant was discarded, 500 μL of 50 mM MES (pH = 6.5) solution was added to reconstitute the fluorescent microspheres, and the mixture was ultrasonically treated for 30-60 s. The operation was repeated twice, and the fluorescent microspheres were centrifuged for 3 times.

[0085] 2. Activation of the fluorescent microspheres: the centrifuged fluorescent microspheres were reconstituted with 500 μL of 50 mM MES (pH = 6.5) solution and ultrasonically treated, 15 μL of EDC (concentration: 100 mg / mL) dissolved in MES (pH = 6.5) solution was added, and the mixture was reacted at 30℃ for 5 min. 15 μL of NHS (concentration: 100 mg / mL) dissolved in MES (pH = 6.5) solution was added, and the mixture was reacted at 30℃ for 30 min. Centrifugation was performed at 15000 r / min for 20 min at 10℃, the supernatant was discarded, the activated microspheres were reconstituted with 500 μL of 50 mM MES (pH = 6.5) solution, and the mixture was ultrasonically treated for 120-180 s. The operation was repeated twice, and the fluorescent microspheres were centrifuged for 3 times. After centrifugation, 500 μL of 50 mM MES (pH = 6.0) solution was added to reconstitute the mixture, and the mixture was uniformly shaken.

[0086] 3, fluorescent microspheres and monoclonal antibody conjugated into fluorescent probe: add the appropriate amount of monoclonal antibody oscillation uniform, and ultrasonic 30-60s, keep 30℃ constant temperature reaction 120 minutes. 10℃ 15000r / min centrifugal 20 minutes, discard supernatant, add 500μL 50mM MES (pH = 6.0) solution, ultrasonic 120-180s, repeat operation two times, a total of 3 times. After centrifugation, add 1mL blocking solution.

[0087] 4, fluorescent probe blocking: constant temperature 30℃ blocking reaction 60 minutes, 10℃ 15000r / min centrifugal 20 minutes, discard supernatant, add 500μL fluorescent preservation solution, ultrasonic 30-60s, repeat operation 1 times, a total of 2 times. After centrifugation, add 500μL fluorescent preservation solution ultrasonic dissolution, 4℃ preservation.

[0088] Example 5 fluorescent immunoassay test strip preparation

[0089] 1, draw membrane: toluene imidazole coated and goat anti mouse IgG secondary antibody were diluted to the appropriate concentration with 0.2mol / L PB buffer (containing 2% trehalose), after the gold spraying membrane instrument was cleaned, the diluted toluene imidazole coated and goat anti mouse IgG secondary antibody were respectively sucked in the pipeline. Nitrocellulose membrane was pasted on the corresponding position of PVC bottom plate, the draw membrane speed in the draw membrane parameter was set to 1μL / cm, the diluted toluene imidazole coated was sprayed on the detection line T, and the diluted goat anti mouse IgG secondary antibody was sprayed on the quality control line C. The distance between the detection line T and the quality control line C was 5mm, and the oven was dried at 37℃ for standby. The nitrocellulose membrane used in the application was CN95 membrane.

[0090] 2, handle the binding pad: soak the whole glass fiber with the binding pad treatment solution, squeeze out the excess liquid, and then lay it on the metal grid plate. Dry in the oven at 37℃ overnight. Cut the treated glass fiber into 30mm×300mm size binding pad with a cutting machine. Add the appropriate amount of prepared fluorescent probe to 60μL fluorescent diluent, oscillate and ultrasonic uniformly, and use the gold spraying membrane instrument to spray the fluorescent probe diluent uniformly on the binding pad at a distance of 2cm from the bottom. The spraying amount is set to 2μL / cm, and the oven is dried at 37℃ for standby.

[0091] 3, test strip assembly: paste the binding pad and water absorption paper on the PVC bottom plate with dried nitrocellulose membrane. The water absorption paper and binding pad overlap the nitrocellulose membrane by 2mm, and a layer of PE film is covered on the surface of the nitrocellulose membrane at a distance of 1.4cm from the bottom of the binding pad. After assembly, cut the fluorescent immunoassay test strip board into 3.78mm wide test strips with a cutting machine, and assemble with the corresponding card shell.

[0092] Example 6 application of fluorescent immunoassay test strip

[0093] 1. Pretreatment of the sample for detection

[0094] Weigh 2 g of the sample, add 20 mL of methanol, vortex for 5 min, centrifuge at 4000 rpm for 5 min, take 100 μL of the supernatant, dilute it ten times with the sample loading diluent, mix well, take 100 μL of the sample to be tested, and test after 15 min of reaction.

[0095] 2. Principle of the fluorescent immunochromatographic test strip for detection

[0096] The detection principle is shown in Figure 5 The sample to be tested is added to the sample loading hole, incubated at 30°C for 15 min, and if it does not contain the target detection substance, the fluorescent microsphere-labeled monoclonal antibody on the conjugate pad specifically binds to the antigen fixed on the detection line through chromatography, so that the T line shows a bright fluorescent band under the ultraviolet lamp. The remaining fluorescent microsphere-labeled monoclonal antibody continues to move forward and is captured by the goat anti-mouse IgG secondary antibody fixed on the C line, and the C line also shows a fluorescent band, which is negative. If the sample to be tested contains the target detection substance, the target detection substance first binds to the antibody labeled with fluorescent microspheres on the conjugate pad to form an antigen-antibody complex, and the remaining fluorescent microsphere-labeled antibody binds to the fixed artificial antigen through the T line, and the fluorescence intensity of the T line is negatively correlated with the content of the target detection substance. With the chromatography, the antigen-antibody complex and the unbound fluorescent microsphere-labeled antibody continue to move forward and bind to the goat anti-mouse IgG secondary antibody fixed on the C line to form an antigen-antibody-secondary antibody complex and an antibody-secondary antibody complex, and finally the C line shows fluorescence and the T line shows weak color or no color, which is positive. When the C line does not show color, it indicates that the test result is invalid.

[0097] 3. Establishment of the standard curve of the toluene imidazole fluorescent immunochromatographic test strip

[0098] The T / C ratio of the test strip is read at a wavelength of 365 nm by a fluorescent immunoassay instrument, wherein T is the fluorescence value of the detection line T and C is the fluorescence value of the quality control line C. The Origin software is used for Logistic function fitting, the logarithmic value of the toluene imidazole drug concentration is used as the abscissa, and B / B0 (B0 is the negative T / C value without the drug to be tested, and B is the positive T / C value with the drug to be tested) is used as the ordinate to draw the standard curve.

[0099] 4. Interpretation of quantitative results

[0100] The standard curve of the toluene imidazole fluorescent immunochromatographic test strip is shown in Figure 11 The quantitative detection limit of the toluene imidazole fluorescent immunochromatographic test strip is 0.51 ng / mL, and the rapid quantitative detection of toluene imidazole can be realized.

[0101] Influence of each condition in the preparation process of fluorescent probe

[0102] 1. Influence of coupling pH value on labeling process

[0103] The fluorescent probe was prepared according to the procedure of Example 4, and after the final centrifugation in the activation step, the activated fluorescent microspheres were reconstituted with MES buffer of different pH values (6.0, 7.0, 8.0, and 9.0). The same sample was detected according to the method of Example 6, and the results are shown in Figure 6 As the coupling pH value increased, the fluorescence intensity of the test strip decreased sharply, and the T line color became weaker and weaker. The inhibition rate first increased and then decreased with the increase of pH value, so 50 mM MES buffer with a pH value of 8.0 was selected as the optimal coupling pH value.

[0104] 2. Influence of antibody amount on fluorescent immunochromatographic method

[0105] The fluorescent probe was prepared according to the procedure of Example 4, and in the coupling step, 20, 30, 40, and 50 μg of toluene imidazole monoclonal antibody diluted with 0.01 mol / L PBS solution were coupled with the fluorescent microspheres, respectively. The prepared test strips were detected according to the method of Example 6. As shown in Figure 7 When the amount of toluene imidazole monoclonal antibody added was 40 μg, the coloration was more obvious, and the inhibition rate did not increase significantly with the further addition of antibody amount, so 40 μg was selected as the optimal antibody labeling amount.

[0106] 3. Influence of fluorescent probe amount on fluorescent immunochromatographic method

[0107] In the preparation of fluorescent immunochromatographic test strips in Example 5, 1, 3, 5, 8, and 10 μL of fluorescent probe were added to 60 μL of fluorescent diluent, and then fixed on the conjugate pad. Then the prepared test strips were detected according to the method of Example 6. The results are shown in Figure 8 If the amount of probe is too small, the coloration will be too shallow and difficult to identify, and if the amount of probe is too large, the T line coloration will be too deep and the detection sensitivity will be reduced, so the optimal amount of toluene imidazole fluorescent probe is determined to be 5 μL.

[0108] Example 8: Influence of the concentration of toluene imidazole coated antigen on the detection line T and the concentration of goat anti-mouse IgG secondary antibody on the control line C on the detection results

[0109] Different combinations of toluene imidazole coated antigen and goat anti-mouse IgG secondary antibody concentrations were set to test the negative sample, as shown in Table 3. When the concentration of coated antigen on the detection line T was 1.4 mg / mL and the concentration of goat anti-mouse secondary antibody on the control line C was 0.05 mg / mL, the test strip line coloration and sensitivity were optimal.

[0110] Table 3: Effect of different T, C line concentrations on fluorescence intensity

[0111]

[0112] Example 9 Effect of binding pad treatment liquid on fluorescence immunoassay method

[0113] 1. As shown in Table 4, under the condition that other components of the binding pad treatment liquid are fixed, the sensitivity of the test strip prepared by using 20 mM sodium citrate, Tris and HEPES as the ion component of the buffer respectively was compared and analyzed for the same test sample, and the results were as follows Figure 9a It can be seen that, compared with Tris and HEPES, the sensitivity of the test strip prepared by using sodium citrate treatment is the best, and therefore 20 mM sodium citrate buffer is preferred as the binding pad treatment liquid.

[0114] Table 4: Effect of different ion components of the buffer on test strip detection

[0115] Ion composition Trehalose content w / w Casein content w / w Tween-20 content w / w 20 mM sodium citrate 5％ 0.5％ 0.1％ 20 mM Tris 5％ 0.5％ 0.1％ 20 mM HEPES 5％ 0.5％ 0.1％

[0116] 2. As shown in Table 5, under the condition that other components of the binding pad treatment liquid are fixed, the detection effect of the test strip prepared by using different contents of trehalose on the same sample was analyzed, and the results were as follows Figure 9b It can be seen that, when no trehalose is added, the T / C ratio is relatively low and the inhibition rate is reduced, and the stability of the fluorescence probe is also difficult to guarantee without a sugar as a protective agent. When the content of trehalose is higher than 5% (w / w), the color development of the test strip has no obvious change, but the inhibition rate gradually decreases, and therefore the preferred addition amount of trehalose is 5% (w / w).

[0117] Table 5: Effect of trehalose content in the binding pad treatment liquid on test strip detection

[0118] Trehalose content w / w Buffer ion composition Casein content w / w Tween-20 content w / w 0 20 mM sodium citrate 0.5％ 0.1％ 3％ 20 mM sodium citrate 0.5％ 0.1％ 5％ 20 mM sodium citrate 0.5％ 0.1％ 7％ 20 mM sodium citrate 0.5％ 0.1％ 10％ 20 mM sodium citrate 0.5％ 0.1％

[0119] 3. As shown in Table 6, under the condition that other components of the binding pad treatment liquid are fixed, the detection effect of the test strip prepared by using different contents of casein on the same sample was analyzed, and the results were as follows Figure 9c It can be seen that, the addition of casein in the binding pad will reduce the non-specific adsorption, and the test strip basically has no color development without the addition of casein, and the fluorescence signal value is almost zero. When the content of casein is higher than 0.5% (w / w), the color development of the test strip has no obvious change, but the inhibition rate gradually decreases, and therefore the preferred addition amount of casein is 0.5% (w / w).

[0120] Table 6: Effect of casein content in the binding pad treatment liquid on test strip detection

[0121] Casein content w / w Buffer ion composition Trehalose content w / w Tween-20 content w / w 0 20 mM sodium citrate 3％ 0.1％ 0.5％ 20 mM sodium citrate 3％ 0.1％ 1％ 20 mM sodium citrate 3％ 0.1％ 1.5％ 20 mM sodium citrate 3％ 0.1％ 2％ 20 mM sodium citrate 3％ 0.1％

[0122] 4. As shown in Table 7, with other components of the conjugate pad treatment solution fixed, the detection effect of test strips prepared with different contents of Tween-20 on the same sample was analyzed. Figure 9d It can be seen that, taking into account both color development and inhibition rate, the optimal addition amount of Tween-20 in the treatment solution is 0.1% (w / w).

[0123] Table 7: Comparative Analysis of the Influence of Tween-20 Content in Binding Pad Treatment Solution on Test Strip Detection

[0124] Tween-20 content w / w Buffer ion composition Trehalose content w / w Casein content w / w 0 20 mM sodium citrate 3％ 0.5％ 0.1％ 20 mM sodium citrate 3％ 0.5％ 0.2％ 20 mM sodium citrate 3％ 0.5％ 0.4％ 20 mM sodium citrate 3％ 0.5％

[0125] Therefore, the preferred composition of the conjugated pad treatment solution is a 20 mM sodium citrate buffer solution with 5% trehalose, 0.5% casein, and 0.1% Tween-20 added.

[0126] Example 10: Effect of sample loading dilution on fluorescence immunochromatography method

[0127] Increasing the PBS ion concentration did not significantly change the color of the T line on the test strip. Figure 10a As shown, the fluorescence signal value of line C is significantly affected, almost indicating that the test strip is invalid. The positive inhibition rate also decreases. Therefore, 0.01M PBS is the optimal ion concentration. Furthermore, as... Figure 10b and 10c As shown, adding 1% PVP and 10% methanol has the best effect on the stability of the detection system and the color development and sensitivity of the test strip.

[0128] Example 11 Sample Addition and Recovery Results

[0129] The concentrations of mebendazole in negative samples were set at 250, 500, and 1000 ng / g. Extraction and loading were performed according to the pretreatment method described in Example 8, and the recovery rate was calculated by substituting the T / C value into the curve. The results are shown in Table 8. The sample spiked recoveries ranged from 95.7% to 127.1%, with coefficients of variation ranging from 3.0% to 14.6%, indicating that the fluorescence immunochromatographic method presented in this study can be used for the quantitative detection of mebendazole in actual samples.

[0130] Table 8: Recovery results of mebendazole in actual samples detected by fluorescent immunochromatographic test strips (n=3)

[0131]

[0132] Obviously, the above-described embodiments are only some embodiments but not all the embodiments of the present application, the preferred embodiments of the present application are shown in the drawings, but do not limit the patent scope of the present application. The present application can be implemented in many different forms, and conversely, the purpose of providing these embodiments is to make the disclosure of the present application more thorough and comprehensive. Although the present application has been described in detail with reference to the foregoing embodiments, those skilled in the art can still modify the technical solutions described in the foregoing specific embodiments, or make equivalent replacements to some technical features therein. Any equivalent structure made by using the content of the specification and drawings, directly or indirectly applied to other related technical fields, is also within the patent protection scope of the present application.

Claims

1. A tolmepiride hapten, characterized in that, The structural formula is shown in Formula I; 。 2. The method for preparing the mebendazole hapten according to claim 1, characterized in that, Includes the following steps: S1. Using DMSO as solvent, tolueneimidazole, potassium tert-butoxide and tert-butyl 4-bromobutyrate reacted completely, and the organic phases were extracted and combined to obtain the tolueneimidazole hapten intermediate. S2. The intermediate product obtained in step S1 is dissolved in a mixed solution of trifluoroacetic acid and dichloromethane, hydrolyzed and rotary evaporated to remove the solvent, and then purified and dried to obtain toluene-imidazol hapten.

3. The method for preparing the tolmepiride hapten according to claim 2, characterized in that, The molar ratio of tolueneimidazole, potassium tert-butoxide, and tert-butyl 4-bromobutyrate is 34:41:34; the volume ratio of trifluoroacetic acid to dichloromethane in the mixed solution of trifluoroacetic acid and dichloromethane is 1:

2.

4. A tolmepiride artificial antigen, characterized in that, It is obtained by conjugating the mebendazole hapten of claim 1 with a carrier protein at a molar ratio of 1:100; the structural formula of the mebendazole artificial antigen is shown in formula (II). 。 5. The mebendazole artificial antigen according to claim 4, characterized in that, The carrier protein is either ovalbumin or lactoferrin.

6. A mebendazole monoclonal antibody prepared from the mebendazole artificial antigen according to claim 5, characterized in that, The carrier protein is lactoferrin.

7. A method for preparing a test strip for detecting mebendazole by using the mebendazole artificial antigen of claim 5 and the mebendazole monoclonal antibody of claim 6, characterized in that, Includes the following steps: S1. After washing and activating the time-resolved fluorescent microspheres, they were reconstituted with 50 mM MES buffer solution at pH 8.0 and then conjugated with mebendazole monoclonal antibody to prepare a fluorescent probe. S2. The mebendazole artificial antigen with chicken ovalbumin as the carrier protein and the goat anti-mouse IgG secondary antibody were respectively sprayed onto the detection line T and the control line C on the nitrocellulose membrane. S3. Spray the fluorescent probe onto the conjunctival pad after it has been soaked in the conjunctival pad treatment solution, wherein the conjunctival pad treatment solution is prepared by; S4. The conjugate pad, nitrocellulose membrane, and absorbent paper are sequentially overlapped on the base plate to form a test strip; S5. Prepare the sample diluent, which is made by adding 1% w / w PVP and 10% w / w methanol to 0.01 mol / L PBS buffer solution.

8. The method for preparing a test strip for detecting mebendazole according to claim 7, characterized in that, In step S1, 40 µg of mebendazole monoclonal antibody is conjugated to every 50 µL of time-resolved fluorescent microspheres.

9. The method for preparing a test strip for detecting mebendazole according to claim 7, characterized in that, In step S2, 1.4 mg / mL of mebendazole artificial antigen and 0.05 mg / mL of goat anti-mouse IgG secondary antibody were sprayed onto the detection line T and the control line C respectively at a scratching speed of 1 µL / cm; in step S3, 5 µL of fluorescent probe was added to 60 µL of fluorescent diluent and then sprayed onto the conjugate pad at a scratching speed of 2 µL / cm.

10. The method for preparing a test strip for detecting mebendazole according to claim 7, characterized in that, In step S5, the conjugation pad treatment solution is prepared by adding 0%~10% w / w trehalose, 0%~2% w / w casein and 0%~0.4% w / w Tween-20 to a 20mM sodium citrate buffer solution.

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