Sulfonamide haptens, artificial antigens and uses thereof

Abstract

The application discloses a precise sulfonamide hapten, an artificial antigen and application thereof. The application provides two kinds of precise sulfonamide haptens, and an antibody of precise sulfonamide is prepared, which has high titer, high stability and high affinity, and provides core raw materials for establishing an immunodetection method of thiazide drugs. The application also establishes an indirect competitive ELISA immunological analysis method of a broad-spectrum thiazide drug, which has high sensitivity, the minimum detection limit (LOD) of the precise sulfonamide is 0.015 ng / mL, and the IC 50 of the six kinds of thiazide drugs ranges from 0.76 ng / ml to 358.76 ng / ml, and the method has a good application prospect in the residual detection of thiazide drugs.

Description

TECHNICAL FIELD

[0001] The present application relates to the technical field of detection, in particular, to a precise sulfonamide hapten, an artificial antigen and application thereof. BACKGROUND

[0002] Thiazide drugs, including precise sulfonamide, hydrochlorothiazide, chlorothiazide, buthiazide, trichlormethiazide, cyclopenthiazide and other representative drugs, are mainly used for cardiac edema, hepatic edema, renal edema and hypertension, etc. By inhibiting the reabsorption of sodium in the distal renal tubule, the excretion of urine is promoted, and the diuretic effect is achieved. However, the abuse of thiazide drugs may cause some adverse reactions, such as hypotension, cholecystitis and cutaneous lupus. In addition, continuous overuse of such drugs may lead to water and electrolyte imbalance, hyperuricemia, hyperglycemia and even death. Therefore, in order to strengthen the supervision of thiazide drugs, it is necessary to establish a rapid detection method which is simple to operate, high in sensitivity and strong in accuracy.

[0003] Precise sulfonamide (4-amino-6-chlorobenzene-1, 3-disulfonamide) has the chemical formula of C6H8ClN3O4S2, is a key pharmaceutical intermediate, can be used for synthesizing hydrochlorothiazide, cyclopenthiazide, trichlormethiazide and chlorothiazide, and as a degradation product of hydrochlorothiazide, precise sulfonamide is often detected simultaneously with hydrochlorothiazide in food safety sampling. The prior art CN110240576A discloses a hydrochlorothiazide hapten and artificial antigen, and a preparation method and application thereof. Although the detection of hydrochlorothiazide can be realized quickly and conveniently, the detection of a single drug of hydrochlorothiazide can be realized, and it is difficult to meet the detection needs of illegal addition of multiple drugs of thiazide in reality. Therefore, it is urgent to develop a rapid and accurate on-site detection method for thiazide drugs such as precise sulfonamide. SUMMARY

[0004] In order to solve the above problems in the prior art, the present application provides a precise sulfonamide hapten, an artificial antigen and application thereof.

[0005] A first object of the present application is to provide a precise sulfonamide hapten.

[0006] A second object of the present application is to provide an application of a compound with a structural formula as shown in formula (I) in the preparation of a precise sulfonamide artificial antigen,

[0007]

[0008] Formula (I).

[0009] A third object of the present application is to provide another precise sulfonamide hapten.

[0010] The fourth object of the present application is to provide the use of the compound with the structural formula as shown in formula (II) in the preparation of the sulfonamide artificial antigen,

[0011]

[0012] Formula (II).

[0013] The fifth object of the present application is to provide a sulfonamide artificial antigen.

[0014] The sixth object of the present application is to provide another sulfonamide artificial antigen.

[0015] The seventh object of the present application is to provide the use of the sulfonamide artificial antigen in the preparation of the antibody for detecting the thiazide drugs.

[0016] The eighth object of the present application is to provide a sulfonamide artificial antigen combination.

[0017] The ninth object of the present application is to provide the use of the sulfonamide artificial antigen combination in the preparation of the product for detecting the thiazide drugs.

[0018] In order to achieve the above objects, the present application is achieved by the following scheme:

[0019] A sulfonamide hapten, namely, a sulfonamide hapten DIS-2C, with the structural formula as shown in formula (I),

[0020]

[0021] Formula (I).

[0022] The sulfonamide hapten DIS-2C is named as 2,2'-((4-amino-6-chloro-1,3-phenylenedisulfonyl)bis-(azanediyl))diacetic acid by systematic nomenclature.

[0023] The preparation method of the sulfonamide hapten DIS-2C comprises the following steps: dissolving sulfonamide in methanol, fully reacting with ethyl bromoacetate in the potassium carbonate environment, separating and purifying the obtained product through column chromatography, fully hydrolyzing the obtained purified product in the alkaline environment, then adjusting the pH of the solution to be acidic, and obtaining the product.

[0024] Preferably, the molar ratio of the sulfonamide and ethyl bromoacetate is 1: (1.4-1.8).

[0025] More preferably, the molar ratio of the refined sulfonamides and ethyl bromoacetate is 1:1.6.

[0026] Specifically, 1 mmol of refined sulfonamides is dissolved in 5 mL of methanol, and then mixed with 1.6 mmol of ethyl bromoacetate and 4 mmol of potassium carbonate, and stirred at 60°C overnight to obtain a crude product of the reactant; the crude product is spin-dried by a rotary evaporator, and then separated and purified by column chromatography, wherein the stationary phase of the column chromatography is silica gel powder, and the developing agent is dichloromethane: ethyl acetate: triethylamine = (160:40:10) by volume; 20 mg of the purified product is dissolved in 2 mL of methanol, and then stirred with a dilute hydrochloric acid aqueous solution at room temperature for 3 h; after the reaction is completed, the pH is adjusted to 6-7, and the solvent is spin-dried to remove; and then the obtained precipitate is collected to obtain the product.

[0027] The application of the compound with the structural formula as shown in formula (I) in the preparation of a refined sulfonamide artificial antigen should also be within the protection scope of the present application,

[0028]

[0029] Formula (I).

[0030] Another refined sulfonamide hapten, i.e., refined sulfonamide hapten DIS-OH, has a structural formula as shown in formula (II),

[0031]

[0032] Formula (II).

[0033] The refined sulfonamide hapten DIS-OH is named as ((4-amino-2-chloro-5-sulfamoylphenyl)sulfonyl) glycine by systematic nomenclature.

[0034] The preparation method of the refined sulfonamide hapten DIS-OH is basically the same as that of the refined sulfonamide hapten DIS-2C, except that sodium hydride is used to replace potassium carbonate, and the developing agent is dichloromethane: ethyl acetate: triethylamine = 180:30:10 by volume.

[0035] The application of the compound with the structural formula as shown in formula (II) in the preparation of a refined sulfonamide artificial antigen should also be within the protection scope of the present application,

[0036]

[0037] Formula (II).

[0038] A refined sulfonamide artificial antigen is obtained by coupling the refined sulfonamide hapten DIS-2C with a carrier protein, and has a structural formula as shown in formula (III).

[0039]

[0040] Formula (III),

[0041] wherein Protein is a carrier protein.

[0042] Preferably, the carrier protein is bovine serum albumin or chicken egg albumin.

[0043] A precision sulfonamide artificial antigen, which is obtained by coupling the precision sulfonamide hapten DIS-OH with a carrier protein, has a structural formula as shown in Formula (IV),

[0044]

[0045] Formula (IV),

[0046] wherein Protein is a carrier protein.

[0047] Preferably, the carrier protein is bovine serum albumin or chicken egg albumin.

[0048] The use of any of the precision sulfonamide artificial antigens in the preparation of an antibody for detecting a thiazide drug should also be within the protection scope of the present application.

[0049] The use of any of the precision sulfonamide artificial antigens in the detection of a thiazide drug should also be within the protection scope of the present application, and the detection is for the purpose of non-disease treatment diagnosis.

[0050] A precision sulfonamide artificial antigen combination, which comprises a coating agent and an immunogen, the coating agent is obtained by coupling the precision sulfonamide hapten DIS-2C or the precision sulfonamide hapten DIS-OH with chicken egg albumin; and the immunogen is obtained by coupling the precision sulfonamide hapten DIS-OH with bovine serum albumin.

[0051] Preferably, the coating agent is obtained by coupling the precision sulfonamide hapten DIS-2C with chicken egg albumin.

[0052] The use of the precision sulfonamide artificial antigen combination in the preparation of a product for detecting a thiazide drug should also be within the protection scope of the present application.

[0053] The use of the precision sulfonamide artificial antigen combination in the detection of a thiazide drug should also be within the protection scope of the present application, and the detection is for the purpose of non-disease treatment diagnosis.

[0054] An immunoassay method for detecting a thiazide drug, which uses the precision sulfonamide artificial antigen combination for detection, and the detection is for the purpose of non-disease treatment diagnosis.

[0055] An ELISA kit for detecting thiazide drugs, comprising a sulfonamide artificial antigen combination, wherein the sulfonamide artificial antigen combination comprises a coating antigen and an immunogen, the coating antigen is obtained by coupling chicken ovalbumin with the sulfonamide hapten DIS-2C or the sulfonamide hapten DIS-OH; and the immunogen is obtained by coupling bovine serum albumin with the sulfonamide hapten DIS-OH.

[0056] Preferably, the coating antigen is obtained by coupling chicken ovalbumin with the sulfonamide hapten DIS-2C.

[0057] Preferably, the kit further comprises an antibody, which is obtained by immunizing an animal with the immunogen.

[0058] More preferably, the antibody is a monoclonal antibody.

[0059] Preferably, the kit further comprises an enzyme-labeled plate, a standard sample of thiazide drugs, and a substrate developing solution. The thiazide drugs involved in the present application include but are not limited to sulfonamide, hydrochlorothiazide, buthiazide, cyclopenthiazide, trichlormethiazide, and chlorothiazide.

[0060] More preferably, the enzyme-labeled plate is coated with the coating antigen.

[0061] More preferably, the substrate developing solution comprises urea peroxide and tetramethyl benzidine.

[0062] More preferably, the kit further comprises a termination solution, a washing solution, a blocking solution, an enzyme-labeled secondary antibody, and an enzyme-labeled secondary antibody diluent.

[0063] Further preferably, the termination solution has a volume fraction of 8% to 12% H2SO4.

[0064] Most preferably, the termination solution has a volume fraction of 10% H2SO4.

[0065] Further preferably, the washing solution comprises 0.5% to 1.0% Tween-20 by volume fraction, 0.01% to 0.03% sodium azide preservative by mass fraction, 0.1 mol / L to 0.3 mol / L phosphate buffer, and has a pH value of 7.2 to 7.6.

[0066] Most preferably, the washing solution comprises 0.8% Tween-20 by volume fraction, 0.02% sodium azide preservative by mass fraction, 0.2 mol / L phosphate buffer, and has a pH value of 7.4.

[0067] Further preferably, the blocking solution comprises 1% to 3% casein by mass fraction, 0.1 mol / L to 0.3 mol / L phosphate buffer, and has a pH value of 7.1 to 7.5.

[0068] Most preferably, the closed liquid is a phosphate buffer solution containing 2% casein by mass fraction, 0.2 mol / L, and the pH value is 7.3.

[0069] Further preferably, the enzyme-labeled secondary antibody is a horseradish peroxidase-labeled goat anti-rabbit antibody or a goat anti-mouse antibody.

[0070] Further preferably, the enzyme-labeled secondary antibody diluent is a 0.1 mol / L-0.3 mol / L phosphate buffer solution.

[0071] Most preferably, the enzyme-labeled secondary antibody diluent is a 0.2 mol / L phosphate buffer solution.

[0072] Compared with the prior art, the present application has the following beneficial effects:

[0073] The present application provides two thiazide haptens, and antibodies of thiazide are prepared, which have high titer, strong stability and high affinity, and provide core raw materials for establishing an immune detection method of thiazide drugs. 50 The present application also establishes an indirect competitive ELISA immune analysis method of thiazide drugs, which has high sensitivity, and the minimum detection limit LOD of thiazide is 0.015 ng / mL, and the IC BRIEF DESCRIPTION OF DRAWINGS

[0074] Figure 1 The synthesis route of thiazide hapten DIS-2C.

[0075] Figure 2 The synthesis route of thiazide hapten DIS-OH.

[0076] Figure 3 The ultraviolet scanning identification result diagram of thiazide haptens DIS-2C-BSA and DIS-2C-OVA, a is DIS-2C-BSA, and b is DIS-2C-OVA.

[0077] Figure 4 The ultraviolet scanning identification result diagram of thiazide hapten DIS-OH-BSA.

[0078] Figure 5 The standard curve of the indirect competitive ELISA of thiazide drugs. DETAILED DESCRIPTION

[0079] The application will be further described in conjunction with the accompanying drawings and specific embodiments, which are only used to explain the application and not to limit the scope of the application. The test methods used in the following examples are conventional methods unless otherwise specified; the materials, reagents, etc. used are commercially available reagents and materials unless otherwise specified.

[0080] Example 1 Synthesis and identification of sulfonamide hapten

[0081] I. Synthesis and identification of sulfonamide hapten DIS-2C

[0082] 1. Synthesis of sulfonamide hapten DIS-2C

[0083] The synthesis route of sulfonamide hapten DIS-2C is shown in Figure 1 The specific steps are as follows:

[0084] Take 285 mg (1 mmol) of sulfonamide and 156 μL (1.2 mmol) of ethyl bromoacetate in a 50 ml single-necked round-bottom flask, dissolve in 5 mL of chromatographic grade methanol, then add 553 mg of K2CO3 (4 mmol), and stir-react at 60°C for 12 h to obtain the crude product of the reaction; spin-dry the methanol of the crude product with a rotary evaporator, mix with 15 mL of ethyl acetate and 100 g of silica gel powder, and spin-dry, then purify by column chromatography using the developing agent (volume ratio, dichloromethane: ethyl acetate: triethylamine = 160:40:10) to obtain the pure intermediate product. Dissolve the target product obtained in 5 mL of methanol, add 1 M dilute hydrochloric acid solution according to the volume ratio of methanol to dilute hydrochloric acid solution 1:1, stir-react at room temperature, and monitor the product with a TCL plate to determine whether the ester group is completely hydrolyzed, and stop the reaction when the ester group of the product is completely hydrolyzed. Spin-evaporate the organic solvent in the reaction solution, adjust the pH to 5-6, add an appropriate amount of saturated brine, extract with ethyl acetate and water at a volume ratio of 1:1, shake thoroughly, collect the organic phase, dry it with anhydrous sodium sulfate, and spin-evaporate the organic solvent to obtain the sulfonamide hapten DIS-2C.

[0085] 2. Identification of sulfonamide hapten DIS-2C

[0086] The nuclear magnetic resonance hydrogen spectrum of sulfonamide hapten DIS-2C is as follows: 1H NMR (600 MHz, Methanol-d4) δ 8.20-8.14 (m, 2H), 8.10 (d, J = 16.5 Hz, 1H), 7.41 (s, 1H), 6.71 (d, J = 6.2 Hz, 1H), 6.65 (d, J = 5.9 Hz, 1H), 3.84 (dd, J = 8.1, 1.5 Hz, 4H).

[0087] The mass spectrometry result of the precise sulfonamide hapten DIS-2C is: MS: C 10 H 12 ClN3O8S2: 401.79, ESI-[M-H] + : 403.10.

[0088] As can be seen from the mass spectrometry and nuclear magnetic resonance results, the mass spectrometry result corresponds to the molecular weight of DIS-2C, and the number of hydrogen spectrum in the nuclear magnetic resonance can correspond to the number of hydrogen spectrum on the structure of DIS-2C, indicating that the precise sulfonamide hapten DIS-2C is successfully prepared, and its structural formula is shown as formula (I),

[0089]

[0090] Formula (I).

[0091] The precise sulfonamide hapten DIS-2C is named by systematic nomenclature as: 2,2'-((4-amino-6-chloro-1,3-phenylenedisulfonyl)-bis(azanediyl))diacetic acid.

[0092] II. Synthesis and identification of the precise sulfonamide hapten DIS-OH

[0093] 1. Synthesis of the precise sulfonamide hapten DIS-OH

[0094] The synthesis route of the precise sulfonamide hapten DIS-OH is shown in Figure 2 The specific steps are basically the same as those of the synthesis of the precise sulfonamide hapten DIS-2C in this embodiment, except that sodium carbonate is replaced by sodium hydride, and the developing agent used in column chromatography is replaced by: dichloromethane: ethyl acetate: triethylamine = 180:30:10 by volume.

[0095] 2. Identification of the precise sulfonamide hapten DIS-OH

[0096] The nuclear magnetic resonance hydrogen spectrum result of the precise sulfonamide hapten DIS-OH is: 1H NMR (600 MHz, Methanol-d4) δ 8.17 (t, J = 8.2 Hz, 1H), 8.14 (s, 1H), 6.67 (d, J = 5.9 Hz, 1H), 6.52 (d, J = 5.9 Hz, 1H), 3.83 (d, J = 8.1 Hz, 2H).

[0097] From the results of mass spectrometry and nuclear magnetic resonance, it can be seen that the mass spectrometry result corresponds to the molecular weight of DIS-OH, and the hydrogen spectrum number of nuclear magnetic resonance corresponds to the hydrogen spectrum number on the structure of DIS-OH, which indicates that the refined sulfonamide hapten DIS-OH is successfully prepared, and the structural formula is shown as formula (II):

[0098]

[0099] Formula (II).

[0100] The refined sulfonamide hapten DIS-OH is named by systematic nomenclature as: 4-(((7-chloro-2-methyl-4-oxo-3-(o-tolyl)-1,2,3,4-tetrahydroquinazoline)-6-sulfonamid-o)methyl)benzoic acid.

[0101] Example 2 Synthesis and identification of refined sulfonamide artificial antigen

[0102] 1. Synthesis of refined sulfonamide hapten DIS-2C artificial antigen

[0103] The refined sulfonamide hapten DIS-2C prepared in Example 1 is coupled with bovine serum albumin (BSA) by active ester method, and the specific steps are as follows:

[0104] Take 1 mol of fine sulfonamide hapten DIS-2C, 0.8 mol of N-hydroxysuccinimide (NHS) and 1.9 mol of 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (EDC) dissolved in 200 μL of N,N-dimethylformamide (DMF), stirring at room temperature (25°C) for 3 h in the dark, to obtain the hapten DIS-NC activation solution, marked as A solution; 10 mg of BSA was dissolved in 1 mL of PBS buffer (0.01 mol / L, pH=7.4) to obtain a BSA solution, marked as B solution; 200 μL of A solution was slowly added dropwise to 1 mL of B solution, and reacted at 4°C for 12 h; dialysis was performed with PBS buffer for 3 days, 3 times a day, and after dialysis, fine sulfonamide artificial antigen DIS-2C-BSA was obtained, which was aliquoted in centrifuge tubes and stored at -20°C for use.

[0105] According to the above method, the only difference is that chicken ovalbumin (OVA) is used instead of BSA to prepare fine sulfonamide artificial antigen DIS-2C-OVA.

[0106] 2. Identification of fine sulfonamide artificial antigens DIS-2C-BSA and DIS-2C-OVA

[0107] BSA, OVA, DIS-2C, DIS-2C-BSA and DIS-2C-OVA were scanned and identified by ultraviolet full wavelength method (200-350 nm).

[0108] As shown in a of Figure 3 By comparing the highest absorbance of each substance before and after coupling, it was found that the absorption curve of DIS-2C-BSA was significantly different from that of the carrier protein BSA. DIS-NC had a strong absorption peak above 345 nm, while after coupling with BSA, the absorption peak of DIS-NC-BSA at 280 nm was significantly higher than that of BSA, and the curve was significantly shifted compared with that of hapten DIS-NC. Since all small molecule components such as drugs that have not reacted have been removed during the dialysis process after coupling, the drug characteristic peak of the coupling product is contributed by the drug molecules bound to the protein, indicating that the reaction product is a complex of carrier protein BSA and DIS-NC.

[0109] As shown in a of Figure 3As shown in b of FIG. 1, by comparing the highest light absorption values of each substance before and after coupling, it is found that the absorption curve of DIS-2C-OVA is obviously different from the carrier protein OVA, the absorption peak of DIS-NC above 345 nm is strong, after coupling OVA, DIS-NC-OVA has obvious absorption peaks at 230 mm and 280 mm, and the peak shape and height at 280 mm are significantly different from the carrier protein OVA, and the curve is significantly shifted compared with the relative hapten DIS-NC. Since all the small molecule components such as unreacted drugs are removed by dialysis after coupling, the drug characteristic peak of the coupling product is contributed by the protein-bound drug molecules, which indicates that the reaction product is a complex of the carrier protein OVA and DIS-NC.

[0110] The above results show that the precision sulfonamide artificial antigen DIS-2C-BSA and DIS-2C-OVA are successfully prepared, and the structural formula is shown as formula (III),

[0111]

[0112] Formula (III),

[0113] In the formula, Protein is a carrier protein BSA or OVA.

[0114] II. Synthesis and identification of precision sulfonamide hapten DIS-OH artificial antigen

[0115] 1. Synthesis of precision sulfonamide hapten DIS-OH artificial antigen

[0116] The precision sulfonamide hapten DIS-OH prepared in Example 1 is coupled with bovine serum albumin (BSA) by the carbonyl dimidazole method, and the specific steps are as follows:

[0117] 1 mol of precision sulfonamide hapten DIS-OH, 0.8 mol of N-hydroxysuccinimide (NHS) and 1.9 mol of 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (EDC) are dissolved in 200 μL of N,N-dimethylformamide (DMF), and stirred at room temperature (25℃) for 3 h in the dark to obtain an activated solution of hapten DIS-OH, which is denoted as solution C; 10 mg of BSA is dissolved in 1 mL of PBS buffer (0.01 mol / L, pH=7.4) to obtain a BSA solution, which is denoted as solution D; 200 μL of solution C is slowly added dropwise to 1 mL of solution D, and reacted at 4℃ for 12 h; dialysis is performed with PBS buffer for 3 days, 3 times a day, and after dialysis, the precision sulfonamide artificial antigen DIS-OH-BSA is obtained, which is stored in centrifuge tubes at -20℃ for use.

[0118] According to the above method, the difference is that chicken ovalbumin (OVA) is used instead of BSA to prepare the fine sulfonamide artificial antigen DIS-OH-OVA.

[0119] 2. Identification of fine sulfonamide hapten DIS-OH artificial antigen

[0120] The above BSA, DIS-OH and DIS-OH-BSA are scanned and identified by ultraviolet full wavelength method (200-350 nm), and the results are shown in Figure 4 By comparing the highest light absorption values of each substance before and after coupling, it is found that the absorption curve of DIS-OH-BSA is obviously different from that of the carrier protein BSA, the hapten DIS-OH has a characteristic peak at 345 nm, and after coupling with BSA, DIS-OH-BSA has obvious absorption peaks at 230 mm and 280 mm, and the absorption at 280 nm is obviously higher than that of BSA, and the curve is significantly shifted compared with that of the hapten DIS-OH. Since all the small molecule components such as drugs that are not reacted are removed during the dialysis process of the coupling reaction, the drug characteristic peak of the coupling product is contributed by the drug molecules combined with the protein, which indicates that the reaction product is a complex of the carrier protein BSA and the hapten DIS-OH, and the fine sulfonamide artificial antigen DIS-OH-BSA is successfully prepared. DIS-OH-OVA also has similar identification results.

[0121] The above results show that the fine sulfonamide artificial antigen DIS-OH-BSA is successfully prepared, and its structural formula is shown in formula (IV),

[0122]

[0123] Formula (IV),

[0124] Protein is the carrier protein BSA or OVA.

[0125] Example 3 Preparation of monoclonal antibody

[0126] The fine sulfonamide artificial antigen DIS-OH-BSA prepared in Example 2 is used as an immunogen to immunize female Balb / c mice. DIS-OH-BSA is emulsified with an equal volume of immunoadjuvant (complete Freund's adjuvant is used for the first immunization, and Freund's incomplete adjuvant is used for the booster immunization), and then the mice are immunized by subcutaneous injection at multiple points on the abdomen. After each booster immunization for 1 week, the tail blood is taken to determine the titer of the antiserum.

[0127] When the titer is stable and unchanged, the mouse with the best immunization effect is selected for booster immunization, and the spleen cells of the mouse are taken 3 days later to fuse with myeloma cells, the positive hybridoma cells selected are injected into the abdominal cavity of the mouse, and the ascites is collected to further purify the monoclonal antibody.

[0128] According to the above method, monoclonal antibody with DIS-2C-BSA as immunogen was prepared.

[0129] Example 4 Combination of immunogen and coating antigen

[0130] The serum titer and inhibition rate obtained by indirect competitive ELISA method were used to select the best combination of immunogen and coating antigen by using the DIS-2C-OVA or DIS-OH-OVA artificial antigen prepared in Example 2 as coating antigen and the monoclonal antibody prepared in Example 3. The specific operation steps are as follows:

[0131] 1. DIS-2C-OVA or DIS-OH-OVA was diluted to a concentration of 1000 ng / mL with coating solution (0.05M carbonate buffer solution, pH 9.6), 100 μL / well was coated on a 96-well enzyme-labeled plate, and incubated in a 37°C constant temperature water bath overnight. The coating solution was discarded, and PBST (0.01M PBS, 0.06% Tween-20 (v / v)) was used to wash twice;

[0132] 2. 120 μL blocking solution (pH 7.3, containing 2% casein by mass fraction, 0.2 mol / L phosphate buffer) was added to each well, and incubated at 37°C for 3 h. The blocking solution was discarded, and the plate was dried in a drying oven at 37°C for standby;

[0133] 3. The monoclonal antibody prepared in Example 3 was diluted to 1:1000, 1:2000, 1:4000, 1:8000, 1:16000, 1:32000 and 1:64000 with PBST, and a blank control well (replaced with PBST) was set. The 1 mg / mL sulfonamide standard was diluted 1000 times to obtain a sulfonamide dilution solution with a concentration of 1 μg / mL;

[0134] 4. Titer column setting: 50 μL PBST was added to each well, then 50 μL monoclonal antibody with different dilution ratios was added to each well, and finally 50 μL PBST was added to the last well instead of antibody;

[0135] 5. Inhibition column setting: 50 μL sulfonamide dilution solution was added to each well, then 50 μL monoclonal antibody with different dilution ratios was added to each well, and finally 50 μL PBST was added to the last well instead of antibody;

[0136] 6. Incubate at 37°C for 40 min, and wash 5 times, and tap the plate;

[0137] 7. Add goat anti-mouse secondary antibody-HRP (diluted 5000 times with PBST), incubate at 37°C for 30 min, wash 5 times, and tap the plate;

[0138] 8. Add chromogenic solution, incubate for 10 min at 37℃, then read OD value at 450 nm;

[0139] 9. Add 10% v / v H2SO4 to terminate the reaction, and read OD value at 450 nm; calculate titer and inhibition rate, titer is OD value corresponding to 1.0, inhibition rate = (OD value of titer - OD value of inhibition) / OD value of inhibition x 100%. 450

[0140] Results of different combinations of immunogens and coating agents are shown in Table 1.

[0141] Table 1. ELISA detection results of immunogens and coating agents

[0142]

[0143] As shown in Table 1, antisera of mice immunized with DIS-2C-BSA and DIS-OH-BSA as immunogens have certain titers, and DIS-OH-BSA immunogen shows stronger recognition ability against two coating agents. The combination of immunogen No. 3 and coating agent has the highest titer (1:32000) and the highest inhibition rate (89.72%), which is the best combination of immunogen and coating agent. Under this combination, the monoclonal antibody can not only specifically recognize the target analyte dapsone, but also has good sensitivity and can be used for specific recognition of the target analyte dapsone.

[0144] Example 5. Establishment of indirect competitive ELISA method for detecting thiazide drugs

[0145] I. Operation steps of detection method

[0146] The present embodiment provides an indirect competitive ELISA method for detecting thiazide drugs, which comprises the following steps:

[0147] 1. Dilute DIS-2C-OVA to a concentration of 250 ng / mL with coating solution (0.05M carbonate buffer solution, pH 9.6), 100 μL / well of 96-well enzyme-labeled plate, incubate in a 37℃ constant temperature water bath overnight, discard the coating solution, wash twice with PBST (0.01M PBS, 0.06% Tween-20 (v / v));

[0148] 2. Add 120 μL blocking solution (pH 7.3, containing 2% casein by mass fraction, 0.2 mol / L phosphate buffer) to each well, block at 37℃ for 3 h, discard the blocking solution, tap the plate, and dry in a drying oven at 37℃ for standby;

[0149] ​3. The monoclonal antibody with the DIS-OH-BSA prepared in Example 3 as the immunogen was diluted with PBST to 1:1000, 1:2000, 1:4000, 1:8000, 1:16000, 1:32000 and 1:64000, and a blank control well (replaced with PBST) was set; 1 mg / mL sulfonamides standard was diluted 1000 times to obtain a sulfonamides dilution solution with a concentration of 1 μg / mL;

[0150] 4. The titer column was set up as follows: 50 μL PBST was added to each well, then 50 μL of the monoclonal antibody with different dilution ratios was added to each well, and finally 50 μL PBST was added to the last well instead of the antibody;

[0151] 5. The inhibition column was set up as follows: 50 μL of the sulfonamides dilution solution was added to each well, then 50 μL of the monoclonal antibody with different dilution ratios was added to each well, and finally 50 μL PBST was added to the last well instead of the antibody;

[0152] 6. Incubation was performed at 37°C for 40 min, and washing and plate tapping were performed 5 times;

[0153] 7. Goat anti-mouse secondary antibody-HRP (diluted 5000 times with PBST) was added, incubation was performed at 37°C for 30 min, and washing and plate tapping were performed 5 times;

[0154] 8. Color developing solution was added, and color development was performed at 37°C for 10 min;

[0155] 9. 10% v / v H2SO4 was added to terminate the reaction, and the OD value was read at 450 nm;

[0156] 10. ELISA standard curve drawing: B / B0 was taken as the ordinate (B is the absorbance OD 450 of the standard of different concentrations of sulfonamides, and B0 is the absorbance OD 450 of the blank control well), and the logarithm of the concentration of the sulfonamides standard was taken as the abscissa, a Logistic function was used for curve fitting, a standard curve was prepared, and the formula of the standard curve and the IC 50 value were obtained.

[0157] II. Performance evaluation of the detection method

[0158] The detection was performed according to the above operation steps, except that in step 3, hydrogenchlorothiazide dilution solution, buthiazide dilution solution, cyclopenthiazide dilution solution, trichlormethiazide dilution solution and chlorothiazide dilution solution were also prepared at the same concentration; and in step 5, 50 μL of the sulfonamides dilution solution, hydrogenchlorothiazide dilution solution, buthiazide dilution solution, cyclopenthiazide dilution solution, trichlormethiazide dilution solution or chlorothiazide dilution solution was added to each well.

[0159] 4-amino-6-chlorobenzene-1,3-disulfonamide (CAS# 121-30-2) indirect competition ELISA standard curve was prepared as shown in Figure 1. Figure 5 The IC 50 was 0.76 ng / mL, the linear detection range was 0.075-6.729 ng / mL, and the lowest detection limit LOD (IC 10 ) was 0.02 ng / mL.

[0160] Table 2. Detection results of thiazide drugs

[0161]

[0162] As shown in Table 2, the indirect competition ELISA detection method established using the monoclonal antibody and the coating antigen prepared by the present application has broad spectrum recognition ability for thiazide drugs, high sensitivity, and the IC 50 is all lower than 1 μg / ml level, which meets the needs of qualitative detection application in reality.

[0163] Example 6. An ELISA kit for detecting thiazide drugs

[0164] 1. Composition

[0165] (1) An enzyme-labeled plate coated with a coating antigen, which is prepared by the following method:

[0166] The fine sulfonamide artificial antigen DIS-2C-OVA or DIS-OH-OVA prepared in Example 2 is used as the coating antigen, which is diluted to 750 ng / mL with a coating antigen solution (0.05M carbonate buffer solution, pH 9.6), and 100 μL / well is coated on a 96-well enzyme-labeled plate, which is incubated at 37°C overnight in the dark; the liquid in the wells is poured off, and the wells are washed twice with the washing solution in the kit, each for 30s, and then dried; then 200 μL / well of blocking solution in the kit is added, and incubated at 25°C for 2 hours in the dark; the liquid in the wells is poured off and dried, and then stored in an aluminum film under vacuum sealing.

[0167] (2) Standard: 8 different concentrations of thiazide drug (fine sulfonamide, hydrochlorothiazide, buthiazide, cyclopenthiazide, trichlormethiazide and chlorothiazide) standard, respectively 1000 ng / mL, 125 ng / mL, 15.63 ng / mL, 1.95 ng / mL, 0.24 ng / mL, 0.031 ng / mL, 0.0038 ng / mL and 0.00048 ng / mL.

[0168] (3) Antibody: the monoclonal antibody prepared in Example 3.

[0169] (4) Enzyme-labeled secondary antibody: horseradish peroxidase-labeled goat anti-rabbit secondary antibody or horseradish peroxidase-labeled goat anti-mouse secondary antibody.

[0170] (5) Substrate solution: composed of A liquid and B liquid, A liquid is urea peroxide, B liquid is tetramethyl benzidine.

[0171] (6) Stop solution: 2 mol / L H2SO4.

[0172] (7) Washing solution: pH value is 7.4, containing 0.8% Tween-20 by volume fraction, 0.02% sodium azide preservative by mass fraction, 0.2 mol / L phosphate buffer; before use, dilute the washing solution 20 times with water (i.e. 1 part of washing solution is added to 19 parts of water, prepared and used immediately), to obtain washing solution working solution.

[0173] (8) Dilution solution: 0.2 mol / L phosphate buffer; before use, dilute the dilution solution 20 times with water (i.e. 1 part of dilution solution is added to 19 parts of water, prepared and used immediately), to obtain dilution solution working solution.

[0174] (9) Blocking solution: pH value is 7.3, containing 2% casein by mass fraction in 0.2 mol / L phosphate buffer.

[0175] 2. Method of use

[0176] (1) Sample detection

[0177] Corresponding microwells of samples and standard samples of the kit are numbered in order, 2 wells in parallel are made for each sample and standard sample, and the positions of standard wells and sample wells are recorded. The antibody is diluted with the dilution solution working solution according to the required amount at a volume ratio of 1:40 (i.e. 1 part of antibody is added to 40 parts of dilution solution working solution, prepared and used immediately), to obtain the antibody working solution. The enzyme-labeled secondary antibody is diluted with the dilution solution working solution according to the required amount at a volume ratio of 1:10 (i.e. 1 part of enzyme-labeled secondary antibody is added to 10 parts of dilution solution working solution, prepared and used immediately), to obtain the enzyme-labeled secondary antibody working solution.

[0178] Add 50 μL of standard sample or sample to the corresponding microwell, then add 50 μL of antibody working solution to the corresponding microwell, shake gently to mix, cover with a cover film, and place in a 25℃ dark environment for reaction for 40 min.

[0179] Spin dry the liquid in the well, add 250 μL / well of washing solution working solution. Wash thoroughly for 4-5 times, with an interval of 10 s each time, discard the washing solution working solution in the well, and dry with a water-absorbing paper (after drying, the bubbles that are not clearly visible can be poked with a unused gun head).

[0180] Add 100 μL / well of enzyme-labeled secondary antibody working solution to the corresponding microwell, shake gently to mix, cover with a cover film, and place in a 25℃ dark environment for reaction for 30 min.

[0181] Shake off the liquid in the well and add 250 μL of washing working solution per well. Wash thoroughly 4-5 times, with 10 s intervals between each wash. Discard the washing working solution in the well and pat dry with absorbent paper (any air bubbles not removed after patting can be punctured with an unused pipette tip).

[0182] Add 50 μL of substrate chromogenic solution A per well, then add 50 μL of substrate chromogenic solution B per well, gently shake to mix, cover with a cover plate and incubate at 25°C in the dark for 10 min.

[0183] Add 50 μL of stop solution per well, gently vortex to mix, set the microplate reader to 450 nm, and measure the OD value of each well.

[0184] (2) Drawing the standard curve

[0185] Plot B / B0 as the ordinate (B represents the absorbance OD of standards at different concentrations). 450 B0 is the absorbance value (OD) of the blank control well. 450 The standard curve is obtained by using the logarithm of the concentration of the standard as the abscissa and performing curve fitting with the Logistic function. The formula for the standard curve is then derived.

[0186] (3) Calculation of sample concentration

[0187] OD of the sample 450 Substituting the average value into the formula of the standard curve above, we obtain the concentration of the sample. Multiplying this by the corresponding dilution factor gives the actual concentration of thiazide drugs in the sample.

[0188] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention and are not intended to limit the scope of protection of the present invention. For those skilled in the art, other variations or modifications can be made based on the above description and ideas, and it is neither necessary nor possible to exhaustively describe all implementation methods here. Any modifications, equivalent substitutions, and improvements made within the spirit and principles of the present invention should be included within the scope of protection of the claims of the present invention.

Claims

1. A refined sulfonamide hapten, characterized in that, A structure formula is shown as formula (I), Formula (I).

2. Application of the compound with a structure formula shown as formula (I) in preparing a precise sulfonamide artificial antigen, Formula (I).

3. A refined sulfonamide hapten, characterized in that, A structure formula is shown as formula (II), Formula (II).

4. Application of the compound with a structure formula shown as formula (II) in preparing a precise sulfonamide artificial antigen, Formula (II).

5. A refined sulfonamide artificial antigen, characterized in that, Obtained from the precise sulfonamide hapten conjugated carrier protein in claim 1, a structure formula is shown as formula (III), Formula (III), Wherein, Protein is a carrier protein, and the carrier protein is bovine serum albumin or chicken egg albumin.

6. A refined sulfonamide artificial antigen, characterized in that, Obtained from the precise sulfonamide hapten conjugated carrier protein in claim 3, a structure formula is shown as formula (IV), Formula (IV), Wherein, Protein is a carrier protein, and the carrier protein is bovine serum albumin or chicken egg albumin.

7. Application of the precise sulfonamide artificial antigen in claim 5 or 6 in preparing an antibody for detecting a thiazide drug.

8. A fine sulfonamide artificial antigen combination, characterized by, Comprising a coating agent and an immunogen, the coating agent is obtained from the precise sulfonamide hapten conjugated chicken egg albumin in claim 1 or 3; and the immunogen is obtained from the precise sulfonamide hapten conjugated bovine serum albumin in claim 3.

9. Application of the precise sulfonamide artificial antigen combination in claim 8 in preparing a product for detecting a thiazide drug.

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