Buthiazine hapten, artificial antigen and their application

Abstract

The present invention discloses a buthiazine hapten, an artificial antigen and its application. The present invention provides a buthiazine hapten and prepares an antibody against buthiazine, which has high sensitivity for buthiazine detection and IC 50 The minimum detection limit (LOD (IC 10 )) was 3.11 ng / mL, with a linear detection range of 5.68 ng / mL to 44.59 ng / mL, and had strong specificity and no obvious cross-reaction with the structural analogs of buthiazine.

Description

Technical Field

[0001] The present invention relates to the technical field of immunoassay, and in particular to a buthiazine hapten, an artificial antigen and applications thereof. Background Art

[0002] 6-chloro-3,4-dihydro-3-isobutyl-1,2,4-benzothiadiazine-7-sulphonamide1,1-dioxide, C 11 H 16 N3O4S2Cl) is a thiazide-type medium-acting diuretic, mainly used in the treatment of hypertension in clinical medicine. It acts on the epithelial cells of the distal convoluted tubule luminal membrane. + 、Cl - Co-transporter, inhibits Na + 、Cl - However, long-term use of butiazide can affect uric acid excretion, leading to elevated uric acid levels and the development of gout, impaired glucose tolerance, and even diabetes. Therefore, to strengthen the regulation of butiazide, it is necessary to establish a simple, sensitive, and accurate rapid butiazide detection method.

[0003] Immunochromatography offers high sensitivity and specificity, enabling quantitative and high-throughput detection of target analytes. It is widely used in the field of rapid diagnostics due to its rapid visual detection, on-site convenience, and low cost. Haptens are crucial for establishing immunoassays, but currently, no buthiazide haptens are known to generate specific antibodies. Summary of the Invention

[0004] In order to solve the above problems existing in the prior art, the present invention provides a buthiazine hapten, an artificial antigen and applications thereof.

[0005] The first object of the present invention is to provide a buthiazine hapten.

[0006] The second object of the present invention is to provide the use of a compound having a structural formula (I) in the preparation of a buthiazine artificial antigen.

[0007]

[0008]

[0009] The third object of the present invention is to provide a buthiazine artificial antigen.

[0010] The fourth object of the present invention is to provide another butiazine hapten.

[0011] The fifth object of the present invention is to provide the use of a compound having a structural formula as shown in formula (II) in the preparation of a buthiazine artificial antigen.

[0012]

[0013] The sixth object of the present invention is to provide another artificial antigen of butiazide.

[0014] The seventh object of the present invention is to provide the use of the butiazine artificial antigen in the preparation of antibodies for detecting butiazine.

[0015] The eighth object of the present invention is to provide a buthiazine artificial antigen combination.

[0016] The ninth objective of the present invention is to provide the use of the butiazine artificial antigen combination in detecting butiazine or in preparing a product for detecting butiazine.

[0017] The tenth object of the present invention is to provide a kit for detecting buthiazine.

[0018] In order to achieve the above object, the present invention is implemented through the following scheme:

[0019] The butiazine-based structural analogue trichlorothiazide of the present invention is chemically modified and connected to an ethyl bromopropionate carbon chain arm to construct a butiazine hapten, which can be directly coupled to a carrier protein for use as an immunogen. At the same time, the connecting arm has a moderate length when connected to the carrier protein, thereby fully exposing its specific binding site after connection to the carrier protein, and the generated antibody has high sensitivity and strong specificity.

[0020] A buthiazine hapten, namely BTZ-3C, has a structural formula as shown in formula (I).

[0021]

[0022] The preparation method of the buthiazine hapten BTZ-3C comprises the following steps: fully reacting trichloromethiazine and ethyl bromopropionate at a molar ratio of 1:(1.2-1.6) under alkaline conditions, separating and purifying the obtained product by column chromatography, and fully hydrolyzing the obtained purified product under an acidic environment; the developing solvent for the column chromatography is: petroleum ether: ethyl acetate: triethylamine = 188:36:10 in volume ratio.

[0023] Preferably, the molar ratio of trichloromethiazine to ethyl bromopropionate is 1:1.4.

[0024] Preferably, before the reaction begins, the trichloromethylthiazine is activated with sodium hydride, and the molar ratio of the sodium hydride to the trichloromethylthiazine is (1.2-1.6):1.

[0025] More preferably, the molar ratio of the sodium hydride to the trichloromethylthiazide is 1.4:1.

[0026] Specifically, the preparation method of the buthiazine hapten BTZ-3C comprises the following steps:

[0027] 379 mg of trichloromethylthiazine was dissolved in 5 mL of chromatographic grade N,N-dimethylformamide, and then 28.8 mg of sodium hydride was added, and the mixture was stirred in an ice bath at 0°C for 60 min to obtain activated trichloromethylthiazine. The activated trichloromethylthiazine was reacted with 254 μL of ethyl bromopropionate and stirred at room temperature for 12 h. After the reaction, the crude product was extracted with saturated sodium chloride solution, and the oil phase was dried using a rotary evaporator. 15 mL of ethyl acetate and 50 g of silica gel powder were added, mixed, and dried using a developing solvent (volume ratio of petroleum ether: ethyl acetate: triethylamine = 188:36:10). The intermediate product is purified by column chromatography to obtain a pure intermediate product; the intermediate product is dissolved in 5 mL of methanol, and a 1 M dilute hydrochloric acid solution is added in a volume ratio of methanol to dilute hydrochloric acid solution of 1:1. The reaction is stirred at room temperature, and a TCL plate is used to monitor whether the product is completely hydrolyzed. The reaction is stopped after the ester group of the product is completely hydrolyzed; the organic solvent in the obtained reaction solution is rotary evaporated, the pH is adjusted to 5-6, saturated brine is added, and extraction is carried out with ethyl acetate and water in a volume ratio of 1:1, and the product is shaken thoroughly. The organic phase is collected, dried over anhydrous sodium sulfate, and the organic solvent is removed by rotary evaporation to obtain the product.

[0028] The use of the compound represented by the structural formula (I) in the preparation of buthiazine artificial antigens should also be within the scope of protection of the present invention.

[0029]

[0030] A buthiazine artificial antigen is obtained by coupling the buthiazine hapten BTZ-3C with a carrier protein, and its structural formula is shown in formula (III):

[0031]

[0032] Among them, Protein is a carrier protein.

[0033] Preferably, the butiazine artificial antigen is obtained by coupling the butiazine hapten BTZ-3C to a carrier protein through the active ester method.

[0034] Preferably, the carrier protein is bovine serum albumin or chicken ovalbumin.

[0035] Another buthiazine hapten, BTZ-4C, has a structural formula as shown in formula (II).

[0036]

[0037] The preparation method of butiazine hapten BTZ-4C is basically the same as that of BTZ-3C, except that ethyl bromopropionate is replaced by ethyl bromobutyrate; and the developing solvent is replaced by: petroleum ether: ethyl acetate: triethylamine = 188:28:10 by volume.

[0038] The use of the compound represented by the structural formula (II) in the preparation of buthiazine artificial antigens should also be within the scope of protection of the present invention.

[0039]

[0040] Another artificial butiazine antigen is obtained by coupling the butiazine hapten BTZ-4C with a carrier protein, and its structural formula is shown in formula (IV):

[0041]

[0042] Among them, Protein is a carrier protein.

[0043] Preferably, the butiazine artificial antigen is obtained by coupling the butiazine hapten BTZ-4C to a carrier protein through the active ester method.

[0044] Preferably, the carrier protein is bovine serum albumin or chicken ovalbumin.

[0045] The use of any of the above-mentioned artificial butiazine antigens in the preparation of antibodies for detecting butiazine should also be within the scope of protection of the present invention.

[0046] Preferably, the antibody is a polyclonal antibody.

[0047] The use of any of the above-mentioned artificial butiazide antigens in detecting butiazide should also be within the scope of protection of the present invention, and the detection is for the purpose of non-disease treatment and diagnosis.

[0048] The use of any of the above-mentioned butiazide artificial antigens in the preparation of products for detecting butiazide should also be within the scope of protection of the present invention.

[0049] A buthiazine artificial antigen combination comprises a coating agent and an immunogen, wherein the coating agent is obtained by coupling the buthiazine hapten BTZ-3C or BTZ-4C with chicken egg albumin; and the immunogen is obtained by coupling the buthiazine hapten BTZ-3C or BTZ-4C with bovine serum albumin.

[0050] Preferably, the coating agent is obtained by coupling the butiazine hapten BTZ-3C with chicken ovalbumin; and the immunogen is obtained by coupling the butiazine hapten BTZ-3C with bovine serum albumin.

[0051] The use of the butiazide artificial antigen combination in detecting butiazide should also be within the scope of protection of the present invention, and the detection is for the purpose of non-disease treatment and diagnosis.

[0052] The use of the butiazide artificial antigen combination in the preparation of products for detecting butiazide should also be within the scope of protection of the present invention.

[0053] An immunoassay method for detecting buthiazine uses the buthiazine artificial antigen combination for detection, wherein the detection is for the purpose of non-disease treatment diagnosis.

[0054] Preferably, the immunoassay method is ELISA.

[0055] More preferably, the immunoassay method is indirect ELISA and / or competitive ELISA.

[0056] Further preferably, the immunoassay method is indirect competitive ELISA.

[0057] A kit for detecting buthiazine comprises the buthiazine artificial antigen combination.

[0058] Preferably, the kit is an ELISA kit.

[0059] Preferably, the kit further comprises antibodies obtained by immunizing an animal with the immunogen.

[0060] More preferably, the antibody is a polyclonal antibody.

[0061] Preferably, the kit further comprises an ELISA plate, a standard substance of buthiazine and a substrate color development solution.

[0062] More preferably, the ELISA plate is coated with the coating agent.

[0063] More preferably, the substrate color developing solution comprises urea peroxide and tetramethylbenzidine.

[0064] More preferably, the kit further comprises a stop solution, a washing solution, a blocking solution, an enzyme-labeled secondary antibody and an enzyme-labeled secondary antibody diluent.

[0065] More preferably, the volume fraction of the termination liquid is 8% to 12% H2SO4.

[0066] Most preferably, the ending liquid volume fraction is 10% H2SO4.

[0067] Further preferably, the washing solution is a phosphate buffer solution containing 0.5% to 1.0% Tween-20 by volume, 0.01% to 0.03% sodium azide preservative by mass, and 0.1 mol / L to 0.3 mol / L, with a pH value of 7.2 to 7.6.

[0068] Most preferably, the washing solution is a phosphate buffer solution containing 0.8% Tween-20 by volume, 0.02% sodium azide preservative by mass, and 0.2 mol / L, with a pH value of 7.4.

[0069] Further preferably, the blocking solution is a phosphate buffer solution containing 1% to 3% casein by mass, 0.1 mol / L to 0.3 mol / L, and a pH value of 7.1 to 7.5.

[0070] Most preferably, the blocking solution is a phosphate buffer solution containing 2% casein by mass, 0.2 mol / L, and a pH value of 7.3.

[0071] More preferably, the enzyme-labeled secondary antibody is a goat anti-rabbit antibody or goat anti-mouse antibody labeled with horseradish peroxidase.

[0072] Further preferably, the enzyme-labeled secondary antibody diluent is 0.1 mol / L to 0.3 mol / L phosphate buffer.

[0073] Most preferably, the enzyme-labeled secondary antibody diluent is 0.2 mol / L phosphate buffer.

[0074] Compared with the prior art, the present invention has the following beneficial effects:

[0075] The present invention provides a buthiazine hapten and prepares an antibody against buthiazine, which has high sensitivity for buthiazine detection and IC 50 The minimum detection limit (LOD (IC 10 )) was 3.11 ng / mL, with a linear detection range of 5.68 ng / mL-44.59 ng / mL, and strong specificity, with no obvious cross-reaction with the structural analogs of buthiazine. BRIEF DESCRIPTION OF THE DRAWINGS

[0076] Figure 1 This is the synthetic route of BTZ-hapten, a is BTZ-3C, b is BTZ-4C.

[0077] Figure 2 These are the UV scanning identification results of BTZ-3C-BSA artificial antigens, a is BTZ-3C-BSA, and b is BTZ-3C-OVA.

[0078] Figure 3 This is the standard curve of the indirect competition ELISA of butiazide polyclonal antibody. DETAILED DESCRIPTION

[0079] The present invention is further described in detail below with reference to the accompanying drawings and specific examples. The examples are intended only to illustrate the present invention and are not intended to limit the scope of the present invention. The experimental methods used in the following examples are conventional methods unless otherwise specified; the materials and reagents used are commercially available unless otherwise specified.

[0080] Example 1 Synthesis and Identification of Buthiazine Hapten

[0081] 1. Synthesis of Buthiazine Hapten BTZ-3C

[0082] The synthetic route of buthiazine hapten is as follows Figure 1 As shown in a, the specific steps are as follows:

[0083] 1. Weigh 379 mg (1 mmol) of trichloromethylthiazine into a 50 mL round-bottom flask, add 5 mL of chromatographic grade N,N-dimethylformamide (DMF) and stir until dissolved, then add 28.8 mg of sodium hydride (NaH), and stir in an ice bath at 0°C for 60 min;

[0084] 2. Add 254 μL (1.4 mmol) of ethyl bromopropionate and stir the reaction at room temperature for 12 h;

[0085] 3. After the reaction, the crude product was extracted with saturated sodium chloride solution to obtain the oil phase, which was then dried using a rotary evaporator. 15 mL of ethyl acetate and 50 g of silica gel powder were added, mixed, and dried. The mixture was then purified by column chromatography using a developing solvent (volume ratio of petroleum ether:ethyl acetate:triethylamine = 188:36:10) to obtain a pure intermediate product. The resulting target product was dissolved in 5 mL of methanol. 1 M dilute hydrochloric acid solution was added at a volume ratio of methanol to dilute hydrochloric acid solution of 1:1. The reaction was stirred at room temperature. The product was monitored for complete hydrolysis using a TLC plate. The reaction was terminated after the ester group of the product was completely hydrolyzed. The organic solvent in the resulting reaction solution was evaporated to dryness, the pH was adjusted to 5-6, an appropriate amount of saturated sodium chloride was added, and the mixture was extracted with ethyl acetate and water in a volume ratio of 1:1. The mixture was shaken thoroughly, and the organic phase was collected and dried over anhydrous sodium sulfate. The organic solvent was then removed by rotary evaporation to obtain the buthiazine hapten BTZ-3C.

[0086] 2. Identification of the Buthiazine Hapten BTZ-3C

[0087] The results of nuclear magnetic resonance hydrogen spectrum of buthiazine hapten are: 1H NMR (600MHz, Methanol-d4) δ

[0088] 8.12(s,1H),7.80(t,J=7.0Hz,1H),7.17(s,1H),6.84(d,J=5.9Hz,1H),6.64( d,J=4.8Hz,1H),5.91(s,0H),5.44(dd,J=6.0,5.0Hz,1H),3.20-3.10(m,2H),

[0089] 2.52 (t, J = 6.2 Hz, 2H).

[0090] The mass spectrometry results of buthiazine hapten are: MS: C 11 H 12 Cl3N3O6S2: 450.92, ESI-[MH]+: 451.35.

[0091] It can be seen from the mass spectrometry and nuclear magnetic resonance results that the mass spectrometry results correspond to the molecular weight of the buthiazine hapten and the hydrogen spectrum number of the nuclear magnetic resonance corresponds to the hydrogen spectrum number on the skeleton structure of the buthiazine hapten, indicating that the buthiazine hapten was successfully prepared and recorded as BTZ-3C. Its structural formula is shown in formula (I):

[0092]

[0093] 3. Synthesis of Buthiazine Hapten BTZ-4C

[0094] The synthetic route of BTZ-4C is as follows: Figure 1 As shown in b, the specific steps are basically the same as the synthesis steps of the buthiazine hapten BTZ-3C in this example, except that: in step 2, ethyl bromopropionate is replaced by ethyl bromobutyrate; in step 3, the developing solvent used for column chromatography is replaced by: petroleum ether: ethyl acetate: triethylamine = 188:28:10 by volume.

[0095] 4. Identification of the Buthiazine Hapten BTZ-4C

[0096] The H NMR spectrum of the buthiazine hapten was as follows: 1H NMR (600 MHz, Methanol-d4) δ 8.12 (s, 1H), 7.98 (t, J = 6.8 Hz, 1H), 7.43 (s, 1H), 6.84 (d, J = 5.9 Hz, 1H), 5.91 (s, 0H), 5.44 (d, J = 10.9 Hz, 0H), 3.11-2.99 (m, 2H), 2.37-2.28 (m, 2H), 1.89-1.78 (m, 2H).

[0097] The mass spectrometry results of buthiazine hapten are: MS: C 12 H 14Cl3N3O6S2, ESI-[MH]+: 466.04. The mass spectrometry and nuclear magnetic resonance results show that the mass spectrometry results correspond to the molecular weight of the butiazine hapten and the hydrogen spectrum number of the nuclear magnetic resonance corresponds to the hydrogen spectrum number of the butiazine hapten skeleton structure, indicating that the butiazine hapten was successfully prepared and recorded as BTZ-4C. Its structural formula is shown in formula (II):

[0098]

[0099] Example 2 Synthesis and Identification of Buthiazine Artificial Antigen

[0100] 1. Synthesis of Buthiazine Artificial Antigen

[0101] 1. BTZ-3C (5.7 mg, 0.012 mol) prepared in Example 1 was added to a brown reaction vial, and 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (EDC) (3.5 mg, 0.018 mol) and NHS (2.1 mg, 0.018 mmol) were added, dissolved in 100 μL DMF, and stirred at room temperature for 3 h to obtain a hapten activation solution;

[0102] 2. Dissolve BSA (10 mg, 0.00015 mol) in 1 mL of PBS buffer (0.01 mol / L, pH = 7.4) to obtain a BSA solution. Stir the BSA solution and hapten activation solution at 4°C overnight.

[0103] 3. Collect the reaction solution, transfer it to the treated dialysis bag, and then place it in 5L of 0.01M PBS buffer;

[0104] 4. Dialyze the cell suspension at 4°C for 3 days, replacing the buffer with pre-cooled 0.01 M PBS every 8 hours.

[0105] 5. After purification by dialysis, the artificial buthiazine antigen was obtained, which was recorded as BTZ-3C-BSA. After concentration determination and UV spectroscopy identification, it was stored in a -20°C refrigerator.

[0106] The above method was followed, with the only difference being that OVA was used instead of BSA to prepare the buthiazine artificial antigen, designated as BTZ-3C-OVA.

[0107] According to the above method, the only difference is that BTZ-4C is used to replace BTZ-3C, and BSA or OVA is used as the carrier protein to prepare the buthiazine artificial antigen, which is recorded as BTZ-4C-BSA and BTZ-4C-OVA respectively.

[0108] 2. Identification of Buthiazine Artificial Antigen

[0109] BSA, OVA, BTZ-3C, BTZ-3C-BSA and BTZ-3C-OVA were scanned and identified using the ultraviolet full wavelength method (200-350nm).

[0110] The identification results of BTZ-3C-BSA are as follows Figure 2 As shown in Figure a, by comparing the maximum absorbance values of each substance before and after coupling, it was found that the absorption curve of BTZ-3C-BSA was significantly different from that of the carrier protein BSA. BTZ-3C had a strong absorption peak above 343nm. After coupling with BSA, the absorption peak of BTZ-3C-BSA was close to that of BSA at 230nm and significantly higher than that of BSA at 280nm. The curve relative to the hapten BTZ-3C was significantly shifted. Since the dialysis process after coupling has completely removed all unreacted components, the characteristic peaks appearing in the coupling product are contributed by the protein-bound drug molecules, indicating that the reaction product is a complex of the carrier protein BSA and BTZ-3C.

[0111] The identification results of BTZ-3C-OVA are as follows Figure 2 As shown in (b), similarly, the absorption curve of BTZ-3C-BSA showed characteristic peaks different from those of OVA and BTZ-3C, indicating that the reaction product was a complex of the carrier protein OVA and BTZ-3C.

[0112] The above results show that the present invention successfully prepared the buthiazine artificial antigens BTZ-3C-BSA and BTZ-3C-OVA, whose structural formula is shown in formula (III):

[0113]

[0114] Wherein, Protein is carrier protein BSA or OVA.

[0115] BSA, OVA, BTZ-4C, BTZ-4C-BSA, and BTZ-4C-OVA were scanned and identified using the full-wavelength UV method (200-350 nm). The results were similar to those of BTZ-3C-BSA and BTZ-3C-OVA. This indicates that the present invention has successfully prepared the buthiazine artificial antigens BTZ-4C-BSA and BTZ-4C-OVA, whose structural formulas are shown in Formula (IV):

[0116]

[0117] Wherein, Protein is carrier protein BSA or OVA.

[0118] Example 3 Preparation of polyclonal antibodies against buthiazine

[0119] 1. Animal Immunization

[0120] Using the BTZ-3C-BSA prepared in Example 2 as the immunogen, BTZ-3C-BSA was diluted to 5 mg / mL with 0.01 mol / L PBS, and then mixed with an equal volume of Freund's complete adjuvant and fully emulsified to immunize 4-week-old New Zealand white rabbits. During the first immunization, two rabbits were inoculated subcutaneously at multiple points on the back of the neck. The antigen dose was 400 μg / rabbit, 0.8 ml per rabbit; 28 days later, the second immunization was performed, and the immunogen was emulsified with an equal volume of Freund's incomplete adjuvant. The immunization dose was the same as the first immunization dose, and the number of booster immunizations was 3. After the immunization, the serum was collected. The serum was purified by ammonium sulfate precipitation to obtain the buthiazine polyclonal antibody, which was frozen at -20°C for future use.

[0121] According to the above method, the difference is that BTZ-4C-BSA prepared in Example 2 is used as the immunogen to prepare buthiazine polyclonal antibodies.

[0122] 2. Sensitivity evaluation of polyclonal antibodies against butiazide (indirect competitive ELISA)

[0123] 1. Use BTZ-3C-OVA prepared in Example 2 as the coating source, dilute it with coating solution (0.05 M carbonate buffer solution, pH 9.6) to a concentration of 62.5 ng / mL, coat a 96-well ELISA plate at 100 μL / well, and incubate at 37°C overnight (12 h);

[0124] 2. Discard the coating solution, wash twice with PBST (0.01M PBS, 0.06% v / v Tween-20), and pat dry;

[0125] 3. Add 120 μL of blocking solution (PBST containing 1 wt% fish skin collagen) to each well and block at 37°C for 3 h;

[0126] 4. Discard the blocking solution, clap the plate, dry it at 37℃ for 30 minutes, then take it out and put it in a ziplock bag for later use;

[0127] 5. The buthiazine polyclonal antibody prepared using BTZ-3C-BSA as the immunogen was diluted with PBST at a volume ratio of 1:4000; the buthiazine standard was diluted with PBST to different concentrations to obtain buthiazine standard dilutions with concentrations of 10000 ng / mL, 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;

[0128] 6. Add 50 μL / well of different concentrations of butiazide standard dilutions (three sets in parallel), then add 50 μL / well of diluted butiazide polyclonal antibody, incubate at 37°C for 40 min, wash five times, and pat dry;

[0129] 7. Add 100 μL / well of goat anti-mouse secondary antibody-HRP (diluted 5000-fold with PBST), incubate at 37°C for 30 min, wash five times, and pat dry;

[0130] 8. Add 100 μL / well color development solution and develop color for 10 minutes;

[0131] 9. Add 50 μL of 10% H2SO4 solution to terminate the reaction and read the OD value at 450 nm;

[0132] 10. ELISA standard curve drawing: use B / B0 as the ordinate (B is the absorbance value OD450 of the standard of different concentrations of butiazine, B0 is the absorbance value OD450 of the blank control well), use the logarithm of the concentration of butiazine standard as the abscissa, use the logistic function to perform curve fitting, and obtain the standard curve formula. Figure 3 As shown, the half-inhibitory concentration (IC 50 ) was 15.92 ng / mL, and the minimum detection limit (LOD (IC 10 )) was 3.11 ng / mL, and the linear detection range was 5.68 ng / mL to 44.59 ng / mL.

[0133] Specificity evaluation of polyclonal antibodies against buthiazine

[0134] Hydrochlorothiazide, chlorothiazide and metolazone are butiazide analogs. The specificity of the butiazide polyclonal antibody prepared in this example was evaluated by cross-reaction experiments.

[0135] The sensitivity evaluation method described above was followed, with the only difference being that the buthiazide standard was replaced with hydrochlorothiazide, chlorothiazide, and metolazone standards, and the IC values of each structural analogue were obtained using the same dilution factor. 50 value.

[0136] The cross-reactivity rate (CR) of buthiazine was calculated according to the following formula: CR (%) = IC 50 (Buthiazine) / IC 50 (Structural analogues)×100%. The smaller the cross-reaction rate, the stronger the specificity.

[0137] Table 1 Cross-reaction results of butiazide polyclonal antibody with butiazide and its analogs

[0138]

[0139]

[0140] Note: NR means no reaction, which means the antibody does not recognize the analog.

[0141] As shown in Table 1, the cross-reactivity rate of the polyclonal antibody against butiazide was 100%, and the IC 50 The antibody for detecting butiazide was 15.92 ng / mL, with no cross-reactivity for hydrochlorothiazide, chlorothiazide, or metolazone. This indicates that the antibody used to detect butiazide has high recognition and specificity for butiazide, effectively eliminating interference from the butiazide analogs hydrochlorothiazide, chlorothiazide, and metolazone, and can be specifically used for the detection of butiazide.

[0142] The above results show that the butiazide polyclonal antibody prepared by the present invention has excellent detection performance for butiazide, high sensitivity and strong specificity.

[0143] Example 4 Combination of Immunogen and Coating Gen

[0144] The buthiazine artificial antigens BTZ-3C-OVA or BTZ-4C-OVA prepared in Example 2 were used as coating agents, and the buthiazine polyclonal antibodies prepared in Example 3 using BTZ-3C-BSA or BTZ-4C-BSA as immunogens were used. The serum titers and inhibition rates obtained by the indirect competitive ELISA method were used to evaluate the effects of different immunogen and coating agent combinations. The specific steps are as follows:

[0145] 1. Dilute the coating agent to a concentration of 1000 ng / mL with coating solution (0.05 M carbonate buffer, pH 9.6). Coat a 96-well ELISA plate at a rate of 100 μL / well. Incubate overnight in a 37°C constant temperature water bath. Discard the coating solution and wash twice with PBST (0.01 M PBS, 0.06% Tween-20 (v / v)).

[0146] 2. Add 120 μL of blocking solution (PBST containing 1 wt% fish skin collagen) to each well and block at 37°C for 3 h. Discard the blocking solution, clap the plate, and dry it in a drying oven at 37°C for later use.

[0147] 3. Dilute the buthiazine polyclonal antibody with PBST at volume ratios of 1:4000, 1:8000, 1:16000, 1:32000, 1:64000, 1:128000, and 1:256000. Set up blank control wells (replaced with PBST). Dilute the 1 mg / mL buthiazine standard 1000-fold with PBST to obtain a buthiazine standard dilution solution with a concentration of 1 μg / mL.

[0148] 4. Titer column setup: first add 50 μL PBST to each well, then add 50 μL of different dilutions of polyclonal antibody to each well, and finally add 50 μL PBST to the last well instead of antibody;

[0149] 5. Inhibition column setup: first add 50 μL of butiazine standard dilution to each well, then add 50 μL of butiazine polyclonal antibody at different dilution ratios to each well, and finally add 50 μL of PBST to the last well instead of antibody;

[0150] 6. Incubate at 37°C for 40 min, wash five times, and clap;

[0151] 7. Add goat anti-rabbit secondary antibody-HRP (diluted 5000 times with PBST), incubate at 37°C for 30 min, wash 5 times, and clap the plate;

[0152] 8. Add color developing solution and incubate at 37°C for 10 minutes;

[0153] 9. Add 10% v / v H2SO4 to terminate the reaction and read the OD value at 450 nm; calculate the titer and inhibition rate. The titer is the antibody dilution factor corresponding to an OD450 of approximately 1.0. The inhibition rate = (OD value of titer - OD value of inhibition) / OD value of inhibition × 100%.

[0154] The results of different combinations of immunogens and coating agents are shown in Table 2.

[0155] Table 2 ELISA test results of immunogen and coating

[0156]

[0157] As shown in Table 2, the antisera of mice using the artificial antigens BTZ-3C-BSA and BTZ-4C-BSA as immunogens have a certain titer, and the BTZ-3C-BSA immunogen shows a stronger recognition ability for both coatings. Among them, the combination of immunogen and coating No. 1 has a high titer (1:256000) and the highest inhibition rate (87.65%), which is the best combination of immunogen and coating. Under this combination, the polyclonal antibody can not only specifically recognize the target analyte butiazine, but also has good antibody sensitivity and can be used to specifically recognize the target analyte butiazine.

[0158] Example 5 An ELISA kit for detecting buthiazine

[0159] 1. Composition

[0160] (1) An ELISA plate coated with a coating agent is prepared by the following method:

[0161] The BTZ-3C-OVA prepared in Example 2 was used as a coating source, and the coating stock solution (0.05 M carbonate buffer solution, pH 9.6) was diluted to 750 ng / mL. 100 μL / well was added to coat a 96-well ELISA plate, and the plate was incubated at 37°C in the dark overnight. The liquid in the wells was poured out, and the plates were washed twice with the washing solution in this kit for 30 seconds each time, and patted dry. Then, 200 μL / well of the blocking solution in this kit was added, and the plates were incubated at 25°C in the dark for 2 hours. The liquid in the wells was poured out, patted dry, and after drying, they were vacuum-sealed with aluminum film for storage.

[0162] (2) Standards: 8 different concentrations of buthiazine standards, namely 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;

[0163] (3) Antibody: the polyclonal antibody against buthiazine prepared in Example 3;

[0164] (4) Enzyme-labeled secondary antibody: horseradish peroxidase-labeled goat anti-rabbit secondary antibody;

[0165] (5) Substrate color development solution: It consists of solution A and solution B. Solution A is urea peroxide and solution B is tetramethylbenzidine.

[0166] (6) Stop solution: 2 mol / L H2SO4;

[0167] (7) Washing solution: pH 7.4, containing 0.8% Tween-20 by volume, 0.02% sodium azide preservative by mass, and 0.2 mol / L phosphate buffer; dilute the washing solution 20 times with water before use (i.e., add 1 part washing solution to 19 parts water, ready for use) to obtain the working solution of washing solution;

[0168] (8) Diluent: 0.2 mol / L phosphate buffer; dilute the diluent 20 times with water before use (i.e., add 1 part diluent to 19 parts water, ready for use) to obtain the diluent working solution;

[0169] (9) Blocking solution: pH 7.3, 0.2 mol / L phosphate buffer containing 2% casein.

[0170] 2. Usage

[0171] (1) Sample testing

[0172] Number the corresponding microwells for the samples and the standard in this kit sequentially. Run two parallel wells for each sample and standard, and record the positions of the standard and sample wells. Dilute the antibody working solution with diluent at a 1:40 volume ratio (i.e., add 1 part antibody to 40 parts diluent working solution, freshly prepared before use) to obtain the antibody working solution. Dilute the enzyme-labeled secondary antibody working solution with diluent at a 1:10 volume ratio (i.e., add 1 part enzyme-labeled secondary antibody to 10 parts diluent working solution, freshly prepared before use) to obtain the enzyme-labeled secondary antibody working solution.

[0173] Add 50 μL of standard or sample to the corresponding microwells, then add 50 μL of antibody working solution to the corresponding microwells, gently shake to mix, cover the plate with a cover film, and react at 25°C in a dark environment for 40 minutes.

[0174] Shake wells dry and add 250 μL / well of working solution. Wash thoroughly 4-5 times, 10 seconds apart each time. Discard the working solution and pat dry with absorbent paper. (Any air bubbles that remain after patting dry can be popped with an unused pipette tip.)

[0175] Add 100 μL / well of enzyme-labeled secondary antibody working solution to the corresponding microwells, gently shake to mix, cover the plate with a cover film, and then react in a dark environment at 25°C for 30 minutes.

[0176] Shake wells dry and add 250 μL / well of working solution. Wash thoroughly 4-5 times, 10 seconds apart each time. Discard the working solution and pat dry with absorbent paper. (Any air bubbles that remain after patting dry can be popped with an unused pipette tip.)

[0177] Add 50 μL / well of substrate color development solution A, then add 50 μL / well of substrate color development solution B, gently shake to mix, cover the plate with cover film, and then react at 25°C in a dark environment for 10 minutes.

[0178] Add 50 μL / well of stop solution, shake gently to mix, set the microplate reader at 450 nm, and measure the OD value of each well.

[0179] (2) Drawing of standard curve

[0180] With B / B0 as the vertical axis (B is the absorbance value OD of the standard with different concentrations 450 , B0 is the absorbance value OD of the blank control well 450 ), with the logarithm of the concentration of the standard as the abscissa, a logistic function was used for curve fitting to obtain a standard curve and the formula of the standard curve was obtained.

[0181] (3) Calculation of sample concentration

[0182] The sample OD 450Substitute the average value into the formula of the standard curve to obtain the concentration of the sample, and then multiply it by the corresponding dilution factor to obtain the actual concentration of buthiazine in the test sample.

[0183] Finally, it should be noted that the above embodiments are intended only to illustrate the technical solutions of the present invention and are not intended to limit the scope of protection of the present invention. Those skilled in the art will readily appreciate that other variations or modifications may be made based on the above descriptions and concepts. It is not necessary and impossible to provide an exhaustive list of all possible implementations. Any modifications, equivalent substitutions, and improvements made within the spirit and principles of the present invention shall be included within the scope of protection of the claims of the present invention.

Claims

1. A buthiazine hapten, characterized in that Its structural formula is shown in formula (I), 2. Use of the compound represented by formula (I) in the preparation of buthiazine artificial antigens, 3. A buthiazine artificial antigen, characterized in that It is obtained by coupling the buthiazine hapten carrier protein according to claim 1, and its structural formula is shown in formula (III): Among them, Protein is a carrier protein.

4. A buthiazine hapten, characterized in that Its structural formula is shown in formula (II), 5. Use of the compound represented by the structural formula (II) in the preparation of buthiazine artificial antigens, 6. A buthiazine artificial antigen, characterized in that It is obtained by coupling the buthiazine hapten carrier protein according to claim 4, and its structural formula is shown in formula (IV): Among them, Protein is a carrier protein.

7. Use of the butiazide artificial antigen according to claim 3 or 6 in the preparation of antibodies for detecting butiazide.

8. A buthiazine artificial antigen combination, characterized in that: The invention comprises a coating agent and an immunogen, wherein the coating agent is obtained by coupling the butiazine hapten according to claim 1 or 4 with chicken egg albumin; and the immunogen is obtained by coupling the butiazine hapten according to claim 1 or 4 with bovine serum albumin.

9. Use of the butiazine artificial antigen combination according to claim 8 in detecting butiazine or in preparing products for detecting butiazine.

10. A kit for detecting buthiazine, characterized in that: Comprising the buthiazine artificial antigen combination according to claim 8.

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