4-Methaminoantipyrine hapten, its preparation method, artificial antigen, and applications

Abstract

This invention provides a 4-methylaminoantipyrine hapten, its preparation method, artificial antigen, and applications. The structural formula of the 4-methylaminoantipyrine hapten is shown in formula (I) or formula (II). The two 4-methylaminoantipyrine haptens provided by this invention have spacer arms of suitable length, allowing the haptens to be fully exposed to the immune system after coupling with a protein, while minimizing recognition of the spacer arm, resulting in superior antibody recognition of 4-methylaminoantipyrine. The 4-methylaminoantipyrine immunoassay detection method based on the artificial antigens and antibodies prepared from these two haptens exhibits high sensitivity and strong specificity.

Description

Technical Field

[0001] This invention belongs to the field of detection technology, specifically relating to a 4-methylaminoantipyrine hapten and its preparation method, and a 4-methylaminoantipyrine artificial antigen and its application. Background Technology

[0002] After entering the human body, aminopyrine is rapidly hydrolyzed to form the main active ingredient, 4-methylaminoantipyrine (chemical formula C10). 12 H 15 (N3O). 4-Methylaminoantipyrine has serious side effects. Due to its definite antipyretic and analgesic effects, low cost, and easy availability, it is often used excessively for extended periods. Furthermore, due to factors such as failure to strictly adhere to withdrawal periods, it easily accumulates in animals, thus endangering human health. Existing methods for detecting 4-methylaminoantipyrine have low sensitivity and poor specificity; therefore, there is an urgent need to develop a highly sensitive and specific method for detecting 4-methylaminoantipyrine. Summary of the Invention

[0003] To solve all or part of the above-mentioned technical problems, the present invention provides the following technical solutions:

[0004] A first aspect of the present invention provides a 4-methylaminoantipyrine hapten, the structural formula of which is shown in formula (I) or formula (II):

[0005] .

[0006] The two 4-methylaminoantipyrine haptens provided by this invention have spacer arms of suitable length. Therefore, after conjugation with a carrier protein, both haptens can be fully exposed while minimizing recognition of the spacer arm, resulting in superior antibody recognition of 4-methylaminoantipyrine. Specifically, the haptens with the structures shown in Formula (I) or (II) have a spacer arm of a certain length connecting the hapten target epitope and the carrier protein, thereby keeping the characteristic structure of the target molecule away from the carrier protein and maximizing the exposure of the hapten to the immune system. If the spacer arm of the hapten is too short, the carrier protein may mask the characteristic structure of the analyte, leading to a poor antibody response. If the spacer arm is too long, the hapten molecule may undergo conformational folding, thus being masked by the carrier protein, making it easier to generate antibodies against the spacer arm. The haptens with spacer arms of suitable length provided by this invention can expose the target molecule to the immune system while minimizing recognition of the spacer arm.

[0007] A second aspect of the present invention provides a method for preparing 4-methylaminoantipyrine hapten.

[0008] The preparation method of the 4-methylaminoantipyrine hapten with the structure shown in formula (I) includes:

[0009] A first mixed reaction system containing 4-aminoantipyrine, propionic acid, thionyl chloride and methanol was reacted at room temperature for a predetermined time, and then sodium borohydride was added. The reaction was continued at a temperature of 0~65℃ to obtain the first intermediate compound.

[0010] The second mixed reaction system containing the first intermediate compound and the Des Martin reagent was reacted at room temperature to obtain the 4-methylaminoantipyrine hapten with the structure shown in formula (I).

[0011] In some embodiments, the molar equivalents of 4-aminoantipyrine, propionic acid, thionyl chloride and sodium borohydride satisfy 1:1~2:0.05~0.5:2~10.

[0012] In some embodiments, the molar equivalent of 4-aminoantipyrine and methanol in the first mixed reaction system is 1:20-100.

[0013] In some embodiments, the preset time is 1 to 12 hours, and the reaction continues for 1 to 12 hours after the sodium borohydride is added.

[0014] In some embodiments, a post-processing step is included after the reaction of the first mixed reaction system is completed. The post-processing may include, for example, adding deionized water to quench the reaction after the reaction is complete, extracting with ethyl acetate; combining the organic phases and washing with saturated brine; adding silica gel powder to the organic phase and drying it using a rotary evaporator to obtain a dry sample containing the first intermediate compound; and purifying the sample using a column chromatography system to obtain the purified first intermediate compound.

[0015] In some embodiments, the molar equivalents of the first intermediate compound and the Des Martin reagent are 1:1 to 3.

[0016] In some embodiments, the solvent of the second mixed reaction system includes dichloromethane, tetrahydrofuran, 1,4-dioxane, etc., but is not limited to these.

[0017] In some embodiments, the second mixed reaction system is reacted at room temperature for 1 to 6 hours.

[0018] In some embodiments, a post-processing step is included after the reaction of the second mixed reaction system is completed. The post-processing may include, for example, adding a saturated sodium bicarbonate solution and shaking it vigorously after the reaction is completed, and then extracting it with ethyl acetate; combining the organic phases and washing them with saturated brine; adding silica gel powder to the organic phase and drying it using a rotary evaporator; purifying the dried silica gel powder containing the sample using a column chromatography system to obtain the purified 4-methylaminoantipyrine hapten with the structure shown in formula (I).

[0019] The preparation method of the 4-methylaminoantipyrine hapten with the structure shown in formula (II) includes:

[0020] The third mixed reaction system containing 4-methylaminoantipyrine, tert-butyl 4-bromobutyrate and an alkaline salt was reacted at a temperature of 70-100°C to obtain the second intermediate compound.

[0021] The fourth mixed reaction system containing the second intermediate compound and the acid was reacted at room temperature to obtain the 4-methylaminoantipyrine hapten with the structure shown in formula (II).

[0022] In some embodiments, the molar equivalents of 4-methylaminoantipyrine and tert-butyl 4-bromobutyrate are 1:1 to 2.

[0023] In some embodiments, the molar equivalent ratio of the 4-methylaminoantipyrine to the alkali salt is 1:1 to 3. The alkali salt is used to provide an alkaline environment, and may be, for example, potassium carbonate, but is not limited thereto.

[0024] In some embodiments, the third mixed reaction system is reacted at 70~100°C for 3~12 hours.

[0025] In some embodiments, the solvent of the third mixed reaction system includes one or more combinations of acetonitrile, DMF, and DMSO, but is not limited thereto.

[0026] In some embodiments, the molar ratio of the second intermediate compound to the acid is 1:3-6.

[0027] In some embodiments, the acid includes hydrochloric acid and / or trifluoroacetic acid, but is not limited thereto.

[0028] In some embodiments, after the reaction in the third mixed reaction system is completed, a post-processing step is further included. The post-processing includes, for example, adding deionized water after the reaction and extracting with ethyl acetate; combining the organic phases and washing with saturated brine; adding silica powder to the organic phase and drying it using a rotary evaporator; and purifying the dried silica powder containing the sample using a column chromatography system to obtain a purified second intermediate compound.

[0029] In some embodiments, the fourth mixed reaction system is reacted at room temperature for 1 to 3 hours.

[0030] In some embodiments, the solvent of the fourth mixed reaction system includes one or more combinations of 1,4-dioxane, tetrahydrofuran, and dichloromethane, but is not limited thereto.

[0031] In some embodiments, after the reaction in the fourth mixed reaction system is completed, a post-processing step is further included. The post-processing includes, for example, drying the reaction product using a rotary evaporator to obtain the 4-methylaminoantipyrine hapten with the structure shown in formula (II).

[0032] A third aspect of the present invention provides a 4-methylaminoantipyrine artificial antigen, wherein the 4-methylaminoantipyrine artificial antigen is obtained by conjugating a carrier protein with the 4-methylaminoantipyrine hapten as described in any of the technical solutions, and its structural formula is shown in formula (III) or formula (IV):

[0033] ;

[0034] Protein is a carrier protein.

[0035] The 4-methylaminoantipyrine artificial antigen of formula (III) is obtained by conjugating the 4-methylaminoantipyrine hapten of formula (I) to a carrier protein. The 4-methylaminoantipyrine artificial antigen of formula (IV) is obtained by conjugating the 4-methylaminoantipyrine hapten of formula (II) to a carrier protein. The conjugation method can be, for example, the active ester method.

[0036] In some embodiments, the carrier protein is any one of bovine serum albumin, ovalbumin, hemocyanin, lactoferrin, and human serum albumin.

[0037] In some embodiments, the 4-methylaminoantipyrine artificial antigen has the structural formula shown in formula (III) and the carrier protein is bovine serum albumin.

[0038] In some embodiments, the 4-methylaminoantipyrine artificial antigen has the structural formula shown in formula (Ⅳ) and the carrier protein is ovalbumin.

[0039] A fourth aspect of the present invention provides a 4-methylaminoantipyrine antibody, which is obtained by immunizing animals with the 4-methylaminoantipyrine artificial antigen described in any of the above-mentioned technical solutions.

[0040] In some embodiments, the 4-methylaminoantipyrine antibody is obtained by immunizing animals with the 4-methylaminoantipyrine artificial antigen of formula (III) and the carrier protein being bovine serum albumin.

[0041] In some embodiments, the antibody is a monoclonal antibody.

[0042] In some embodiments, the antibody is obtained from hybridoma cells.

[0043] A fifth aspect of the present invention provides a 4-methylaminoantipyrine artificial antigen combination, comprising an immunogen and a coating antigen, wherein the structure of the immunogen is shown in formula (III) and the carrier protein is bovine serum albumin, and the structure of the coating antigen is shown in formula (IV) and the carrier protein is ovalbumin.

[0044] The sixth aspect of the present invention provides the use of the 4-methylaminoantipyrine hapten, the 4-methylaminoantipyrine artificial antigen, the 4-methylaminoantipyrine antibody, and the 4-methylaminoantipyrine artificial antigen combination described in any one of the technical solutions in the preparation of 4-methylaminoantipyrine detection products or in the detection of 4-methylaminoantipyrine for the purpose of non-disease treatment diagnosis.

[0045] A seventh aspect of the present invention provides a kit for detecting 4-methylaminoantipyrine, comprising the 4-methylaminoantipyrine artificial antigen and the 4-methylaminoantipyrine antibody as described in any of the above-described technical solutions.

[0046] In some preferred embodiments, the 4-methylaminoantipyrine artificial antigen has the structure shown in formula (IV) and the carrier protein is ovalbumin, and the 4-methylaminoantipyrine antibody is obtained by immunizing animals with the 4-methylaminoantipyrine artificial antigen having the structure shown in formula (III) and the carrier protein being bovine serum albumin. Under these combined conditions, the monoclonal antibody not only specifically recognizes the target analyte 4-methylaminoantipyrine, but also exhibits good antibody sensitivity.

[0047] In some preferred embodiments, the 4-methylaminoantipyrine antibody is a monoclonal antibody, such as a monoclonal antibody obtained by immunizing an animal with an artificial antigen of formula (III) and a carrier protein of bovine serum albumin, thereby further obtaining better specificity.

[0048] In some embodiments, the kit is an enzyme-linked immunosorbent assay kit, i.e., an ELISA kit.

[0049] In some embodiments, the ELISA kit includes: an enzyme-labeled plate coated with the 4-methylaminoantipyrine artificial antigen and a 4-methylaminoantipyrine antibody, and further includes a 4-methylaminoantipyrine standard and a substrate chromogenic solution.

[0050] In some embodiments, the substrate developing solution contains urea peroxide and tetramethylbenzidine.

[0051] In some embodiments, the ELISA kit further includes one or more of the following: stop solution, washing solution, blocking solution, enzyme-labeled secondary antibody, and enzyme-labeled secondary antibody dilution solution.

[0052] In some embodiments, the terminating solution comprises 8% to 12% H2SO4 by volume. In some preferred embodiments, the terminating solution comprises 10% H2SO4 by volume.

[0053] In some embodiments, the washing solution comprises 0.5%–1.0% Tween-20 (v / v), 0.01%–0.03% sodium azide preservative (w / w), and 0.1 mol / L–0.3 mol / L phosphate buffer, with a pH of 7.2–7.6. In some preferred embodiments, the washing solution comprises 0.8% Tween-20 (v / v), 0.02% sodium azide preservative (w / w), and 0.2 mol / L phosphate buffer, with a pH of 7.4.

[0054] In some embodiments, the blocking solution comprises 1%–3% casein by mass and 0.1 mol / L–0.3 mol / L phosphate buffer, with a pH of 7.1–7.5. In some preferred embodiments, the blocking solution comprises 2% casein by mass and 0.2 mol / L phosphate buffer.

[0055] In some embodiments, the enzyme-labeled secondary antibody includes horseradish peroxidase-labeled goat anti-mouse antibody.

[0056] In some embodiments, the enzyme-labeled secondary antibody diluent comprises 0.1 mol / L to 0.3 mol / L phosphate buffer. In some preferred embodiments, the enzyme-labeled secondary antibody diluent comprises 0.2 mol / L phosphate buffer.

[0057] It should be noted that the substrate chromogenic solution, stop solution, washing solution, blocking solution, enzyme-labeled secondary antibody, and enzyme-labeled secondary antibody diluent in the ELISA kit can all be any known substances or formulations in the art. The components of the substrate chromogenic solution, stop solution, washing solution, blocking solution, enzyme-labeled secondary antibody, and enzyme-labeled secondary antibody diluent described above are merely exemplary and are not intended to limit the present invention.

[0058] The eighth aspect of the present invention provides an immunoassay method for detecting 4-methylaminoantipyrine, wherein the immunoassay method uses the 4-methylaminoantipyrine artificial antigen and the 4-methylaminoantipyrine antibody as described in any of the technical solutions for detection. The immunoassay method is for the purpose of non-disease treatment diagnosis.

[0059] In some preferred embodiments, the 4-methylaminoantipyrine artificial antigen has the structure shown in formula (Ⅳ) and the carrier protein is ovalbumin, and the 4-methylaminoantipyrine antibody is obtained by immunizing animals with an artificial antigen having the structure shown in formula (Ⅲ) and the carrier protein being bovine serum albumin.

[0060] In some preferred embodiments, the 4-methylaminoantipyrine antibody is a monoclonal antibody, such as a monoclonal antibody obtained by immunizing an animal with an artificial antigen of formula (III) and a carrier protein of bovine serum albumin, thereby further obtaining better specificity.

[0061] In some embodiments, the immunoassay method is an enzyme-linked immunosorbent assay (ELISA), which can be either an indirect ELISA or a competitive ELISA. In some embodiments, an indirect competitive ELISA is preferred.

[0062] Compared with the prior art, the present invention has at least the following beneficial effects:

[0063] (1) The two 4-methylaminoantipyrine haptens provided by this invention have spacer arms of suitable length, which allows the haptens to be fully exposed to the immune system after being coupled with the protein, while minimizing the recognition of the spacer arms, resulting in better antibody recognition of 4-methylaminoantipyrine. The 4-methylaminoantipyrine immunoassay detection method based on the artificial antigens and antibodies prepared from these two haptens has high sensitivity and strong specificity.

[0064] (2) The ELISA detection method based on the artificial antigen and antibody of 4-methylaminoantipyrine provided in this invention has high sensitivity and strong specificity. The limit of detection (LOD) is 0.19 ng / mL, the half-inhibitory concentration (IC50) is 4.02 ng / mL, and the quantitative detection range is 0.58~28.00 ng / mL. It has no obvious cross-reactivity with similar drugs of 4-methylaminoantipyrine. Attached Figure Description

[0065] To more clearly illustrate the technical solutions in the embodiments of this application or the prior art, the drawings used in the description of the embodiments or the prior art will be briefly introduced below. Obviously, the drawings described below are only some embodiments recorded in this application. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort.

[0066] Figure 1 The image shows the UV scanning identification results of 4-methylaminoantipyrine hapten MAA-1, bovine serum albumin BSA, and 4-methylaminoantipyrine artificial antigen MAA-1-BSA in the embodiments of the present invention.

[0067] Figure 2 The image shows the UV scanning identification results of 4-methylaminoantipyrine hapten MAA-2, ovalbumin OVA, and 4-methylaminoantipyrine artificial antigen MAA-2-OVA in the embodiments of the present invention.

[0068] Figure 3 This document describes a standard curve for detecting 4-methylaminoantipyrine using an indirect competitive ELISA method constructed based on the 4-methylaminoantipyrine artificial antigen and monoclonal antibody described in this invention. Detailed Implementation

[0069] The technical solutions of the present invention will be described in detail below with reference to specific embodiments, so that those skilled in the art can better understand and implement the technical solutions of the present invention. The specific functional details disclosed herein should not be construed as limiting, but are merely intended to form the basis of the claims and to teach those skilled in the art to employ the representative basis of the invention in different ways in any suitable detailed embodiment.

[0070] In addition, unless otherwise specified, all raw materials used in the following embodiments can be purchased from the market or other sources, and all production and testing equipment used are known in the art, as are the testing methods used.

[0071] Unless otherwise specified, the raw material "equivalent" used in the embodiments of the present invention refers to molar equivalent.

[0072] Example 1

[0073] This embodiment provides a 4-methylaminoantipyrine hapten and its synthesis method, and identifies the synthesized hapten.

[0074] Synthesis and Identification of 1,4-Methaminoantipyrine Hapten MAA-1

[0075] (1) The synthetic route of the 4-methylaminoantipyrine hapten MAA-1 is as follows:

[0076]

[0077] Specifically, the steps include the following:

[0078] One equivalent of 4-aminoantipyrine was dissolved in 15 volumes of methanol, and one equivalent of propionic acid and 0.05 equivalents of thionyl chloride were added. The mixture was stirred at room temperature for 2 hours. After the reaction was complete, 5 equivalents of sodium borohydride were added to the reaction mixture in five equal portions, and the mixture was refluxed overnight. After the reaction was complete, deionized water was added to quench the reaction, and the mixture was extracted with ethyl acetate. The organic phases were combined and washed with saturated brine. 15 volumes of silica gel powder were added to the organic phase, and the mixture was evaporated to dryness using a rotary evaporator. The dried silica gel powder containing the sample was purified using a column chromatography system (petroleum ether: ethyl acetate: formic acid: 60:90:1).

[0079] One equivalent of the purified product was dissolved in 15 volumes of dichloromethane, and 1.2 equivalents of Desmond-Martin reagent were added. The mixture was reacted overnight at room temperature. After the reaction was complete, 15 volumes of saturated sodium bicarbonate solution were added and the mixture was shaken vigorously for 1 minute, followed by extraction with ethyl acetate. The organic phases were combined and washed with saturated brine. 15 volumes of silica gel powder were added to the organic phase, and the mixture was evaporated to dryness using a rotary evaporator. The dried silica gel containing the sample was purified using a column chromatography system (petroleum ether: ethyl acetate: formic acid: 30:90:1). This yielded the 4-methylaminoantipyrine hapten MAA-1.

[0080] (2) Identification of 4-methylaminoantipyrine hapten MAA-1

[0081] The 1H NMR spectrum of the 4-methylaminoantipyrine hapten MAA-1 is as follows: 1 H NMR (600 MHz, DMSO- d 6) δ 9.63 (t, J = 5.7 Hz, 1H), 7.63 – 7.45 (m, 2H), 7.41 – 7.22 (m,4H), 3.42 (q, J = 5.6 Hz, 2H), 3.00 (s, 3H), 2.45 (q, J = 5.7 Hz, 2H), 2.18 (s, 3H).

[0082] The mass spectrometry results of the 4-methylaminoantipyrine hapten MAA-1 are as follows: + ESI-MS + [MH] + 298.11.

[0083] The mass spectrometry and NMR results show that the mass spectrometry results correspond to the molecular weight of the 4-methylaminoantipyrine hapten, and the number of proton NMR spectra corresponds to the number of proton NMR spectra on the 4-methylaminoantipyrine hapten backbone structure. This indicates that the 4-methylaminoantipyrine hapten, denoted as MAA-1, was successfully prepared, and its structural formula is shown in formula (I).

[0084] .

[0085] Synthesis and Identification of 2,4-Methaminoantipyrine Hapten MAA-2

[0086] (1) The synthetic route of the 4-methylaminoantipyrine hapten MAA-2 is as follows:

[0087]

[0088] Specifically, the steps include the following:

[0089] Dissolve 1 equivalent of 4-methylaminoantipyrine in 15 volumes of acetonitrile, add 1.2 equivalents of potassium carbonate and 1.2 equivalents of tert-butyl 4-bromoacetate, and reflux overnight. After the reaction is complete, add 15 volumes of deionized water and extract with ethyl acetate. Combine the organic phases and wash with saturated brine. Add 15 volumes of silica gel powder to the organic phase and evaporate to dryness using a rotary evaporator. Purify the dried silica gel containing the sample using a column chromatography system (petroleum ether: ethyl acetate: formic acid: 50:80:1).

[0090] The purified product was dissolved in 15 times its volume of 1,4-dioxane, and then 3 mL of hydrochloric acid was added. The mixture was stirred overnight at room temperature. After the reaction was complete, the product was evaporated to dryness using a rotary evaporator to obtain the 4-methylaminoantipyrine hapten MAA-2.

[0091] (2) Identification of 4-methylaminoantipyrine hapten MAA-2

[0092] The 1H NMR spectrum of the 4-methylaminoantipyrine hapten is as follows: 1 H NMR (600 MHz, DMSO- d 6) δ 12.59 (s, 1H), 7.48 (t, J = 7.7 Hz, 2H), 7.40 – 7.16 (m, 3H), 4.06 (s, 2H), 3.15 (s, 3H), 3.01 (s, 3H), 2.25 (s, 3H).

[0093] The mass spectrometry results of the 4-methylaminoantipyrine hapten are as follows: + ESI-MS + [MH] + 274.17.

[0094] The mass spectrometry and NMR results show that the mass spectrometry results correspond to the molecular weight of the 4-methylaminoantipyrine hapten, and the number of protons in the NMR spectrum corresponds to the number of protons in the 4-methylaminoantipyrine hapten backbone structure. This indicates that the 4-methylaminoantipyrine hapten, denoted as MAA-2, was successfully prepared, and its structural formula is shown in formula (II).

[0095] .

[0096] Example 2

[0097] Example 2 synthesizes 4-methylaminoantipyrine artificial antigen based on the 4-methylaminoantipyrine semi-antibody MAA-1 and MAA-2 synthesized in Example 1, and the synthesized artificial antigen can be identified.

[0098] Synthesis of 1,4-methylaminoantipyrine artificial antigen MAA-1-BSA

[0099] (1) The MAA-1 (3.156 mg, 0.012 mol) prepared in Example 1 was added to a brown reaction vial, and PBS buffer (0.01 mol / L, pH=7.4) containing 10 mg BSA (bovine serum albumin) was added. Two equivalents of sodium borohydride were added and the reaction was carried out overnight at 4°C.

[0100] (2) Collect the reaction solution, transfer it to the prepared dialysis bag, and then put it into 5 L of 0.01 M PBS buffer; dialyze at 4 ℃ for 3 days, and change the solution with pre-cooled 0.01 M PBS buffer every 8 hours.

[0101] (3) After dialysis purification, 4-methylaminoantipyrine artificial antigen, denoted as MAA-1-BSA, was obtained. After concentration determination and ultraviolet spectral scanning identification, it was stored in a -20 ℃ refrigerator.

[0102] 2. Synthesis of 4-methylaminoantipyrine artificial antigen MAA-2-OVA

[0103] (1) The MAA-2 (3.156 mg, 0.012 mol) prepared in Example 1 was added to a brown reaction vial, and EDC (3.5 mg, 0.018 mol) and NHS (2.1 mg, 0.018 mmol) were added and dissolved in 100 μL DMF. The mixture was stirred at room temperature for 3 h to obtain the hapten activation solution.

[0104] (2) Dissolve ovalbumin OVA (10 mg, 0.00015 mol) in 1 mL PBS buffer (0.01 mol / L, pH=7.4) to obtain OVA solution. Stir the OVA solution and hapten activation solution at 4 °C overnight.

[0105] (3) Collect the reaction solution, transfer it to the prepared dialysis bag, and then put it into 5 L of 0.01 M PBS buffer; dialyze at 4 ℃ for 3 days, and change the solution with pre-cooled 0.01 M PBS buffer every 8 hours.

[0106] (4) After dialysis purification, 4-methylaminoantipyrine artificial antigen was obtained, denoted as MAA-2-OVA. After concentration determination and ultraviolet spectral scanning identification, it was stored in a -20 ℃ refrigerator.

[0107] 3. Identification of 4-methylaminoantipyrine artificial antigen

[0108] BSA, OVA, MAA-1, MAA-2, MAA-1-BSA, and MAA-2-OVA were identified by scanning using the ultraviolet full-wavelength method (200–350 nm).

[0109] Figure 1 The image shows the UV scanning identification results of 4-methylaminoantipyrine hapten MAA-1, bovine serum albumin (BSA), and 4-methylaminoantipyrine artificial antigen MAA-1-BSA. By comparing the highest absorbance values ​​of each substance before and after conjugation, it was found that the absorption curve of MAA-1-BSA is significantly different from that of the carrier protein BSA. MAA-1 has a strong absorption peak above 343 nm, while after conjugation with BSA, the absorption peak of MAA-1-BSA is similar to that of BSA at 230 nm, but significantly higher than that of BSA at 280 nm, and the curve relative to the hapten MAA-1 shows a significant shift. Since all unreacted components were removed by dialysis after conjugation, the characteristic peaks of the conjugation product are contributed by the protein-bound drug molecules, indicating that the reaction product is a complex of the carrier protein BSA and MAA.

[0110] Figure 2 The image shows the UV scanning identification results of 4-methylaminoantipyrine hapten MAA-2, ovalbumin OVA, and 4-methylaminoantipyrine artificial antigen MAA-2-OVA. Similarly, the absorption curve of MAA-2-OVA shows characteristic peaks that are different from those of OVA and MAA-2, indicating that the reaction product is a complex of carrier protein OVA and MAA-2.

[0111] The above results demonstrate that the present invention successfully prepared 4-methylaminoantipyrine artificial antigens MAA-1-BSA (its structural formula is shown in formula (III), where protein is BSA) and MAA-2-OVA (its structural formula is shown in formula (IV), where protein is OVA):

[0112] .

[0113] Example 3

[0114] Example 3 uses the MAA-1-BSA obtained in Example 2 as an immunogen to prepare 4-methylaminoantipyrine monoclonal antibody.

[0115] 1. Animal immunization

[0116] Using MAA-1-BSA prepared in Example 2 as the immunogen, MAA-1-BSA was diluted to 5 mg / mL with 0.01 mol / L PBS, then mixed with an equal volume of Freund's complete adjuvant and thoroughly emulsified. This mixture was then used to immunize 8-week-old female BALB / c mice. For the first immunization, three female BALB / c mice were subcutaneously inoculated at multiple sites in the abdomen, with an antigen dose of 100 μg / mouse, administered in 0.1 ml per mouse. A second immunization was performed 14 days later, emulsified with an equal volume of Freund's incomplete adjuvant, using the same dose as the first immunization. Three booster immunizations were administered. After immunization, tail serum was collected. Its titer and inhibition rate were determined. Mice with the best results were selected for a pulse immunization, with an antigen dose of 100 μg / mouse.

[0117] 2. Cell fusion

[0118] Three days after the sprint immunization, cell fusion was performed using the standard PEG (polyethylene glycol, molecular weight 1450) method, with the following specific steps:

[0119] a. After euthanizing mice by blood collection from the eyeballs, immediately disinfect them in 75% alcohol for about 5 minutes. Aseptically remove the spleen of the mice, grind it moderately with the rubber tip of a syringe and pass it through a 200-mesh cell sieve to obtain a spleen cell suspension. Collect the suspension, centrifuge (1000 rpm, 7 min), wash the spleen cells three times with RPMI-1640 medium, and after the last centrifugation, dilute the spleen cells to a certain volume, count them, and set them aside for later use.

[0120] b. Collection of SP2 / 0 cells: 7-10 days before fusion, SP2 / 0 tumor cells were cultured in RPMI 1640 medium containing 10% FBS (fetal bovine serum) in a 5% CO2 incubator. The number of SP2 / 0 tumor cells should reach (1-4) × 10⁻⁶ cells before fusion. 7 To ensure that SP2 / 0 tumor cells are in the logarithmic growth phase before fusion, tumor cells are collected and suspended in RPMI-1640 basal culture medium for cell counting during fusion.

[0121] c. Mix the two cells together, bring the volume to 15 mL, centrifuge (1000 rpm / min, 7 min), and discard the supernatant.

[0122] Remove the centrifuge tubes, rotate them in a circular motion at a medium speed of 60°, and then begin the fusion process. The fusion time should be controlled as follows:

[0123] First minute: Slowly add PEG dropwise;

[0124] Second minute: Continue rotating (1 minute);

[0125] Third minute: Add 1 mL of basal culture medium;

[0126] Fourth minute: Add 3 mL of basal culture medium;

[0127] Fifth minute: Add 8 mL of basal culture medium;

[0128] Sixth minute: Add 8 mL of basal culture medium;

[0129] After fusion, the cell culture was centrifuged (1000 rpm / min, 7 min), and the 200 mL HAT culture medium was removed and placed in a clean bench for later use.

[0130] d. After centrifuging the hybridoma cells, discard the supernatant, aspirate 2 mL of HAT medium into a centrifuge tube, gently and slowly pipette to mix evenly, then transfer the cell solution in the centrifuge tube to 200 mL of HAT medium, gently shake the medium to disperse the cells evenly, and then plate them.

[0131] 3. Cell selection and cell line establishment:

[0132] On day 3 of cell fusion, the fused cells were screened with RPMI-1640 medium with a half-replacement. On day 5, the medium was completely replaced with RPMI-1640 transition medium containing 20% ​​fetal bovine serum and 2% 50×HT. On day 7, the cell supernatant was collected for screening.

[0133] The screening process consisted of two steps: First, positive cell wells were selected using ic-ELISA. Second, 4-methylaminoantipyrine was used as a standard, and the inhibitory effect on positive cells was determined using ic-ELISA. Cell wells that showed good inhibition of the 4-methylaminoantipyrine standard were selected and subcloned using the limiting dilution method. The cells were then tested using the same method after seven days. Subcloning was performed three times using the above method to finally obtain the 4-methylaminoantipyrine monoclonal antibody cell line.

[0134] 4. Preparation and identification of 4-methylaminoantipyrine monoclonal antibody

[0135] BALB / c mice aged 8-10 weeks were injected intraperitoneally with 1 mL of sterile paraffin oil; 7 days later, each mouse was injected intraperitoneally with 1×10 6Analgin hybridoma cells were used, and ascites fluid was collected starting on day 7. The ascites fluid was then purified using immunoaffinity chromatography. Under neutral conditions, Protein G bound to IgG immunoglobulins in the ascites fluid, causing other impurities to flow out and be discarded. Then, IgG was desorbed from Protein G under acidic conditions using 0.01 M glycine buffer to obtain high-purity IgG immunoglobulins. The pH of the IgG immunoglobulin solution was adjusted to neutral using 0.01 M Tris-HCl buffer, followed by dialysis to desalt, ultimately yielding high-purity 4-methylaminoantipyrine monoclonal antibody. The purified monoclonal antibody was stored at -20 °C.

[0136] 5. Sensitivity evaluation of 4-methylaminoantipyrine monoclonal antibody (indirect competitive ELISA method)

[0137] The MAA-2-OVA prepared in Example 2 was used as the coating agent. It was diluted with coating buffer (0.05 M carbonate buffer, pH 9.6) to a concentration of 500 ng / mL. 100 μL / well was used to coat a 96-well microplate and incubated at 4 °C overnight (12 h).

[0138] Discard the coating solution, wash twice with PBST (0.01 M PBS, 0.06 % v / v Tween-20), and blot dry; add 120 μL of blocking solution (PBST containing 7% skim milk powder) to each well, and block at 37 ℃ for 1 h; discard the blocking solution, blot the plate, dry at 37 ℃, and then pack it in a resealable bag for later use.

[0139] 4-Methaminoantipyrine monoclonal antibody prepared using MAA-1-BSA as an immunogen was diluted with PBST at a volume ratio of 1:128000. 4-Methaminoantipyrine standard was diluted with PBST to different concentrations, yielding 4-Methaminoantipyrine standard dilutions with concentrations of 10000 ng / mL, 5000 ng / mL, 500 ng / mL, 80 ng / mL, 15 ng / mL, 5 ng / mL, 1 ng / mL, 0.1 ng / mL, 0.05 ng / mL, 0.01 ng / mL, 0.001 ng / mL, and 0 ng / mL.

[0140] Add 50 μL / well of 4-methylaminoantipyrine standard dilution buffer at different concentrations (three sets in parallel), then add 50 μL / well of diluted 4-methylaminoantipyrine monoclonal antibody, incubate at 37 ℃ for 40 min, wash five times, and pat dry;

[0141] Add 100 μL / well of goat anti-mouse secondary antibody-HRP (diluted 5000 times with PBST), incubate at 37 ℃ for 30 min, wash five times, and pat dry;

[0142] Add 100 μL / well of colorimetric reagent and develop for 10 min;

[0143] The reaction was terminated by adding 50 μL of 10% H2SO4 solution, and the OD value was read at 450 nm.

[0144] ELISA standard curve plotting: Plot B / B0 as the ordinate (B is the absorbance OD of different concentrations of 4-methylaminoantipyrine standards). 450 B0 is the absorbance value (OD) of the blank control well. 450 The standard curve was obtained by fitting the logarithm of the concentration of 4-methylaminoantipyrine standard to the logarithm on the x-axis using the Logistic function, and the formula for the standard curve was derived.

[0145] Figure 3 This is a standard curve of the indirect competitive ELISA method constructed based on the aforementioned 4-methylaminoantipyrine artificial antigen and monoclonal antibody, as shown below. Figure 3 As shown, the limit of detection (LOD) was 0.19 ng / mL, and the half-inhibitory concentration (IC50) was [missing value]. 50 The concentration was 4.02 ng / mL, and the quantitative detection range was 0.58~28.00 ng / mL.

[0146] 6. Specificity evaluation of 4-methylaminoantipyrine monoclonal antibody

[0147] Naproxen, ibuprofen, and piroxicam are analogues of 4-methylaminoantipyrine. The specificity of the 4-methylaminoantipyrine monoclonal antibody prepared in this embodiment was evaluated by cross-reactivity assay.

[0148] Following the sensitivity evaluation method described above, the only difference was that the 4-methylaminoantipyrine standard was replaced with naproxen, ibuprofen, and piroxicam standards, and the detection was performed at the same dilution factor to obtain the IC50 values ​​for each structural analog. 50 value.

[0149] The cross-reactivity rate (CR) was calculated using the following formula: CR (%) = IC50 (4-methylaminoantipyrine) / IC50 (similar drug) × 100%. A lower cross-reactivity rate indicates higher specificity, as shown in Table 1.

[0150] Table 1. Cross-reactivity results of monoclonal antibodies with 4-methylaminoantipyrine and its analogues

[0151] ;

[0152] Note: NR in Table 1 indicates no reaction, meaning the antibody does not recognize the analogue.

[0153] As shown in Table 1, the cross-reactivity of the 4-methylaminoantipyrine monoclonal antibody with 4-methylaminoantipyrine was 100%, with an IC50 of 4.02 ng / mL. No cross-reactivity was observed with naproxen, ibuprofen, or piroxicam. This indicates that the antibody used for detecting 4-methylaminoantipyrine has high recognition ability and specificity, effectively eliminating interference from the detection of 4-methylaminoantipyrine analogs such as naproxen, ibuprofen, and piroxicam, and can be specifically used for the detection of 4-methylaminoantipyrine.

[0154] The above results indicate that the 4-methylaminoantipyrine monoclonal antibody prepared in this invention exhibits excellent detection performance, high sensitivity, and strong specificity for 4-methylaminoantipyrine.

[0155] Example 4

[0156] Example 4 provides a 4-methylaminoantipyrine artificial antigen combination.

[0157] Using the 4-methylaminoantipyrine artificial antigen MAA-2-OVA prepared in Example 2 as the coating antigen, and the 4-methylaminoantipyrine monoclonal antibody prepared in Example 3 using MAA-1-BSA as the immunogen, the combined effects of different immunogens and coating antigens were evaluated by serum titers and inhibition rates obtained through an indirect competitive ELISA method. The specific operating steps are as follows:

[0158] 1. Dilute the coating agent to a concentration of 1000 ng / mL with coating buffer (0.05 M carbonate buffer, pH 9.6), add 100 μL / well to coat a 96-well microplate, incubate overnight at 4 ℃, discard the coating buffer, and wash twice with PBST (0.01 M PBS, 0.06% Tween-20 (v / v)).

[0159] 2. Add 120 μL of blocking buffer (PBST containing 7% skim milk powder) to each well, block at 37 ℃ for 1 h, discard the blocking buffer, plate, and dry in a drying oven at 37 ℃ for later use;

[0160] 3. Dilute the 4-methylaminoantipyrine monoclonal antibody with PBST at volume ratios of 1:4000, 1:8000, 1:16000, 1:32000, 1:64000, 1:128000, and 1:256000, and set up blank control wells (using PBST instead); dilute 1 mg / mL 4-methylaminoantipyrine standard 1000 times with PBST to obtain a 1 μg / mL 4-methylaminoantipyrine standard dilution solution;

[0161] 4. Potency column setup: First, add 50 μL of PBST to each well, then add 50 μL of 4-methylaminoantipyrine monoclonal antibody at different dilution ratios to each well, and finally add 50 μL of PBST to the last well instead of the antibody.

[0162] 5. Inhibition column setup: First, add 50 μL of 4-methylaminoantipyrine standard dilution buffer to each well, then add 50 μL of 4-methylaminoantipyrine monoclonal antibody at different dilution ratios to each well, and finally add 50 μL of PBST to the last well instead of antibody.

[0163] 6. Incubate at 37℃ for 40 minutes, wash 5 times, and then plate.

[0164] 7. Add goat anti-mouse secondary antibody-HRP (diluted 5000 times with PBST), incubate at 37 ℃ for 30 min, wash 5 times, and plate.

[0165] 8. Add the color developing solution and incubate at 37 ℃ for 10 min;

[0166] 9. Terminate the reaction by adding 10% v / v H2SO4 and read the OD value at 450 nm; calculate the titer and inhibition rate. The titer is the OD value. 450 The antibody dilution factor corresponding to approximately 1.0 is: inhibition rate = (OD value of titer - OD value of inhibition) / OD value of inhibition × 100%.

[0167] In addition, this invention also uses MAA-1 conjugated with OVA and LF (lactoferrin), and MAA-2 conjugated with LF to prepare artificial antigens MAA-1-OVA, MAA-1-LF, and MAA-2-LF, respectively. Different combinations of immunogens and coating antigens are constructed and evaluated according to the same method as above. The results are shown in Table 2.

[0168] Table 2. ELISA detection results of immunogens and coating antigens.

[0169] ;

[0170] As shown in Table 2, the monoclonal antibody prepared using the artificial antigen MAA-1-BSA of 4-methylaminoantipyrine as an immunogen and MAA-2-OVA is the optimal combination of immunogen and coating agent. Under this combination, the monoclonal antibody can not only specifically recognize the target analyte 4-methylaminoantipyrine, but also has good antibody sensitivity and can be used to specifically recognize the target analyte 4-methylaminoantipyrine.

[0171] Example 5

[0172] Example 5 provides an ELISA kit for detecting 4-methylaminoantipyrine.

[0173] 1. Components of an ELISA kit

[0174] (1) The enzyme-labeled plate coated with the coating agent is prepared by the following method:

[0175] The MAA-2-OVA prepared in Example 2 was used as the coating agent. It was diluted to 500 ng / mL with the coating stock solution (0.05 M carbonate buffer, pH 9.6). 100 μL of the solution was added to coat 96-well microplates and incubated overnight at 4 °C in the dark. The liquid in the wells was discarded, and the plates were washed twice with the washing buffer provided in this kit for 30 s each time. The plates were then patted dry. Then, the blocking buffer provided in this kit was added at 120 μL / well and incubated at 37 °C in the dark for 1 h. The liquid in the wells was discarded, and the plates were patted dry. After drying, the plates were vacuum sealed with aluminum foil for storage.

[0176] (2) Standards: 12 different concentrations of 4-methylaminoantipyrine standards at concentrations of 10000 ng / mL, 5000 ng / mL, 500 ng / mL, 80 ng / mL, 15 ng / mL, 5 ng / mL, 1 ng / mL, 0.1 ng / mL, 0.05 ng / mL, 0.01 ng / mL, 0.001 ng / mL and 0 ng / mL.

[0177] (3) Antibody: 4-methylaminoantipyrine monoclonal antibody prepared using MAA-1-BSA as an immunogen in Example 3;

[0178] (4) Enzyme-labeled secondary antibody: Horseradish peroxidase-labeled goat anti-mouse secondary antibody;

[0179] (5) Substrate colorimetric solution: composed of solution A and solution B, where solution A is urea peroxide and solution B is tetramethylbenzidine;

[0180] (6) Termination solution: 2 mol / L H2SO4;

[0181] (7) Washing solution: pH value is 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, prepare fresh before use) to obtain the washing solution working solution;

[0182] (8) Diluent: 0.2 mol / L phosphate buffer; dilute the diluent 20 times with water before use (i.e., add 1 part of diluent to 19 parts of water, prepare fresh before use) to obtain the working solution of the diluent;

[0183] (9) Blocking solution: pH 7.3, 0.2 mol / L phosphate buffer containing 2% casein.

[0184] 2. Use the above ELISA kit to test the samples.

[0185] (1) Sample testing

[0186] Number the wells of the samples and standards from the ELISA kit sequentially, performing two parallel wells for each sample and standard, and record the positions of the standard and sample wells. Dilute the antibody with the required amount of diluent at a 1:40 volume ratio (i.e., add 1 part antibody to 40 parts diluent, prepare fresh before use) to obtain the antibody working solution. Dilute the enzyme-labeled secondary antibody with the required amount of diluent at a 1:10 volume ratio (i.e., add 1 part enzyme-labeled secondary antibody to 10 parts diluent, prepare fresh before use) to obtain the enzyme-labeled secondary antibody working solution.

[0187] Add 50 μL of standard or sample to the corresponding well, then add 50 μL of antibody working solution to the corresponding well, gently shake to mix, cover with a cover plate and incubate at 25 ℃ in the dark for 40 min.

[0188] 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).

[0189] Add 100 μL / well of enzyme-labeled secondary antibody working solution to the corresponding microwell, gently shake to mix, cover with a cover plate membrane, and incubate at 25 ℃ in the dark for 30 min.

[0190] 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).

[0191] 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.

[0192] Add 50 μL of stop solution per well, gently shake to mix, set the microplate reader to 450 nm, and measure the OD value of each well.

[0193] (2) Drawing the standard curve

[0194] 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. 450The standard curve was obtained by using the logarithm of the concentration of the standard as the abscissa and performing curve fitting with the Logistic function.

[0195] (3) Calculation of the concentration of 4-methylaminoantipyrine in the sample

[0196] OD of the sample 450 Substituting the average value into the formula of the standard curve above, we can obtain the concentration of 4-methylaminoantipyrine in the sample. Multiplying this by the corresponding dilution factor gives the actual concentration of 4-methylaminoantipyrine in the test sample.

[0197] In summary, this invention provides two 4-methylaminoantipyrine haptens with appropriately sized spacer arms, and uses them to prepare 4-methylaminoantipyrine artificial antigens and antibodies. The obtained 4-methylaminoantipyrine monoclonal antibodies exhibit high titer, strong specificity, and high affinity, providing core raw materials for establishing a specific immunoassay method for 4-methylaminoantipyrine. The immunoassay method based on the 4-methylaminoantipyrine artificial antigen and antibody provided by this invention has high specificity and high sensitivity, with a limit of detection (LOD) of 0.19 ng / mL and an IC50 half-maximum inhibitory concentration (IC50). 50 The concentration is 4.02 ng / mL, and the quantitative detection range is 0.58~28.00 ng / mL. It has no cross-reactivity with similar drugs of 4-methylaminoantipyrine. It can be used for rapid qualitative and quantitative detection of 4-methylaminoantipyrine in samples. The operation is simple and the detection results are accurate and reliable.

[0198] All aspects, embodiments, features, and examples of this invention are to be regarded as illustrative in all respects and are not intended to limit the invention, the scope of which is defined only by the claims. Other embodiments, modifications, and uses will become apparent to those skilled in the art without departing from the spirit and scope of the invention as claimed.

[0199] In addition, the inventors of this case also conducted experiments with other raw materials, process operations, and process conditions described in this specification, referring to the aforementioned embodiments, and obtained relatively ideal results in all cases.

[0200] Although the invention has been described with reference to illustrative embodiments, those skilled in the art will understand that various other changes, omissions, and / or additions can be made without departing from the spirit and scope of the invention, and that elements of the embodiments can be substituted with substantially equivalents. Furthermore, many modifications can be made without departing from the scope of the invention to adapt particular situations or materials to the teachings of the invention. Therefore, this invention is not intended to be limited to the specific embodiments disclosed for carrying out the invention, but rather is intended to encompass all embodiments falling within the scope of the appended claims. Moreover, unless specifically stated otherwise, any use of the terms first, second, etc., does not indicate any order or importance, but is used to distinguish one element from another.

Claims

1. A 4-methylaminoantipyrine hapten, characterized in that, Its structure is shown in equation (Ⅰ): 。 2. The method for preparing the 4-methylaminoantipyrine hapten according to claim 1, characterized in that, include: A first mixed reaction system containing 4-aminoantipyrine, propionic acid, thionyl chloride and methanol was reacted at room temperature for a predetermined time, and then sodium borohydride was added. The reaction was continued at a temperature of 0~65℃ to obtain a first intermediate compound. A second mixed reaction system containing the first intermediate compound and the Dess-Martin reagent was reacted at room temperature to obtain the 4-methylaminoantipyrine hapten with the structure shown in formula (I). The structure of the first intermediate compound is as follows: 。 3. The preparation method according to claim 2, characterized in that: The molar equivalents of 4-aminoantipyrine, propionic acid, thionyl chloride and sodium borohydride satisfy the ratio of 1:1~2:0.05~0.5:2~10.

4. The preparation method according to claim 2, characterized in that: The preset time is 1 to 12 hours, and the reaction continues for another 1 to 12 hours after the sodium borohydride is added.

5. The preparation method according to claim 2, characterized in that: The molar equivalents of the first intermediate compound and the Dess-Martin reagent are 1:1 to 3.

6. The preparation method according to claim 2, characterized in that: The second mixed reaction system was allowed to react at room temperature for 1 to 6 hours.

7. A 4-methylaminoantipyrine artificial antigen, characterized in that, The 4-methylaminoantipyrine artificial antigen is obtained by conjugating the 4-methylaminoantipyrine hapten with a carrier protein as described in claim 1, and its structure is shown in formula (III): ； Protein is a carrier protein.

8. The 4-methylaminoantipyrine artificial antigen according to claim 7, characterized in that: The carrier protein is any one of bovine serum albumin, ovalbumin, hemocyanin, lactoferrin, or human serum albumin.

9. A 4-methylaminoantipyrine artificial antigen combination, characterized in that, It includes an immunogen and a coating antigen, wherein the structure of the immunogen is as shown in formula (III) of claim 7 and the carrier protein is bovine serum albumin, and the structure of the coating antigen is as shown in formula (IV), where Protein represents ovalbumin; 。 10. The use of the 4-methylaminoantipyrine hapten of claim 1, the 4-methylaminoantipyrine artificial antigen of claim 7 or 8, and the 4-methylaminoantipyrine artificial antigen combination of claim 9 in the detection of 4-methylaminoantipyrine for the purpose of non-disease treatment diagnosis.

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