An aminopyrine hapten and its preparation method, artificial antigen, antibody and application

Abstract

This invention provides an aminopyrine hapten, its preparation method, an artificial antigen, an antibody, and its applications. The structure of the aminopyrine hapten is shown in formula (I) or formula (II). The two aminopyrine haptens provided by this invention have spacer arms of suitable length. After being coupled with a protein, the hapten can be fully exposed to the immune system while minimizing recognition of the spacer arm, resulting in superior antibody recognition of aminopyrine. This invention uses the aforementioned aminopyrine hapten to prepare artificial antigens and antibodies. The obtained aminopyrine monoclonal antibodies have high titers and strong specificity. The immunoassay method constructed based on it has high specificity and sensitivity, enabling rapid qualitative and quantitative detection of aminopyrine in samples. The operation is simple, and the detection results are accurate and reliable.

Description

Technical Field

[0001] This invention belongs to the field of detection technology, specifically relating to an aminopyrine hapten and its preparation method, artificial antigen, antibody and application. Background Technology

[0002] Metamizole Sodium (chemical formula C) 13 H 18 N3NaO5S was first synthesized by Hoechst AG in Germany in 1911 and officially marketed as a non-opioid analgesic in 1922. Metamizole has serious side effects; patients taking it have frequently been reported to experience hematologic disorders and allergic reactions, such as agranulocytosis, aplastic anemia, and anaphylactic shock. Due to the high risk of serious side effects, the China Food and Drug Administration (CFDA) completely banned the production and use of metamizole-related preparations in human diseases on March 17, 2023. However, due to its low cost and significant efficacy, metamizole is often illegally added by unscrupulous manufacturers. Therefore, there is an urgent need for a highly specific and sensitive detection method for metamizole. 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 an aminopyrine hapten, the structure of which is shown in formula (I) or formula (II):

[0005] .

[0006] The metamizole hapten provided by this invention, as shown in formula (I) or formula (II), has a spacer arm of suitable length. After conjugation with a carrier protein, the hapten is fully exposed while minimizing recognition of the spacer arm, thereby generating better antibody recognition of metamizole. If the spacer arm of the hapten is too short, the carrier protein will mask the characteristic structure of the analyte, resulting in a poor antibody response. If the spacer arm of the hapten is too long, the conformation of the hapten molecule will fold, thus being masked by the carrier protein, making it easier to generate antibodies against the spacer arm.

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

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

[0009] The first mixed reaction system containing tert-butyl gamma-butyrate, triethylamine and thioyl chloride was subjected to a first reaction at a temperature of 0~35℃ to obtain the first intermediate product;

[0010] The second mixed reaction system containing the first intermediate product, 4-methylaminoantipyrine and triethylamine was subjected to a second reaction at a temperature of 0~35°C to obtain the second intermediate product.

[0011] The third mixed reaction system containing the second intermediate product and acid was subjected to a third reaction at a temperature of 0~35℃ to obtain the metamizole hapten with the structure shown in formula (I).

[0012] In some embodiments, the molar equivalents of tert-butyl aminobutyrate, triethylamine, and thioyl chloride in the first mixed reaction system satisfy 1:1-3:1-3.

[0013] In some embodiments, the molar equivalents of the first intermediate, 4-methylaminoantipyrine, and triethylamine in the second mixed reaction system satisfy 1:1-3:1-3.

[0014] In some embodiments, the first mixed reaction system further includes a first organic solvent, which includes, but is not limited to, dichloromethane.

[0015] In some embodiments, the second mixed reaction system further includes a second organic solvent, which includes, but is not limited to, acetonitrile.

[0016] In some embodiments, the third mixed reaction system further includes a third organic solvent, which includes, but is not limited to, 1,4-dioxane.

[0017] In some embodiments, the molar equivalent of the acid and the second intermediate product satisfies a ratio of 1-6:1.

[0018] In some embodiments, the acid includes hydrochloric acid, but is not limited thereto.

[0019] In some embodiments, the first reaction takes 1-3 hours.

[0020] In some embodiments, the second reaction takes 1-3 hours.

[0021] In some embodiments, the third reaction takes 1-3 hours.

[0022] In some typical embodiments, the preparation method of the metamizole hapten with the structure shown in formula (I) includes:

[0023] The first reaction was carried out by dissolving tert-butyl aminobutyrate in dichloromethane and adding triethylamine and thioyl chloride.

[0024] After the first reaction is completed, dichloromethane in the reaction product of the first reaction is distilled off, then acetonitrile is added to dissolve it, and then the 4-methylaminoantipyrine and triethylamine are added to carry out the second reaction;

[0025] After the second reaction is completed, silica gel powder is added to the reaction product of the second reaction, and the product is dried by rotary evaporation. The silica gel powder containing the second intermediate is then purified by column chromatography.

[0026] The purified second intermediate was dissolved in 1,4-dioxane and hydrochloric acid was added to carry out the third reaction to obtain the metamizole hapten with the structure shown in formula (I).

[0027] The preparation method of the metamizole hapten with the structure shown in formula (II) includes: carrying out a fourth reaction in a fourth mixed reaction system containing antipyrine and phosphorus oxychloride at a temperature of 80-120℃ to obtain the metamizole hapten with the structure shown in formula (II).

[0028] In some embodiments, the molar equivalents of antipyrine and phosphorus oxychloride in the fourth mixed reaction system satisfy 1:1-3.

[0029] In some embodiments, the fourth reaction takes 1-6 hours.

[0030] In some embodiments, the fourth mixed reaction system further includes a solvent, which may include, for example, N,N-dimethylformamide, but is not limited thereto.

[0031] A third aspect of the present invention provides an artificial aminopyrine antigen, which is obtained by conjugating an aminopyrine hapten with a carrier protein as described in any of the technical solutions, wherein the structure of the artificial aminopyrine antigen is shown in formula (III) or formula (IV):

[0032] ;

[0033] Protein is a carrier protein.

[0034] The metamizole artificial antigen with the structure shown in formula (III) is obtained by conjugating the metamizole hapten with a carrier protein with the structure shown in formula (I), for example, by using the active ester method to conjugate the carrier protein.

[0035] The metamizole artificial antigen with the structure shown in formula (Ⅳ) is obtained by conjugating the metamizole hapten with a carrier protein as described in formula (Ⅱ), for example by conjugating the carrier protein by the amine reduction method.

[0036] In some embodiments, the carrier protein is any one of bovine serum albumin, lactoferrin, chicken ovalbumin, and human serum albumin, but is not limited thereto.

[0037] In some embodiments, the structure of the metamizole artificial antigen is as shown in formula (Ⅲ) and the carrier protein is bovine serum albumin.

[0038] In some embodiments, the structure of the metamizole artificial antigen is as shown in formula (Ⅳ) and the carrier protein is lactoferrin.

[0039] A fourth aspect of the present invention provides an aminopyrine antibody obtained by immunizing animals with the aminopyrine artificial antigen described in any of the technical solutions.

[0040] In some embodiments, the aminopyrine antibody is a monoclonal antibody.

[0041] In some embodiments, the aminopyrine antibody is obtained by immunizing animals with an aminopyrine artificial antigen of formula (III) and whose carrier protein is bovine serum albumin.

[0042] A fifth aspect of the present invention provides an aminopyrine 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 lactoferrin.

[0043] The sixth aspect of the present invention provides the use of any of the following technical solutions: metamizole hapten, metamizole artificial antigen, metamizole antibody, or metamizole artificial antigen combination in the preparation of metamizole detection products or in the detection of metamizole for non-disease treatment diagnosis purposes.

[0044] A seventh aspect of the present invention provides a kit for detecting metamizole, the kit comprising the metamizole artificial antigen and the metamizole antibody as described in any of the technical solutions.

[0045] In some embodiments, the structure of the metamizole artificial antigen is as shown in formula (IV) and the carrier protein is lactoferrin, and the metamizole antibody is obtained by immunizing animals with the metamizole artificial antigen as shown in formula (III) and the carrier protein is bovine serum albumin.

[0046] In some embodiments, the aminopyrine antibody is a monoclonal antibody. For example, it can be obtained through hybridoma cells.

[0047] In some embodiments, the kit is an ELISA kit.

[0048] In some embodiments, the ELISA kit further includes an enzyme-labeled plate, an aminopyrine standard, and a substrate chromogenic solution, wherein the aminopyrine artificial antigen is coated on the enzyme-labeled plate.

[0049] In some embodiments, the substrate developing solution includes urea peroxide and tetramethylbenzidine.

[0050] In some embodiments, the ELISA kit further includes a stop solution, a washing solution, a blocking solution, an enzyme-labeled secondary antibody, and an enzyme-labeled secondary antibody dilution solution.

[0051] 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.

[0052] 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 contains 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.

[0053] In some embodiments, the blocking solution comprises 1% to 3% casein by mass and 0.1 mol / L to 0.3 mol / L phosphate buffer, with a pH of 7.1 to 7.5. In some preferred embodiments, the blocking solution comprises 2% casein by mass and 0.2 mol / L phosphate buffer, with a pH of 7.3.

[0054] In some embodiments, the enzyme-labeled secondary antibody is a horseradish peroxidase-labeled goat anti-mouse antibody.

[0055] In some embodiments, the enzyme-labeled secondary antibody dilution solution is a 0.1 mol / L to 0.3 mol / L phosphate buffer. In some preferred embodiments, the enzyme-labeled secondary antibody dilution solution is a 0.2 mol / L phosphate buffer.

[0056] 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.

[0057] The eighth aspect of the present invention provides an immunoassay method for detecting metamizole, wherein the immunoassay method uses the metamizole artificial antigen and the metamizole antibody as described in any of the technical solutions to detect metamizole in a sample.

[0058] In some embodiments, the structure of the metamizole artificial antigen is as shown in formula (IV), and the carrier protein is lactoferrin. The metamizole antibody is obtained by immunizing animals with the metamizole artificial antigen of formula (III), where the carrier protein is bovine serum albumin. The detection method based on this combination has high specificity and good antibody sensitivity.

[0059] In some embodiments, the aminopyrine antibody is a monoclonal antibody.

[0060] In some embodiments, the immunoassay detection method is ELISA.

[0061] In some embodiments, the ELISA is an indirect ELISA or a competitive ELISA, and is more preferably an indirect competitive ELISA.

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

[0063] (1) The two metamizole haptens provided by the present invention have spacer arms of suitable length, so that after the two haptens are coupled with the protein, the haptens can be fully exposed to the immune system, while the spacer arms can be avoided as much as possible, resulting in better antibody recognition of metamizole.

[0064] (2) The present invention uses an aminopyrine hapten with a spacer arm of appropriate length to prepare an artificial antigen, and then uses it to immunize animals to obtain aminopyrine monoclonal antibodies. The obtained antibodies have high titer, strong specificity and high affinity, providing core raw materials for establishing a specific aminopyrine immunoassay method.

[0065] (3) The immunoassay method for aminopyrine based on artificial antigen and antibody provided by the present invention has high sensitivity and strong specificity. The limit of detection (LOD) is 2.13 ng / mL, the half-inhibitory concentration (IC50) is 18.34 ng / mL, and the quantitative detection range is 4.72~71.19 ng / mL. It has no cross-reactivity with similar drugs of aminopyrine. It can perform rapid qualitative and quantitative detection of aminopyrine in samples. The operation is simple and the detection results are accurate and reliable. Attached Figure Description

[0066] 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.

[0067] Figure 1The image shows the ultraviolet scanning identification results of the aminopyrine hapten MS-1, aminopyrine artificial antigen MS-1-BSA and BSA prepared in the embodiments of the present invention.

[0068] Figure 2 The image shows the ultraviolet scanning identification results of the aminopyrine hapten MS-2, aminopyrine artificial antigen MS-2-LF, and LF prepared in the embodiments of the present invention.

[0069] Figure 3 This is a standard curve for detecting metamizole using an indirect competitive ELISA method constructed from metamizole monoclonal antibody obtained by immunizing animals with metamizole artificial antigen MS-1-BSA and metamizole artificial antigen MS-2-LF, as described in this embodiment of the invention. Detailed Implementation

[0070] 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.

[0071] 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.

[0072] Unless otherwise specified, the "equivalent" of raw material usage in the following specific embodiments refers to molar equivalent.

[0073] Example 1

[0074] 1. Synthesis and identification of metamizole hapten MS-1

[0075] 1.1 Synthesis of Analgin Hapten MS-1

[0076] The synthetic route for the metamizole hapten MS-1 is as follows:

[0077] ;

[0078] The specific steps include: dissolving 1 equivalent of tert-butyl aminobutyrate in dichloromethane, adding 1.1 equivalents of triethylamine and 1 equivalent of thioyl chloride, and stirring overnight; after the reaction, dichloromethane in the reaction system is evaporated off using a rotary evaporator; acetonitrile is added back to the reaction flask to dissolve the sample, followed by 1 equivalent of 4-methylaminoantipyrine and 1.1 equivalents of triethylamine, and the mixture is refluxed overnight; after the reaction, 15 times the volume of silica gel powder is added to the organic phase, and the mixture is evaporated to dryness using a rotary evaporator; the dried silica gel powder containing the sample is purified using a column chromatography system (petroleum ether: ethyl acetate: 1:1); the purified product is dissolved in 15 times the volume of 1,4-dioxane, and 3 mL of hydrochloric acid is added, and the mixture is stirred overnight at room temperature. After the reaction, the mixture is evaporated to dryness using a rotary evaporator to obtain the aminopyrine hapten MS-1.

[0079] 1.2 Identification of Analgin Hapten MS-1

[0080] The 1H NMR spectrum of the metamizole hapten MS-1 is as follows: 1 H NMR (600 MHz, DMSO- d 6) δ 11.73(s, 1H), 7.63 – 7.45 (m, 3H), 7.40 – 7.21 (m, 3H), 3.18 (s, 3H), 3.11 (s,3H), 2.97 (q, J = 6.4 Hz, 2H), 2.30 (t, J = 8.5 Hz, 2H), 2.24 (s, 3H), 1.79(tt, J = 8.5, 6.7 Hz, 2H).

[0081] The mass spectrometry results of the metamizole hapten MS-1 are as follows: + ESI-MS + [MH] + 381.10.

[0082] The mass spectrometry and NMR results show that the mass spectrometry results correspond to the molecular weight of the metamizole hapten, and the proton NMR spectra correspond to the proton NMR spectra on the metamizole hapten backbone structure. This indicates that the metamizole hapten, denoted as MS-1, was successfully prepared, and its structural formula is shown in formula (I).

[0083] .

[0084] 2. Synthesis and identification of metamizole hapten MS-2

[0085] 2.1 Synthesis of Analgin Hapten MS-2

[0086] The synthetic route for the metamizole hapten MS-2 is shown below:

[0087]

[0088] The specific steps include: dissolving 1 equivalent of antipyrine in 15 volumes of DMF, adding 2 equivalents of phosphorus oxychloride; stirring the reaction overnight at 100 °C; after the reaction, adding 10 volumes of deionized water and extracting with ethyl acetate; combining the organic phases and washing with saturated brine; adding 15 volumes of silica gel powder to the organic phase and drying it using a rotary evaporator. The dried silica gel powder containing the sample was purified using a column chromatography system (petroleum ether: ethyl acetate: 1:3) to obtain purified metamizole hapten MS-2.

[0089] 2.2 Identification of Analgin Hapten MS-2

[0090] The 1H NMR spectrum of the metamizole hapten MS-2 is as follows: 1 H NMR (600 MHz, DMSO- d 6) δ 10.00– 9.72 (m, 1H), 7.48 (t, J = 7.6 Hz, 2H), 7.40 – 7.19 (m, 3H), 3.18 (s, 3H), 2.50 (s, 3H).

[0091] The mass spectrometry results of the metamizole hapten MS-2 are as follows: + ESI-MS + [MH] + 215.08.

[0092] The mass spectrometry and NMR results show that the mass spectrometry results correspond to the molecular weight of the metamizole hapten, and the number of protons in the NMR spectrum corresponds to the number of protons in the metamizole hapten backbone structure. This indicates that the metamizole hapten, denoted as MS-2, was successfully prepared, and its structural formula is shown in formula (II).

[0093] .

[0094] Example 2

[0095] 1. Synthesis of Analgin Artificial Antigen

[0096] 1.1 Synthesis of Analgin Artificial Antigen MS-1-BSA

[0097] (1) The aminopyrine hapten MS-1 (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. The mixture was dissolved in 100 μL DMF and stirred at room temperature for 3 h to obtain the hapten activation solution.

[0098] (2) Bovine serum albumin BSA (10 mg, 0.00015 mol) was dissolved in 1 mL of PBS buffer (0.01 mol / L, pH=7.4) to obtain BSA solution. The BSA solution and hapten activation solution were stirred overnight at 4 °C.

[0099] (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;

[0100] (4) After dialysis purification, the artificial antigen of aminopyrine, denoted as MS-1-BSA, was obtained. After concentration determination and ultraviolet spectroscopy identification, it was stored in a -20 ℃ refrigerator.

[0101] 1.2 Synthesis of Analgin Artificial Antigen MS-2-LF

[0102] (1) The aminopyrine hapten MS-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. The mixture was dissolved in 100 μL DMF and stirred at room temperature for 3 h to obtain the hapten activation solution.

[0103] (2) Dissolve lactoferrin LF in 1 mL PBS buffer (0.01 mol / L, pH=7.4);

[0104] (3) The MS-2 (3.1 mg, 0.012 mol) prepared in Example 1 was added to the protein solution along with 2 equivalents of sodium borohydride, and stirred overnight at 4 °C;

[0105] (4) 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] (5) After dialysis purification, the artificial antigen of aminopyrine, denoted as MS-2-LF, was obtained. After concentration determination and ultraviolet spectroscopy identification, it was stored in a -20 ℃ refrigerator.

[0107] 2. Identification of Analgin Artificial Antigen

[0108] BSA, LF, MS-1, MS-2, MS-1-BSA, and MS-2-LF were scanned and identified using the ultraviolet full-wavelength method (200–350 nm).

[0109] Figure 1 The images show the UV scanning identification results of metamizole hapten MS-1, metamizole artificial antigen MS-1-BSA, and BSA, as shown below. Figure 1 As shown, by comparing the highest absorbance values ​​of each substance before and after conjugation, it was found that the absorption curve of MS-1-BSA was significantly different from that of the carrier protein BSA. MS-1 had a strong absorption peak above 343 nm, while after conjugation with BSA, the absorption peak of MS-1-BSA was 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 MS-1 showed a significant shift. Since all unreacted components were removed by dialysis during the post-conjugation dialysis process, the characteristic peaks of the conjugation product were contributed by the protein-bound drug molecules, indicating that the reaction product is a complex of the carrier protein BSA and MS-1.

[0110] Figure 2 These are UV scan identification results for Analgin hapten MS-2, Analgin artificial antigen MS-2-LF, and LF, as shown in the image. Figure 2 As shown, the absorption curve of MS-2-LF showed characteristic peaks that were different from those of LF and MS-2, indicating that the reaction product was a complex of the carrier protein LF and MS-2.

[0111] The above results indicate that the present invention successfully prepared aminopyrine artificial antigens MS-1-BSA and MS-2-LF. The structure of aminopyrine artificial antigen MS-1-BSA is shown in formula (III) and the carrier protein is BSA. The structure of MS-2-LF is shown in formula (IV) and the carrier protein is lactoferrin.

[0112] .

[0113] Example 3

[0114] 1. Animal immunization

[0115] MS-1-BSA, prepared in Example 2, was used as the immunogen. MS-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 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 and 0.1 ml per mouse. A second immunization was performed 14 days later, using an equal volume of Freund's incomplete adjuvant emulsified with the immunogen, at 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.

[0116] 2. Cell fusion

[0117] 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:

[0118] 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.

[0119] 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.

[0120] c. Mix the two cell lines, bring the volume to 15 mL, centrifuge (1000 rpm / min, 7 min), and discard the supernatant. Remove the centrifuge tube, gently shake at 60°C in a circular motion, and then begin preparation for fusion. The fusion time should be controlled as follows:

[0121] First minute: Slowly add PEG dropwise;

[0122] Second minute: Continue rotating (1 minute);

[0123] Third minute: Add 1 mL of basal culture medium;

[0124] Fourth minute: Add 3 mL of basal culture medium;

[0125] Fifth minute: Add 8 mL of basal culture medium;

[0126] Sixth minute: Add 8 mL of basal culture medium;

[0127] After fusion, the cell culture was centrifuged (1000 rpm / min, 7 min), and the 200 mL HAT medium incubated was removed and placed in a clean bench for later use.

[0128] 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.

[0129] 3. Cell selection and cell line establishment:

[0130] On day 3 of cell fusion, the fused cells were partially replaced with RPMI-1640 screening medium. 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.

[0131] The screening process consisted of two steps: First, positive cell wells were selected using ic-ELISA. Second, allopurinol was used as a standard, and its inhibitory effect on positive cells was measured using ic-ELISA. Cell wells that showed good inhibition of the allopurinol 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 MS-1 monoclonal antibody cell line containing aminopyrine.

[0132] 4. Preparation and identification of aminopyrine monoclonal antibodies

[0133] 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 6 Ascites fluid was collected from Analgin hybridoma cells starting on day 7. The ascites fluid was then purified for antibody removal 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 Analgin monoclonal antibodies. The purified monoclonal antibodies were stored at -20 °C.

[0134] 5. Sensitivity evaluation of metamizole monoclonal antibody (indirect competitive ELISA method)

[0135] MS-2-LF 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 overnight (12 h) at 4 °C.

[0136] 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.

[0137] Metamizole monoclonal antibody prepared using MS-1-BSA as an immunogen was diluted with PBST at a volume ratio of 1:128000. Metamizole standard was diluted with PBST to different concentrations, resulting in metamizole standard dilutions with concentrations of 1000 ng / mL, 200 ng / mL, 40 ng / mL, 8 ng / mL, 1.6 ng / mL, 0.32 ng / mL, 0.064 ng / mL, 0.0128 ng / mL, and 0 ng / mL.

[0138] Add 50 μL / well of different concentrations of aminopyrine standard dilution buffer (three parallel groups), then add 50 μL / well of diluted aminopyrine monoclonal antibody, incubate at 37 ℃ for 40 min, wash five times, and pat dry;

[0139] 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;

[0140] Add 100 μL / well of colorimetric reagent and develop for 10 min;

[0141] The reaction was terminated by adding 50 μL of 10% H2SO4 solution, and the OD value was read at 450 nm.

[0142] ELISA standard curve plotting: Plot B / B0 as the ordinate (B represents the absorbance OD of standards at different concentrations of aminopyrine). 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 aminopyrine standard to the x-axis using the Logistic function, and the formula for the standard curve was derived.

[0143] Figure 3To establish a standard curve for detecting metamizole using an indirect competitive ELISA method constructed from metamizole monoclonal antibody obtained by immunizing animals with metamizole artificial antigen MS-1-BSA and metamizole artificial antigen MS-2-LF, as shown in the figure... Figure 3 As shown, the limit of detection (LOD) of this indirect competitive ELISA method is 2.13 ng / mL, and the half-maximum inhibitory concentration (IC50) is [missing value]. 50 The concentration was 18.34 ng / mL, and the quantitative detection range was 4.72~71.19 ng / mL.

[0144] 6. Specificity evaluation of metamizole monoclonal antibody

[0145] Naproxen, ibuprofen, and piroxicam are analogues of metamizole. The specificity of the metamizole monoclonal antibody prepared in this embodiment was evaluated by cross-reactivity assay.

[0146] Following the sensitivity evaluation method described above, the only difference is that allopurinol standards are replaced with naproxen, ibuprofen, and piroxicam standards, and detection is performed at the same dilution factor to obtain the IC50 values ​​for each structural analog. 50 value.

[0147] The cross-reactivity rate (CR) of Analgin is calculated using the following formula: CR (%) = IC50 (Analgin) / IC50 (similar drug) × 100%. The lower the cross-reactivity rate, the stronger the specificity.

[0148] Table 1. Cross-reactivity results of metamizole monoclonal antibody with metamizole and its analogues.

[0149] ;

[0150] Note: NR indicates no reaction, meaning the antibody does not recognize the analogue.

[0151] As shown in Table 1, the cross-reactivity rate of the aminopyrine monoclonal antibody prepared in this invention to aminopyrine is 100%, and the IC50 value is [missing information]. 50 The concentration was 18.34 ng / mL, with no cross-contamination with naproxen, ibuprofen, and piroxicam. This indicates that the antibody used to detect metamizole has high recognition ability and specificity for metamizole, effectively eliminating interference from the detection of metamizole analogs such as naproxen, ibuprofen, and piroxicam, and can be specifically used for the detection of metamizole.

[0152] The above results indicate that the monoclonal antibody for aminopyrine prepared in this invention exhibits excellent detection performance, high sensitivity, and strong specificity for aminopyrine.

[0153] Example 4

[0154] Example 4 provides an aminopyrine artificial antigen combination, comprising a combination of an immunogen and a coating antigen:

[0155] Using the aminopyrine artificial antigen MS-2-LF prepared in Example 2 as the coating antigen, and the aminopyrine monoclonal antibody prepared in Example 3 using MS-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:

[0156] 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)).

[0157] 2. Add 120 μL of blocking solution (PBST containing 7% skim milk powder) to each well, block at 37 ℃ for 1 h, discard the blocking solution, plate, and dry in a drying oven at 37 ℃ for later use;

[0158] 3. Dilute the metamizole 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 metamizole standard 1000 times with PBST to obtain a 1 μg / mL metamizole standard dilution solution;

[0159] 4. Potency column setup: First, add 50 μL of PBST to each well, then add 50 μL of aminopyrine monoclonal antibody at different dilution ratios to each well, and finally add 50 μL of PBST to the last well instead of the antibody.

[0160] 5. Inhibition column setup: First, add 50 μL of aminopyrine standard dilution buffer to each well, then add 50 μL of aminopyrine monoclonal antibody at different dilution ratios to each well, and finally add 50 μL of PBST to the last well instead of antibody.

[0161] 6. Incubate at 37℃ for 40 minutes, wash 5 times, and then plate.

[0162] 7. Add goat anti-mouse secondary antibody-HRP (diluted 5000 times with PBST), incubate at 37 ℃ for 30 min, wash 5 times, and plate.

[0163] 8. Add the color developing solution and incubate at 37 ℃ for 10 min;

[0164] 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%.

[0165] The present invention also prepared metamizole artificial antigens MS-2-OVA, MS-1-LF, and MS-1-OVA using the same method as described above, and constructed different combinations of immunogens and coating antigens. The combined effects of different immunogens and coating antigens were evaluated using the same method as described above, and the results are shown in Table 2.

[0166] Table 2. ELISA detection results of immunogens and coating antigens.

[0167] ;

[0168] As shown in Table 2, the monoclonal antibody prepared using the artificial antigen MS-1-BSA of metamizole as an immunogen and MS-2-LF is the optimal combination of immunogen and coating agent. Under this combination, the monoclonal antibody can not only specifically recognize the target analyte metamizole, but also has good antibody sensitivity and can be used to specifically recognize the target analyte metamizole.

[0169] Example 5

[0170] Example 5 provides an ELISA kit for detecting aminopyrine, as detailed below:

[0171] 1. Components of an ELISA kit

[0172] (1) The enzyme-labeled plate coated with the coating agent is prepared by the following method:

[0173] MS-2-LF 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, and then patted dry. Then, 120 μL of the blocking buffer provided in this kit was added to each 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, they were vacuum sealed with aluminum foil for storage.

[0174] (2) Standards: Diluted solutions of 9 different concentrations of aminopyrine standards: 1000 ng / mL, 200 ng / mL, 40 ng / mL, 8 ng / mL, 1.6 ng / mL, 0.32 ng / mL, 0.064 ng / mL, 0.0128 ng / mL and 0 ng / mL.

[0175] (3) Antibody: The monoclonal antibody prepared in Example 3 using MS-1-BSA as the immunogen;

[0176] (4) Enzyme-labeled secondary antibody: Horseradish peroxidase-labeled goat anti-mouse secondary antibody;

[0177] (5) Substrate colorimetric solution: composed of solution A and solution B, where solution A is urea peroxide and solution B is tetramethylbenzidine;

[0178] (6) Termination solution: 2 mol / L H2SO4;

[0179] (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;

[0180] (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;

[0181] (9) Blocking solution: pH 7.3, 0.2 mol / L phosphate buffer containing 2% casein.

[0182] 2. Instructions for use

[0183] (1) Sample testing

[0184] Number the wells corresponding to the samples and standards in this kit sequentially. Perform 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.

[0185] 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.

[0186] 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).

[0187] 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.

[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 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.

[0190] 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.

[0191] (2) Drawing the standard curve

[0192] 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 was obtained by using the logarithm of the concentration of the standard as the abscissa and performing curve fitting with the Logistic function.

[0193] (3) Calculation of sample concentration

[0194] 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 aminopyrine in the sample.

[0195] In summary, this invention provides two metamizole haptens with suitable spacer arm lengths, and based on these, prepares artificial antigens and antibodies. The resulting metamizole monoclonal antibodies exhibit high titer, strong specificity, and high affinity, providing core raw materials for establishing a specific immunoassay method for metamizole. This invention also establishes a highly specific and sensitive immunoassay method for metamizole, with a limit of detection (LOD) of 2.13 ng / mL and an IC50 half-inhibition concentration (IC50). 50 The concentration was 18.34 ng / mL, and the quantitative detection range was 4.72~71.19 ng / mL. It showed no cross-reactivity with similar drugs to aminopyrine, enabling rapid qualitative and quantitative detection of aminopyrine in samples. The operation was simple and the detection results were accurate and reliable.

[0196] 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.

[0197] The aspects, embodiments, features, and examples of this invention are to be considered illustrative and are not intended to limit the invention. The scope of the invention 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.

[0198] 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 described 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 document is not intended to limit the invention to the specific embodiments disclosed, but rather to include 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. An aminopyrine artificial antigen, characterized in that, Its structure is shown in equation (Ⅳ): ； Protein is a carrier protein, which is any one of bovine serum albumin, lactoferrin, chicken ovalbumin, or human serum albumin.

2. A combination of aminopyrine artificial antigens, characterized in that, It includes an immunogen and a coating antigen, wherein the structure of the immunogen is as shown in formula (III) and the carrier protein is bovine serum albumin, and the structure of the coating antigen is as shown in formula (IV) of claim 1 and the carrier protein is lactoferrin. 。 3. The use of the aminopyrine artificial antigen of claim 1 or the aminopyrine artificial antigen combination of claim 2 in the preparation of aminopyrine detection products.

4. The use of the aminopyrine artificial antigen of claim 1 or the aminopyrine artificial antigen combination of claim 2 in the detection of aminopyrine for the purpose of non-disease treatment diagnosis.

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