Analgin hapten, preparation method thereof, artificial antigen, antibody and application

Abstract

The invention provides an analgin hapten, a preparation method thereof, an artificial antigen, an antibody and application. The structures of the analgin haptens are as shown in a formula (I) or a formula (II), the two analgin haptens provided by the invention have spacer arms with proper lengths, and after the two analgin haptens are coupled with protein, the haptens can be fully exposed to an immune system, the spacer arms can be prevented from being recognized as much as possible, and a better antibody is generated to recognize analgin. The analgin hapten is used for preparing artificial antigen and antibody, the obtained analgin monoclonal antibody has high titer and strong specificity, an immunoassay method constructed based on the analgin monoclonal antibody has high specificity and sensitivity, analgin in a sample can be rapidly, qualitatively and quantitatively detected, the operation is simple and convenient, and the detection result is accurate and reliable.

Description

Technical Field

[0001] The invention belongs to the field of detection technology, and particularly relates to a metamizole hapten and a preparation method thereof, an artificial antigen, an antibody and applications thereof. Background Art

[0002] Metamizole Sodium (Chemical Formula C 13 H 18 N3NaO5S) was first synthesized by the German Hoechst Chemical Company in 1911 and officially launched on the market as a non-opioid analgesic in 1922. Analgin has serious side effects, and people who take analgin are often reported to have blood system diseases and allergic reactions, such as agranulocytosis, aplastic anemia, anaphylactic shock and other diseases. Because analgin is prone to cause serious side effects, the China Food and Drug Administration has completely banned the production of analgin-related preparations and their use in human diseases on March 17, 2023. However, due to the low cost and significant efficacy of analgin, it is often illegally added by unscrupulous merchants. Therefore, there is an urgent need to provide a highly specific and sensitive detection method for analgin. Summary of the Invention

[0003] In order to solve all or part of the above technical problems, the present invention provides the following technical solutions: The first aspect of the present invention provides a metamizole hapten, the structure of which is shown in formula (I) or formula (II): .

[0004] The analgin hapten of formula (I) or formula (II) provided by the present invention has a spacer arm of appropriate length, which can fully expose the hapten after coupling with the carrier protein and minimize the recognition of the spacer arm, thereby generating better antibodies to recognize analgin. If the spacer arm of the hapten is 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 long, the conformation of the hapten molecule will fold, thereby being masked by the carrier protein, and antibodies against the spacer arm will be easily generated.

[0005] The second aspect of the present invention provides a method for preparing an analgin hapten.

[0006] The preparation method of the analgin hapten having the structure shown in formula (I) comprises: allowing a first mixed reaction system containing tert-butyl aminobutyrate, triethylamine, and sulfuryl chloride to undergo a first reaction at a temperature of 0 to 35° C. to obtain a first intermediate product; allowing a second mixed reaction system containing the first intermediate product, 4-methylaminoantipyrine and triethylamine to undergo a second reaction at a temperature of 0 to 35° C. to obtain a second intermediate product; The third mixed reaction system containing the second intermediate product and the acid is subjected to a third reaction at a temperature of 0-35° C. to obtain the analgin hapten having a structure as shown in formula (I).

[0007] In some embodiments, the molar equivalents of tert-butyl aminobutyrate, triethylamine, and sulfuryl chloride in the first mixed reaction system satisfy 1:1-3:1-3.

[0008] In some embodiments, the molar equivalents of the first intermediate product, 4-methylaminoantipyrine, and triethylamine in the second mixed reaction system satisfy 1:1-3:1-3.

[0009] In some embodiments, the first mixed reaction system further includes a first organic solvent, and the first organic solvent includes dichloromethane, but is not limited thereto.

[0010] In some embodiments, the second mixed reaction system further includes a second organic solvent, and the second organic solvent includes acetonitrile, but is not limited thereto.

[0011] In some embodiments, the third mixed reaction system further includes a third organic solvent, and the third solvent includes 1,4-dioxane, but is not limited thereto.

[0012] In some embodiments, the molar equivalent of the acid to the second intermediate product satisfies 1-6:1.

[0013] In some embodiments, the acid includes hydrochloric acid, but is not limited thereto.

[0014] In some embodiments, the first reaction time is 1-3 hours.

[0015] In some embodiments, the second reaction time is 1-3 hours.

[0016] In some embodiments, the third reaction lasts for 1-3 hours.

[0017] In some typical embodiments, the method for preparing the analgin hapten of the structure shown in formula (I) comprises: Dissolving tert-butyl aminobutyrate in dichloromethane, and adding triethylamine and sulfuryl chloride to carry out the first reaction; After the first reaction is completed, dichloromethane in the reaction product of the first reaction is distilled off, acetonitrile is added to dissolve it, and then 4-methylaminoantipyrine and triethylamine are added to carry out a second reaction; After the second reaction is completed, silica gel powder is added to the reaction product of the second reaction, the product is dried using a rotary evaporator, and the silica gel powder containing the second intermediate product is purified using a column chromatography system; The purified second intermediate product is dissolved in 1,4-dioxane, and hydrochloric acid is added to carry out a third reaction to obtain the analgin hapten with the structure shown in formula (I).

[0018] The preparation method of the analgin hapten having a structure as shown in formula (II) comprises: subjecting a fourth mixed reaction system containing antipyrine and phosphorus oxychloride to a fourth reaction at a temperature of 80-120° C. to obtain the analgin hapten having a structure as shown in formula (II).

[0019] In some embodiments, the molar equivalent of antipyrine to phosphorus oxychloride in the fourth mixed reaction system satisfies 1:1-3.

[0020] In some embodiments, the fourth reaction lasts for 1-6 hours.

[0021] 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.

[0022] The third aspect of the present invention provides an artificial analgin antigen, which is obtained by coupling the analgin hapten with a carrier protein as described in any technical solution. The structure of the analgin artificial antigen is shown in formula (III) or formula (IV): ; Among them, Protein is a carrier protein.

[0023] The analgin artificial antigen with a structure as shown in formula (III) is obtained by coupling an analgin hapten with a structure as shown in formula (I) to a carrier protein. For example, the carrier protein can be coupled using an active ester method.

[0024] The analgin artificial antigen with a structure as shown in formula (IV) is obtained by coupling the analgin hapten with a structure as shown in formula (II) to a carrier protein, for example, the carrier protein can be coupled by a reductive amine method.

[0025] 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.

[0026] In some embodiments, the structure of the analgin artificial antigen is as shown in formula (III) and the carrier protein is bovine serum albumin.

[0027] In some embodiments, the structure of the analgin artificial antigen is as shown in formula (IV) and the carrier protein is lactoferrin.

[0028] The fourth aspect of the present invention provides an analgin antibody, which is obtained by immunizing an animal with the analgin artificial antigen described in any one of the technical solutions.

[0029] In some embodiments, the analgin antibody is a monoclonal antibody.

[0030] In some embodiments, the analgin antibody is obtained by immunizing an animal with an analgin artificial antigen having a structure represented by formula (III) and a carrier protein being bovine serum albumin.

[0031] The fifth aspect of the present invention provides an artificial antigen combination of analgin, comprising an immunogen and a coating agent, 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 agent is as shown in formula (IV) and the carrier protein is lactoferrin.

[0032] The sixth aspect of the present invention provides the use of the analgin hapten described in any technical solution, the analgin artificial antigen described in any technical solution, the analgin antibody described in any technical solution, or the analgin artificial antigen combination described in any technical solution in the preparation of an analgin detection product or in the detection of analgin for non-disease treatment and diagnosis purposes.

[0033] A seventh aspect of the present invention provides a kit for detecting analgin, which comprises the analgin artificial antigen and the analgin antibody described in any one of the technical solutions.

[0034] In some embodiments, the structure of the analgin artificial antigen is as shown in formula (IV) and the carrier protein is lactoferrin, and the analgin antibody is obtained by immunizing an animal with the analgin artificial antigen shown in formula (III) and the carrier protein is bovine serum albumin.

[0035] In some embodiments, the analgin antibody is a monoclonal antibody, which can be obtained, for example, from hybridoma cells.

[0036] In some embodiments, the kit is an ELISA kit.

[0037] In some embodiments, the ELISA kit further comprises an enzyme labeling plate, an analgin standard and a substrate color development solution, and the analgin artificial antigen is coated on the enzyme labeling plate.

[0038] In some embodiments, the substrate-developing solution includes urea peroxide and tetramethylbenzidine.

[0039] In some embodiments, the ELISA kit further comprises a stop solution, a washing solution, a blocking solution, an enzyme-labeled secondary antibody, and an enzyme-labeled secondary antibody diluent.

[0040] In some embodiments, the stop solution comprises 8% to 12% H2SO4 by volume. In some preferred embodiments, the stop solution comprises 10% H2SO4 by volume.

[0041] In some embodiments, the washing solution comprises 0.5% to 1.0% Tween-20 by volume, 0.01% to 0.03% sodium azide preservative by mass, and 0.1 mol / L to 0.3 mol / L phosphate buffer, and the pH value of the washing solution is 7.2 to 7.6. In some preferred embodiments, the washing solution comprises 0.8% Tween-20 by volume, 0.02% sodium azide preservative by mass, and 0.2 mol / L phosphate buffer, and the pH value of the washing solution is 7.4.

[0042] In some embodiments, the blocking solution comprises 1% to 3% casein by mass, 0.1 mol / L to 0.3 mol / L phosphate buffer, and the pH value of the blocking solution is 7.1 to 7.5. In some preferred embodiments, the blocking solution comprises 2% casein by mass, 0.2 mol / L phosphate buffer, and the pH value is 7.3.

[0043] In some embodiments, the enzyme-labeled secondary antibody is a goat anti-mouse antibody labeled with horseradish peroxidase.

[0044] In some embodiments, the enzyme-labeled secondary antibody diluent is a 0.1 mol / L to 0.3 mol / L phosphate buffer. In some preferred embodiments, the enzyme-labeled secondary antibody diluent is a 0.2 mol / L phosphate buffer.

[0045] It should be noted that the substrate color developing solution, stop solution, washing solution, blocking solution, enzyme-labeled secondary antibody, and enzyme-labeled secondary antibody dilution solution in the ELISA kit can be any substance or formula known in the art. The components of the substrate color developing solution, stop solution, washing solution, blocking solution, enzyme-labeled secondary antibody, and enzyme-labeled secondary antibody dilution solution described above are merely exemplary and are not intended to limit the present invention.

[0046] An eighth aspect of the present invention provides an immunoassay detection method for analgin, wherein the immunoassay detection method uses the analgin artificial antigen described in any one of the technical solutions and the analgin antibody described in any one of the technical solutions to detect analgin in a sample.

[0047] In some embodiments, the structure of the analgin artificial antigen is represented by formula (IV) and the carrier protein is lactoferrin, and the analgin antibody is obtained by immunizing an animal with the analgin artificial antigen represented by formula (III) and the carrier protein is bovine serum albumin. The detection method based on this combination has high specificity and good antibody sensitivity.

[0048] In some embodiments, the analgin antibody is a monoclonal antibody.

[0049] In some embodiments, the immunoassay detection method is ELISA.

[0050] In some embodiments, the ELISA is an indirect ELISA or a competitive ELISA, more preferably an indirect competitive ELISA.

[0051] Compared with the prior art, the present invention has at least the following beneficial effects: (1) The two analgin haptens provided by the present invention have spacer arms of appropriate lengths, so that after the two haptens are coupled to proteins, the haptens can be fully exposed to the immune system while minimizing the recognition of the spacer arms, thereby generating better antibodies that recognize analgin.

[0052] (2) The present invention uses an analgin hapten with a spacer arm of appropriate length to prepare an artificial antigen, and immunizes animals to obtain an analgin monoclonal antibody. The obtained antibody has high titer, strong specificity and high affinity, providing core raw materials for establishing a specific analgin immunoassay method.

[0053] (3) The analgin immunoassay detection method based on the analgin artificial antigen and antibody provided by the present invention has high sensitivity and strong specificity, with a minimum detection limit LOD of 2.13 ng / mL, a half-inhibitory concentration IC50 of 18.34 ng / mL, and a quantitative detection range of 4.72~71.19 ng / mL. It has no cross-reaction with similar drugs of analgin and can perform rapid qualitative and quantitative detection of analgin in samples. The operation is simple and the test results are accurate and reliable. BRIEF DESCRIPTION OF THE DRAWINGS

[0054] In order to more clearly illustrate the embodiments of the present application or the technical solutions in the prior art, the following briefly introduces the drawings required for use in the embodiments or the description of the prior art. Obviously, the drawings described below are only some embodiments recorded in this application. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying any creative work.

[0055] Figure 1 It is a UV scanning identification result diagram of the analgin hapten MS-1, the analgin artificial antigen MS-1-BSA and BSA prepared in the embodiment of the present invention; Figure 2 This is a UV scanning identification result diagram of the analgin hapten MS-2, analgin artificial antigen MS-2-LF and LF prepared in the embodiment of the present invention; Figure 3 This is a standard curve for detecting analgin using an indirect competitive ELISA method constructed based on the analgin monoclonal antibody obtained by immunizing animals with the analgin artificial antigen MS-1-BSA and the analgin artificial antigen MS-2-LF in the embodiments of the present invention. DETAILED DESCRIPTION

[0056] The technical solutions of the present invention are described in detail below in conjunction with 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 interpreted as limiting, but rather as a basis for the claims and as a representative basis for teaching those skilled in the art to employ the present invention in various ways in virtually any appropriate detailed embodiment.

[0057] In addition, unless otherwise specified, the various raw materials used in the following examples can be purchased from the market and other channels, the various production and testing equipment used are also equipment known in the art, and the detection methods used are also detection methods known in the art.

[0058] Unless otherwise specified, the "equivalents" of the amounts of raw materials used in the following specific embodiments are all molar equivalents.

[0059] Example 1 1. Synthesis and identification of analgin hapten MS-1 1.1 Synthesis of Metamizole Hapten MS-1 The synthetic route of analgin hapten MS-1 is as follows: ; The method comprises the following steps: dissolving 1 equivalent of tert-butyl aminobutyrate in dichloromethane, adding 1.1 equivalents of triethylamine and 1 equivalent of sulfuryl chloride, and stirring overnight. The dichloromethane in the reaction system is removed using a rotary evaporator. Acetonitrile is then added to the reaction flask to dissolve the product, followed by the addition of 1 equivalent of 4-methylaminoantipyrine and 1.1 equivalents of triethylamine, and the reaction is refluxed overnight. After the reaction, 15 volumes of silica gel powder are added to the organic phase and the product is dried 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 ratio). The purified product is dissolved in 15 volumes of 1,4-dioxane, followed by the addition of 3 mL of hydrochloric acid, and stirred at room temperature overnight. After the reaction, the product is dried using a rotary evaporator to obtain the analgin hapten MS-1.

[0060] 1.2 Identification of the Analgin Hapten MS-1 The results of the nuclear magnetic resonance hydrogen spectrum of analgin hapten MS-1 are: 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). The mass spectrometry results of Analgin hapten MS-1 are: + ESI-MS + [MH] + :381.10. It can be seen from the mass spectrometry and nuclear magnetic resonance results that the mass spectrometry results can correspond to the molecular weight of the analgin hapten, and the hydrogen spectrum number of the nuclear magnetic resonance can correspond to the hydrogen spectrum number on the skeleton structure of the analgin hapten, indicating that the analgin hapten was successfully prepared and recorded as MS-1. Its structural formula is shown in formula (I): .

[0061] 2. Synthesis and identification of analgin hapten MS-2 2.1 Synthesis of Metamizole Hapten MS-2 The synthetic route of analgin hapten MS-2 is as follows:

[0062] The method specifically involves dissolving 1 equivalent of antipyrine in 15 volumes of DMF and adding 2 equivalents of phosphorus oxychloride. The reaction is stirred at 100°C overnight. After completion of the reaction, 10 volumes of deionized water are added and the mixture is extracted with ethyl acetate. The organic phases are combined and washed with saturated brine. 15 volumes of silica gel powder are added to the organic phase and the mixture is dried using a rotary evaporator. The dried silica gel powder containing the sample is purified using a column chromatography system (petroleum ether:ethyl acetate:1:3) to obtain the purified metamizole hapten MS-2.

[0063] 2.2 Identification of the Analgin Hapten MS-2 The results of the nuclear magnetic resonance hydrogen spectrum of analgin hapten MS-2 are: 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). The mass spectrometry results of Analgin hapten MS-2 are: + ESI-MS + [MH] + :215.08. It can be seen from the mass spectrometry and nuclear magnetic resonance results that the mass spectrometry results can correspond to the molecular weight of the analgin hapten, and the hydrogen spectrum number of the nuclear magnetic resonance can correspond to the hydrogen spectrum number on the skeleton structure of the analgin hapten, indicating that the analgin hapten was successfully prepared and recorded as MS-2. Its structural formula is shown in formula (II): .

[0064] Example 2 1. Synthesis of Analgin Artificial Antigen 1.1 Synthesis of the artificial analgin antigen MS-1-BSA (1) The analgin 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, dissolved in 100 μL DMF, and stirred at room temperature for 3 h to obtain a hapten activation solution; (2) Dissolve bovine serum albumin (BSA) (10 mg, 0.00015 mol) in 1 mL of PBS buffer (0.01 mol / L, pH = 7.4) to obtain a BSA solution. Stir the BSA solution and hapten activation solution at 4 °C overnight. (3) Collect the reaction solution, transfer it to a treated dialysis bag, and then place it in 5 L of 0.01 M PBS buffer; dialyze it at 4 °C for 3 days, and replace the solution with pre-cooled 0.01 M PBS buffer every 8 hours; (4) After purification by dialysis, the artificial antigen of analgin was obtained, which was recorded as MS-1-BSA. After concentration determination and UV spectral scanning identification, it was stored in a -20 ℃ refrigerator.

[0065] 1.2 Synthesis of the Analgin Artificial Antigen MS-2-LF (1) The analgin 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, dissolved in 100 μL DMF, and stirred at room temperature for 3 h to obtain a hapten activation solution; (2) Dissolve lactoferrin LF in 1 mL PBS buffer (0.01 mol / L, pH = 7.4); (3) Add MS-2 (3.1 mg, 0.012 mol) prepared in Example 1 to the protein solution and add 2 equivalents of sodium borohydride, and stir at 4°C overnight; (4) Collect the reaction solution, transfer it to a treated dialysis bag, and then place it in 5 L of 0.01 M PBS buffer; dialyze it at 4 °C for 3 days, and replace the solution with pre-cooled 0.01 M PBS buffer every 8 hours; (5) After purification by dialysis, the artificial analgin antigen was obtained, which was named MS-2-LF. After concentration determination and UV spectral scanning identification, it was stored in a -20 ℃ refrigerator.

[0066] 2. Identification of Analgin Artificial Antigen BSA, LF, MS-1, MS-2, MS-1-BSA and MS-2-LF were scanned and identified using the full-wavelength UV method (200-350 nm).

[0067] Figure 1 This is the UV scanning identification result diagram of Analgin hapten MS-1, Analgin artificial antigen MS-1-BSA and BSA, as shown in the figure below: Figure 1 As shown in the figure, by comparing the maximum absorbance values ​​of each substance before and after conjugation, it is found that the absorption curve of MS-1-BSA is significantly different from that of the carrier protein BSA. MS-1 has a strong absorption peak above 343 nm. However, after conjugation with BSA, the absorption peak of MS-1-BSA is close to that of BSA at 230 nm and significantly higher than BSA at 280 nm. The curve relative to the hapten MS-1 is significantly shifted. Since the dialysis process after conjugation has completely removed all unreacted components, the characteristic peaks of the conjugation product are contributed by the protein-bound drug molecules, indicating that the reaction product is a complex between the carrier protein BSA and MS-1.

[0068] Figure 2 This is the UV scanning identification result diagram of Analgin hapten MS-2, Analgin artificial antigen MS-2-LF and LF, as shown in the figure below: Figure 2 As shown, the absorption curve of MS-2-LF showed characteristic peaks different from those of LF and MS-2, indicating that the reaction product was a complex of carrier protein LF and MS-2.

[0069] The above results show that the present invention successfully prepared the analgin artificial antigens MS-1-BSA and MS-2-LF. The structure of the analgin 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: .

[0070] Example 3 1. Animal immunization Using the MS-1-BSA prepared in Example 2 as the immunogen, the MS-1-BSA was diluted to 5 mg / mL with 0.01 mol / L PBS. The mixture was then mixed with an equal volume of Freund's complete adjuvant and fully emulsified. Eight-week-old BALB / c female mice were immunized with the vaccine. For the first immunization, three BALB / c female mice were inoculated subcutaneously at multiple points in the abdomen at a dose of 100 μg / mouse antigen, with 0.1 ml administered to each BALB / c female mouse. Fourteen days later, a second immunization was performed using an equal volume of the immunogen emulsified with Freund's incomplete adjuvant at the same dose as the first immunization. Three booster immunizations were performed. Following immunization, tail serum was collected. The titer and inhibition rate were determined. Mice with the best response were selected for a boost immunization at a dose of 100 μg / mouse antigen.

[0071] 2. Cell fusion Three days after the sprint immunization, cell fusion was performed according to the conventional PEG (polyethylene glycol, molecular weight 1450) method. The specific steps are as follows: After killing the mouse by drawing blood from the eyeball, the mouse was immediately sterilized in 75% alcohol and soaked for about 5 minutes. The spleen of the mouse was removed aseptically, and the spleen cell suspension was obtained by moderate grinding with the rubber tip of a syringe and passing through a 200-mesh cell sieve. The spleen cell suspension was collected and centrifuged (1000 rpm, 7 minutes). The spleen cells were washed three times with RPMI-1640 medium. After the final centrifugation, the spleen cells were diluted to a certain volume, counted, and set aside. b. Collect SP2 / 0 cells: 7-10 days before fusion, SP2 / 0 tumor cells were cultured in RPMI1640 medium containing 10% FBS (fetal bovine serum) in a 5% CO2 incubator. Before fusion, the number of SP2 / 0 tumor cells should reach (1-4) × 10 7 , ensure that SP2 / 0 tumor cells are in the logarithmic growth phase before fusion. When fusion occurs, tumor cells are collected, suspended in RPMI-1640 basal culture medium, and cell counts are performed; c. Mix the two cells, adjust the volume to 15 mL, centrifuge (1000 rpm / min, 7 min), and discard the supernatant. Remove the centrifuge tube, shake it in a 60° medium-speed circular motion, and then prepare for fusion. The fusion time is controlled as follows: First minute: slowly add PEG; Second minute: continue to rotate (1 min); Third minute: add 1 mL of basal culture medium; Fourth minute: add 3 mL of basal culture medium; Fifth minute: add 8 mL of basal culture medium; Sixth minute: add 8 mL of basal culture medium; After fusion, the cell fluid was centrifuged (1000 rpm / min, 7 min) and the incubated 200 mL HAT medium was taken out and placed in a clean bench for use.

[0072] d. After centrifugation, discard the supernatant of the hybridoma cells, pipette 2 mL of HAT medium into a centrifuge tube, and gently pipette to evenly distribute the cells. Then transfer the cell solution in the centrifuge tube to 200 mL of HAT medium. Gently shake the medium to evenly disperse the cells, and then plate the cells.

[0073] 3. Cell screening and cell line establishment: On the third day of cell fusion, the fused cells were subjected to a half-change of RPMI-1640 screening medium. On the fifth day, the medium was fully changed with RPMI-1640 transition medium containing 20% ​​fetal bovine serum and 2% 50×HT. On the seventh day, the cell supernatant was collected for screening. The screening process was divided into two steps: the first step was to identify positive cell wells using the ic-ELISA method; the second step was to use allopurinol as a standard and determine the inhibitory effect of the positive cells using the ic-ELISA method; the cell wells that showed good inhibition to the allopurinol standard were selected and subcloned using the limiting dilution method, and then tested using the same method seven days later; three subcloning steps were performed according to the above method, and finally the analgin monoclonal antibody cell line MS-1 was obtained.

[0074] 4. Preparation and identification of analgin monoclonal antibody 8-10 week old BALB / c mice were injected intraperitoneally with 1 mL of sterile paraffin oil. Seven days later, 1×10 6 Starting on the seventh day, ascites fluid was collected from the analgin hybridoma cells and purified by immunoaffinity chromatography. Under neutral conditions, protein G binds to IgG immunoglobulins in the ascites, allowing other impurities to flow out and be discarded. IgG and protein G were then desorbed under acidic conditions using 0.01 M glycine buffer, yielding highly pure IgG immunoglobulins. The IgG immunoglobulin solution was adjusted to a neutral pH using 0.01 M Tris-HCl buffer and then dialyzed for desalination, ultimately yielding highly pure analgin monoclonal antibodies. The purified monoclonal antibodies were stored at -20°C.

[0075] 5. Sensitivity evaluation of analgin monoclonal antibody (indirect competitive ELISA method) The MS-2-LF prepared in Example 2 was used as the coating agent and diluted with coating solution (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 ELISA plate and incubated at 4°C overnight (12 h). Discard the coating solution, wash twice with PBST (0.01 M PBS, 0.06% v / v Tween-20), and pat dry. Add 120 μL of blocking solution (PBST containing 7% skim milk powder) to each well and block at 37°C for 1 h. Discard the blocking solution, clap the plate, dry it at 37°C, remove it from the well, and store it in a ziplock bag for later use. Analgin monoclonal antibodies prepared using MS-1-BSA as an immunogen were diluted with PBST at a volume ratio of 1:128,000. Analgin standards were diluted with PBST to different concentrations, obtaining analgin 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. Add 50 μL / well of different concentrations of analgin standard dilution solution (three sets in parallel), then add 50 μL / well of diluted analgin monoclonal antibody, incubate at 37°C for 40 min, wash five times, and pat dry; Add 100 μL / well of goat anti-mouse secondary antibody-HRP (diluted 5000-fold with PBST), incubate at 37°C for 30 min, wash five times, and pat dry; Add 100 μL / well color development solution and develop for 10 min; The reaction was terminated by adding 50 μL of 10% H2SO4 solution and the OD value was read at 450 nm; ELISA standard curve drawing: take B / B0 as the vertical axis (B is the absorbance value OD of the standard of different concentrations of analgin 450 , B0 is the absorbance value OD of the blank control well 450 ), with the logarithm of the concentration of the analgin standard as the horizontal axis, the logistic function was used for curve fitting to obtain the standard curve and the formula of the standard curve.

[0076] Figure 3 The standard curve for the indirect competitive ELISA method for detecting analgin is constructed based on the analgin monoclonal antibody obtained by immunizing animals with the analgin artificial antigen MS-1-BSA and the analgin artificial antigen MS-2-LF. Figure 3 As shown in the figure, the minimum detection limit LOD of the indirect competitive ELISA method was 2.13 ng / mL, and the half-inhibitory concentration IC 50 The concentration of nitric oxide in the serum was 18.34 ng / mL, and the quantitative detection range was 4.72~71.19 ng / mL.

[0077] 6. Specificity evaluation of analgin monoclonal antibody Naproxen, ibuprofen, and piroxicam are similar drugs to analgin. The specificity of the analgin monoclonal antibody prepared in this example was evaluated by a cross-reaction experiment.

[0078] The sensitivity evaluation method described above was followed, with the only difference being that the allopurinol standard was replaced with naproxen, ibuprofen, and piroxicam standards, and the IC values ​​of each structural analogue were obtained using the same dilution factor. 50 value.

[0079] The cross-reaction rate (CR) of analgin was calculated according to the following formula: CR (%) = IC50 (analgin) / IC50 (similar drug) × 100%. The smaller the cross-reaction rate, the stronger the specificity.

[0080] Table 1 Cross-reaction results of analgin monoclonal antibody with analgin and its analogs ; Note: NR means no reaction, which means the antibody does not recognize the analog.

[0081] As shown in Table 1, the cross-reactivity rate of the analgin monoclonal antibody prepared by the present invention to analgin is 100%, IC 50 The concentration of the antibody was 18.34 ng / mL, with no cross-reactivity for naproxen, ibuprofen, or piroxicam. This indicates that the antibody used to detect analgin has a high recognition ability and specificity for analgin, effectively eliminating interference from analgin-like drugs such as naproxen, ibuprofen, and piroxicam, and can be specifically used for the detection of analgin.

[0082] The above results show that the analgin monoclonal antibody prepared by the present invention has excellent detection performance for analgin, high sensitivity and strong specificity.

[0083] Example 4 Example 4 provides a metamizole artificial antigen combination, including a combination of an immunogen and a coating agent: The analgin artificial antigen MS-2-LF prepared in Example 2 was used as the coating agent, and the analgin monoclonal antibody prepared using MS-1-BSA as the immunogen in Example 3 was used to evaluate the combination effects of different immunogens and coating agents by serum titers and inhibition rates obtained by indirect competitive ELISA. The specific steps are as follows: 1. Dilute the coating agent to a concentration of 1000 ng / mL with coating solution (0.05 M carbonate buffer, pH 9.6). Coat a 96-well microtiter plate at a rate of 100 μL / well. Refrigerate at 4°C overnight. Discard the coating solution and wash twice with PBST (0.01 M PBS, 0.06% Tween-20 (v / v)). 2. Add 120 μL of blocking solution (PBST containing 7% skim milk powder) to each well and block at 37°C for 1 h. Discard the blocking solution, clap the plate, and dry it in a drying oven at 37°C until ready for use. 3. Dilute the analgin monoclonal antibody with PBST at volume ratios of 1:4000, 1:8000, 1:16000, 1:32000, 1:64000, 1:128000, and 1:256000. Set up blank control wells (use PBST instead). Dilute the 1 mg / mL analgin standard 1000-fold with PBST to obtain an analgin standard dilution solution with a concentration of 1 μg / mL. 4. Titer column setup: first add 50 μL PBST to each well, then add 50 μL of analgin monoclonal antibody at different dilution ratios to each well, and finally add 50 μL PBST to the last well instead of the antibody; 5. Inhibition column setup: first add 50 μL of analgin standard dilution to each well, then add 50 μL of analgin monoclonal antibody with different dilution ratios to each well, and finally add 50 μL of PBST to the last well instead of the antibody; 6. Incubate at 37°C for 40 min, wash five times, and clap the plate; 7. Add goat anti-mouse secondary antibody-HRP (diluted 5000-fold with PBST), incubate at 37°C for 30 min, wash 5 times, and clap the plate; 8. Add color developing solution and incubate at 37°C for 10 min; 9. Add 10% v / v H2SO4 to terminate the reaction and read the OD value at 450 nm; calculate the titer and inhibition rate. The titer is OD 450 The antibody dilution factor corresponding to about 1.0, inhibition rate = (OD value of titer - OD value of inhibition) / OD value of inhibition × 100%.

[0084] The present invention also uses the same method as above to prepare the analgin artificial antigens MS-2-OVA, MS-1-LF, and MS-1-OVA, and constructs different combinations of immunogens and coatings. The same method as above is used to evaluate the effects of the combinations of different immunogens and coatings. The results are shown in Table 2.

[0085] Table 2 ELISA test results of immunogen and coating ; As shown in Table 2, the monoclonal antibody prepared with the artificial antigen MS-1-BSA as the immunogen and MS-2-LF is the best immunogen and coating combination. 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.

[0086] Example 5 Example 5 provides an ELISA kit for detecting analgin, as follows: 1. Composition of ELISA kit (1) The ELISA plate coated with the coating agent is prepared by the following method: The MS-2-LF prepared in Example 2 was used as a coating source and diluted to 500 ng / mL with a coating stock solution (0.05 M carbonate buffer, pH 9.6). 100 μL / well of the solution was added to a 96-well ELISA plate and incubated overnight at 4°C in the dark. The liquid in the wells was poured off, and the plates were washed twice with the washing solution in this kit for 30 seconds each time, and patted dry. Then, 120 μL / well of the blocking solution in this kit was added, and the plates were incubated at 37°C in the dark for 1 hour. The liquid in the wells was poured off, patted dry, and after drying, vacuum-sealed with aluminum film for storage. (2) Standards: 9 different concentrations of analgin 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 analgin standard dilutions.

[0087] (3) Antibody: the monoclonal antibody prepared using MS-1-BSA as the immunogen in Example 3; (4) Enzyme-labeled secondary antibody: horseradish peroxidase-labeled goat anti-mouse secondary antibody; (5) Substrate color development solution: It consists of solution A and solution B. Solution A is urea peroxide and solution B is tetramethylbenzidine. (6) Stop solution: 2 mol / L H2SO4; (7) Washing solution: pH 7.4, containing 0.8% Tween-20 by volume, 0.02% sodium azide preservative by mass, and 0.2 mol / L phosphate buffer; dilute the washing solution 20 times with water before use (i.e., add 1 part washing solution to 19 parts water, ready for use) to obtain the working solution of washing solution; (8) Diluent: 0.2 mol / L phosphate buffer; dilute the diluent 20 times with water before use (i.e., add 1 part diluent to 19 parts water, ready for use) to obtain the diluent working solution; (9) Blocking solution: pH 7.3, 0.2 mol / L phosphate buffer containing 2% casein.

[0088] 2. Usage (1) Sample testing Number the corresponding microwells for the samples and the standard in this kit sequentially. Run two parallel wells for each sample and standard, and record the locations of the standard and sample wells. Dilute the antibody working solution at a 1:40 volume ratio (i.e., add 1 part antibody to 40 parts dilution working solution, ready for use) to obtain the antibody working solution. Dilute the enzyme-labeled secondary antibody working solution at a 1:10 volume ratio (i.e., add 1 part enzyme-labeled secondary antibody to 10 parts dilution working solution, ready for use) to obtain the enzyme-labeled secondary antibody working solution.

[0089] Add 50 μL of standard or sample to the corresponding microwells, then add 50 μL of antibody working solution to the corresponding microwells, gently shake to mix, cover the plate with a cover film, and react at 25°C in a dark environment for 40 min.

[0090] Pour the liquid from the wells dry and add 250 μL / well of working wash solution. Wash thoroughly 4–5 times, 10 seconds apart. Discard the working wash solution and pat dry with absorbent paper. (Use an unused pipette tip to pop any bubbles that remain after patting dry.)

[0091] Add 100 μL / well of enzyme-labeled secondary antibody working solution to the corresponding microwells, gently shake to mix, cover the plate with a cover film, and then react in a dark environment at 25°C for 30 min.

[0092] Pour the liquid from the wells dry and add 250 μL / well of working wash solution. Wash thoroughly 4–5 times, 10 seconds apart. Discard the working wash solution and pat dry with absorbent paper. (Use an unused pipette tip to pop any bubbles that remain after patting dry.)

[0093] Add 50 μL / well of substrate color development solution A, then add 50 μL / well of substrate color development solution B, gently shake to mix, cover the plate with cover film, and then react at 25°C in a dark environment for 10 min.

[0094] Add 50 μL / well of stop solution, gently shake to mix, set the microplate reader at 450 nm, and measure the OD value of each well.

[0095] (2) Drawing of the standard curve Take B / B0 as the vertical axis (B is the absorbance value OD of the standard with different concentrations 450 , B0 is the absorbance value OD of the blank control well 450 ), the logarithm of the concentration of the standard was used as the horizontal axis, and the logistic function was used for curve fitting to obtain the standard curve.

[0096] (3) Calculation of sample concentration The sample OD 450Substitute the average value into the formula of the standard curve to obtain the concentration of the sample, and then multiply it by the corresponding dilution factor to obtain the actual concentration of analgin in the test sample.

[0097] In summary, the present invention provides two analgin haptens with spacer arms of appropriate lengths, and based on them, prepares artificial antigens and antibodies. The obtained analgin monoclonal antibodies have high titer, strong specificity, and high affinity, providing core raw materials for establishing a specific analgin immunoassay method. The present invention also establishes an analgin immunoassay method with high specificity and sensitivity, with a minimum detection limit LOD of 2.13 ng / mL and a half inhibitory concentration IC of 2.13 ng / mL. 50 The concentration of analgin is 18.34 ng / mL, and the quantitative detection range is 4.72~71.19 ng / mL. It has no cross reaction to similar drugs of analgin. It can perform rapid qualitative and quantitative detection of analgin in samples. The operation is simple and the test results are accurate and reliable.

[0098] In addition, the inventors of this case also referred to the aforementioned embodiments and conducted experiments using other raw materials, process operations, and process conditions described in this specification, and obtained relatively ideal results.

[0099] The various aspects, embodiments, features and examples of the present invention should be considered as illustrative and not intended to limit the present invention, the scope of which is defined solely by the claims. Other embodiments, modifications and uses will be apparent to those skilled in the art without departing from the spirit and scope of the present invention.

[0100] Although the present invention has been described with reference to illustrative embodiments, it will be understood by those skilled in the art that various other changes, omissions and / or additions may be made without departing from the spirit and scope of the present invention, and that substantial equivalents may be substituted for the elements of the embodiments. Additionally, many modifications may be made to adapt specific circumstances or materials to the teachings of the present invention without departing from the scope of the present invention. Therefore, it is not intended herein to limit the present invention to the specific embodiments disclosed, but rather to include all embodiments within the scope of the appended claims. Furthermore, unless specifically stated, any use of the terms first, second, etc. does not indicate any order or importance, but rather uses the terms first, second, etc. to distinguish one element from another.

Claims

1. A metamizole hapten, characterized in that: Its structure is shown in formula (I) or formula (II): 。 2. The method for preparing the analgin hapten according to claim 1, wherein include: A first mixed reaction system containing tert-butyl aminobutyrate, triethylamine and sulfuryl chloride is subjected to a first reaction at a temperature of 0-35° C. to obtain a first intermediate product; a second mixed reaction system containing the first intermediate product, 4-methylaminoantipyrine and triethylamine is subjected to a second reaction at a temperature of 0-35° C. to obtain a second intermediate product; and a third mixed reaction system containing the second intermediate product and an acid is subjected to a third reaction at a temperature of 0-35° C. to obtain a metamizole hapten having a structure represented by formula (I); Alternatively, the fourth mixed reaction system containing antipyrine and phosphorus oxychloride is subjected to a fourth reaction at a temperature of 80-120° C. to obtain the analgin hapten having a structure as shown in formula (II).

3. The method for preparing the analgin hapten according to claim 2, wherein: The molar equivalents of tert-butyl aminobutyrate, triethylamine, and sulfuryl chloride in the first mixed reaction system satisfy 1:1-3:1-3; And / or, the molar equivalents of the first intermediate product, 4-methylaminoantipyrine, and triethylamine in the second mixed reaction system satisfy 1:1-3:1-3; and / or, the molar equivalent of the acid to the second intermediate product satisfies 1-6:1; And / or, the first reaction time is 1-3h; And / or, the second reaction time is 1-3h; And / or, the third reaction time is 1-3h; And / or, the molar equivalent of antipyrine to phosphorus oxychloride in the fourth mixed reaction system satisfies 1:1-3; And / or, the fourth reaction time is 1-6 hours.

4. An artificial analgin antigen, characterized in that: It is obtained by coupling the analgin hapten-carrier protein according to claim 1, and the structure of the analgin artificial antigen is shown in formula (III) or formula (IV): ； Among them, Protein is a carrier protein.

5. The analgin artificial antigen according to claim 4, characterized in that: The carrier protein is any one of bovine serum albumin, lactoferrin, chicken ovalbumin, and human serum albumin.

6. A metamizole antibody, characterized in that: The method is obtained by immunizing an animal with the analgin artificial antigen as claimed in claim 4 or 5.

7. The analgin antibody according to claim 6, characterized in that: The analgin antibody is a monoclonal antibody; And / or, the analgin antibody is obtained by immunizing an animal with an analgin artificial antigen having a structure represented by formula (III) and a carrier protein being bovine serum albumin.

8. A metamizole artificial antigen combination, characterized in that: The invention comprises an immunogen and a coating agent, wherein the structure of the immunogen is as shown in formula (III) of claim 4 and the carrier protein is bovine serum albumin, and the structure of the coating agent is as shown in formula (IV) of claim 4 and the carrier protein is lactoferrin.

9. Use of the analgin hapten according to claim 1, the analgin artificial antigen according to claim 4 or 5, the analgin antibody according to claim 6 or 7, or the analgin artificial antigen according to claim 8 in combination in the preparation of an analgin detection product or in the detection of analgin for non-disease treatment and diagnosis purposes.

10. A kit for detecting analgin, characterized in that: The invention comprises the analgin artificial antigen according to claim 4 or 5, and the analgin antibody according to claim 6 or 7.

11. The kit according to claim 10, characterized in that: The structure of the analgin artificial antigen is as shown in formula (IV) of claim 4, and the carrier protein is lactoferrin. The analgin antibody is obtained by immunizing an animal with the analgin artificial antigen as shown in formula (III) of claim 4, and the carrier protein is bovine serum albumin; And / or, the analgin antibody is a monoclonal antibody; And / or, the kit is an ELISA kit.

12. An immunoassay detection method for Analgin, characterized in that: The analgin artificial antigen of claim 4 or 5 and the analgin antibody of claim 6 or 7 are used to detect analgin in a sample.

13. The immunoassay detection method of Analgin according to claim 12, wherein: The structure of the analgin artificial antigen is represented by formula (IV) in claim 4, and the carrier protein is lactoferrin. The analgin antibody is obtained by immunizing an animal with the analgin artificial antigen represented by formula (III) in claim 4, and the carrier protein is bovine serum albumin. And / or, the analgin antibody is a monoclonal antibody; And / or, the immunoassay detection method is ELISA.

Images