Allopurinol hapten and its preparation method, artificial antigen, antibody and its application

Abstract

This invention provides an allopurinol hapten, its preparation method, an artificial antigen, an antibody, and their applications. The structure of the allopurinol hapten is shown below. The allopurinol hapten provided by this invention has a spacer arm of suitable length, which allows the characteristic structure of the target molecule to be far away from the carrier protein, maximizing the exposure of the hapten to the immune system while minimizing recognition of the spacer arm, resulting in better antibody recognition of allopurinol. This invention uses this allopurinol hapten to prepare artificial antigens and antibodies, obtaining allopurinol monoclonal antibodies with high titer, strong specificity, and high affinity. The immunoassay method based on this allopurinol artificial antigen and antibody has high specificity and sensitivity, and no cross-reactivity with allopurinol analogs, enabling rapid qualitative and quantitative detection of allopurinol.

Description

Technical Field

[0001] This invention belongs to the field of detection technology, specifically relating to an allopurinol hapten and its preparation method, artificial antigens, antibodies and their applications. Background Technology

[0002] Allopurinol (1H-pyrazolo[3,4-d]pyrimidin-4-ol; C5H4N4O) is a commonly used drug, primarily used to treat gout and hyperuricemia. It lowers blood uric acid levels by inhibiting xanthine oxidase, thus reducing uric acid production.

[0003] Allopurinol can easily cause various adverse reactions. For example, it can increase the burden on the kidneys by affecting uric acid metabolism and excretion. If patients with renal insufficiency use allopurinol, it may lead to further aggravation of kidney damage. Simultaneously, allopurinol may cause uric acid crystals to accumulate in the kidneys, leading to kidney stones or acute renal failure. Furthermore, research has found that HLA-B... The percentage of carriers of the 58:01 gene is relatively high, and patients carrying this gene have a significantly increased risk of allergic reactions when using allopurinol.

[0004] However, unscrupulous merchants often illegally add allopurinol to functional foods for treating gout. Therefore, in order to strengthen the supervision of allopurinol, it is necessary to establish a simple, sensitive, and accurate detection method for allopurinol. Summary of the Invention

[0005] To solve all or part of the above-mentioned technical problems, the present invention provides the following technical solutions:

[0006] A first aspect of the present invention provides an allopurinol hapten, the structure of which is shown in formula (I) or formula (II):

[0007] .

[0008] The two allopurinol haptens provided by this invention have spacer arms of suitable length. Following the coupling protein, both allow the characteristic structure of the target molecule to be kept away from the carrier protein. This maximizes the exposure of the hapten to the immune system while minimizing recognition of the spacer arm, resulting in superior antibody recognition of allopurinol. If the spacer arm of the hapten is too short, the carrier protein may mask the characteristic structure of the analyte, leading to a poor antibody response. Conversely, if the spacer arm is too long, the hapten molecule may undergo conformational folding, making it more susceptible to masking by the carrier protein and facilitating the generation of antibodies against the spacer arm.

[0009] A second aspect of the present invention provides a method for preparing the allopurinol hapten.

[0010] The preparation method of the allopurinol hapten with the structure shown in formula (I) includes:

[0011] The first mixed reaction system containing allopurinol, sodium hydride and tert-butyl 4-bromobutyrate was subjected to a first reaction at 0-35°C to obtain the first intermediate compound;

[0012] The second mixed reaction system containing the first intermediate compound and the acid was subjected to a second reaction at 0-35°C to obtain the allopurinol hapten with the structure shown in formula (I).

[0013] In some embodiments, the molar equivalents of allopurinol, sodium hydride, and tert-butyl 4-bromobutyrate satisfy a ratio of 1-3:1-3:1.

[0014] In some embodiments, the first reaction takes 1-6 hours.

[0015] In some embodiments, the second reaction takes 1-6 hours.

[0016] In some embodiments, the molar equivalent of the acid and the first intermediate compound is 1-6:1. The acid may be hydrochloric acid, but is not limited thereto.

[0017] In some embodiments, the solvent of the first mixed reaction system includes N,N-dimethylformamide, but is not limited thereto.

[0018] In some embodiments, the solvent of the second mixed reaction system includes, but is not limited to, 1,4-dioxane.

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

[0020] The third reaction was carried out at 0-35°C in a third mixed reaction system containing theophylline, sodium hydride and tert-butyl bromoacetate to obtain the second intermediate compound;

[0021] The fourth mixed reaction system containing the second intermediate compound and the acid was subjected to a fourth reaction at 0-35°C to obtain the allopurinol hapten with the structure shown in formula (II).

[0022] In some embodiments, the molar equivalents of theophylline, sodium hydride, and tert-butyl bromoacetate satisfy a ratio of 1-3:1-3:1.

[0023] In some embodiments, the third reaction takes 1-6 hours.

[0024] In some embodiments, the fourth reaction takes 1-6 hours.

[0025] In some embodiments, the molar equivalent of the acid and the first intermediate compound is 1-6:1. The acid may be hydrochloric acid, but is not limited thereto.

[0026] In some embodiments, the solvent of the third mixed reaction system includes N,N-dimethylformamide, but is not limited thereto.

[0027] In some embodiments, the solvent of the fourth mixed reaction system includes, but is not limited to, 1,4-dioxane.

[0028] A third aspect of the present invention provides an allopurinol artificial antigen, which is obtained by conjugating the allopurinol hapten to a carrier protein, the structure of which is shown in formula (III) or formula (IV):

[0029] ;

[0030] Protein is a carrier protein.

[0031] The allopurinol artificial antigen of formula (III) is obtained by conjugating the allopurinol hapten of formula (I) with a carrier protein. The allopurinol artificial antigen of formula (IV) is obtained by conjugating the allopurinol hapten of formula (II) with a carrier protein. The method of conjugating the carrier protein may be, for example, the active ester method or any method known in the art.

[0032] In some embodiments, the carrier protein is any one of hemocyanin, chicken ovalbumin, bovine lactoferrin, or human serum albumin, but is not limited thereto.

[0033] In some embodiments, the structure of the allopurinol artificial antigen is shown in formula (Ⅲ) and the carrier protein is hemocyanin.

[0034] In some embodiments, the structure of the allopurinol artificial antigen is shown in formula (Ⅳ) and the carrier protein is chicken ovalbumin.

[0035] A fourth aspect of the present invention provides an allopurinol antibody obtained by immunizing animals with the allopurinol artificial antigen described in any of the technical solutions.

[0036] In some embodiments, the allopurinol antibody is a monoclonal antibody.

[0037] In some embodiments, the allopurinol antibody is obtained by immunizing animals with an allopurinol artificial antigen of formula (III) with hemocyanin as the carrier protein.

[0038] A fifth aspect of the present invention provides an allopurinol 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 hemocyanin, and the structure of the coating antigen is shown in Formula (IV) and the carrier protein is chicken ovalbumin. The monoclonal antibody prepared based on this combination not only specifically recognizes the target analyte allopurinol, but also exhibits superior antibody sensitivity and high specificity for recognizing the target analyte allopurinol.

[0039] The sixth aspect of the present invention provides the use of the allopurinol hapten, the allopurinol artificial antigen, the allopurinol antibody, or the allopurinol artificial antigen combination described in any of the technical solutions in the preparation of allopurinol detection products or in the detection of allopurinol for the purpose of non-disease treatment diagnosis.

[0040] A seventh aspect of the present invention provides a kit for detecting allopurinol, the kit comprising the allopurinol artificial antigen and the allopurinol antibody as described in any of the technical solutions.

[0041] In some embodiments, the structure of the allopurinol artificial antigen is as shown in formula (Ⅳ) and the carrier protein is chicken ovalbumin, and the allopurinol antibody is obtained by immunizing animals with the allopurinol artificial antigen of formula (Ⅲ) and the carrier protein is hemocyanin.

[0042] In some embodiments, the allopurinol antibody is a monoclonal antibody.

[0043] In some embodiments, the kit is an ELISA kit.

[0044] In some embodiments, the ELISA kit further includes an enzyme-labeled plate, allopurinol standards, and a substrate chromogenic solution, wherein the allopurinol artificial antigen is coated on the enzyme-labeled plate.

[0045] In some embodiments, the substrate developing solution includes urea peroxide and tetramethylbenzidine.

[0046] 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.

[0047] 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.

[0048] 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.

[0049] In some embodiments, the blocking solution comprises 1%–3% casein and 0.1 mol / L–0.3 mol / L phosphate buffer, with a pH of 7.1–7.5. In some preferred embodiments, the blocking solution comprises 2% casein and 0.2 mol / L phosphate buffer, with a pH of 7.3.

[0050] In some embodiments, the enzyme-labeled secondary antibody is a horseradish peroxidase-labeled goat anti-mouse antibody.

[0051] 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.

[0052] 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.

[0053] An eighth aspect of the present invention provides an immunoassay method for detecting allopurinol, wherein the immunoassay method uses the allopurinol artificial antigen and the allopurinol antibody as described in any of the technical solutions for detection. The immunoassay method is for non-disease treatment diagnosis purposes.

[0054] In some embodiments, the structure of the allopurinol artificial antigen is as shown in formula (Ⅳ) and the carrier protein is chicken ovalbumin, and the allopurinol antibody is obtained by immunizing animals with the allopurinol artificial antigen of formula (Ⅲ) and the carrier protein is hemocyanin.

[0055] In some embodiments, the allopurinol antibody is a monoclonal antibody.

[0056] In some embodiments, the immunoassay method is ELISA. For example, it can be indirect ELISA and / or competitive ELISA. In some preferred embodiments, the immunoassay method is indirect competitive ELISA.

[0057] Compared with the prior art, the present invention has at least the following beneficial effects:

[0058] (1) The two allopurinol 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, so as to generate better antibodies to recognize allopurinol.

[0059] (2) The present invention uses allopurinol hapten with a suitable length of spacer arm to prepare artificial antigen, and immunizes animals to obtain allopurinol monoclonal antibody. The obtained antibody has high titer, strong specificity and high affinity, providing core raw materials for establishing an immunoassay method for specific detection of allopurinol.

[0060] (3) The allopurinol immunoassay method based on the allopurinol artificial antigen and antibody provided by this invention has high sensitivity and strong specificity, with a limit of detection (LOD) of 0.57 ng / mL and a half-inhibitory concentration (IC50) of 1 / 2. 50 The concentration is 5.90 ng / mL, and the quantitative detection range is 1.35~25.67 ng / mL. It has no cross-reactivity with similar drugs of allopurinol. It can perform rapid qualitative and quantitative detection of allopurinol in samples. The operation is simple and the detection results are accurate and reliable. Attached Figure Description

[0061] 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.

[0062] Figure 1 The image shows the ultraviolet full-wavelength scan identification of allopurinol hapten AP-1, allopurinol artificial antigen AP-1-KLH and hemocyanin KLH prepared in the embodiments of the present invention.

[0063] Figure 2 The image shows the ultraviolet full-wavelength scan identification of allopurinol hapten AP-2, allopurinol artificial antigen AP-2-OVA and chicken ovalbumin OVA prepared in the embodiments of the present invention.

[0064] Figure 3This is a standard curve diagram of the indirect competitive ELISA method for detecting allopurinol constructed based on allopurinol monoclonal antibody and allopurinol artificial antigen AP-2-OVA in an embodiment of the present invention. Detailed Implementation

[0065] 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.

[0066] 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.

[0067] Unless otherwise specified, the "equivalent" used to indicate the amount of raw materials in the following specific embodiments of this application refers to molar equivalent.

[0068] Example 1

[0069] This embodiment provides two allopurinol haptens and their synthesis methods, and identifies the synthesized allopurinol haptens.

[0070] 1. Synthesis of allopurinol hapten AP-1

[0071] The synthetic route for the allopurinol hapten AP-1 is as follows:

[0072] ;

[0073] Specifically, the process involved: suspending 1.2 equivalents of allopurinol in 20 volumes of DMF, adding 1.2 equivalents of sodium hydride, and stirring at room temperature until the solution became clear; then adding 1 equivalent of tert-butyl 4-bromobutyrate and stirring overnight at room temperature; after the reaction was complete, quenching the reaction with deionized water and extracting with ethyl acetate; adding 15 volumes of silica gel powder to the organic phase and drying it using a rotary evaporator; purifying the dried silica gel powder containing the sample using a column chromatography system (petroleum ether: ethyl acetate: formic acid: 50:50:1); dissolving the purified product in 15 volumes of 1,4-dioxane and adding 3 mL of hydrochloric acid, stirring overnight. After the reaction was complete, the mixture was filtered and washed with deionized water to obtain the allopurinol hapten AP-1.

[0074] 2. Identification of allopurinol hapten AP-1

[0075] The 1H NMR spectrum of the allopurinol hapten AP-1 prepared above is as follows: 1H NMR (600 MHz, DMSO- d 6) δ 12.59 (s, 1H), 12.06 (d, J = 6.8 Hz, 1H), 8.33 (s, 1H), 8.08 (d, J =6.4 Hz, 1H), 5.10 (s, 2H).

[0076] The mass spectrometry results for the allopurinol hapten are as follows: + ESI-MS + [MH] + 193.04.

[0077] The mass spectrometry and NMR results show that the mass spectrometry results correspond to the molecular weight of the allopurinol hapten, and the number of protons in the NMR spectrum corresponds to the number of protons in the allopurinol hapten backbone structure. This indicates that the allopurinol hapten, denoted as AP-1, was successfully prepared, and its structural formula is shown in formula (I).

[0078]

[0079] 3. Synthesis of allopurinol hapten AP-2

[0080] The synthetic route for the allopurinol hapten AP-2 is shown below:

[0081]

[0082] 1.2 equivalents of theophylline were suspended in 20 volumes of DMF, and 1.2 equivalents of sodium hydride were added. The mixture was stirred at room temperature until the solution became clear. Then, 1 equivalent of tert-butyl bromopropionate was added, and the mixture was stirred overnight at room temperature. After the reaction was complete, the reaction was quenched with deionized water, and the mixture was extracted with ethyl acetate. 15 volumes of silica gel powder were added to the organic phase, and the mixture was evaporated to dryness using a rotary evaporator. The dried silica gel powder containing the sample was purified using a column chromatography system (petroleum ether: ethyl acetate: formic acid: 70:50:1). The purified product was dissolved in 15 volumes of 1,4-dioxane, and 3 mL of hydrochloric acid was added. The mixture was stirred overnight at room temperature. After the reaction was complete, the mixture was filtered and washed with deionized water to obtain the allopurinol hapten AP-2.

[0083] 4. Identification of allopurinol hapten AP-2

[0084] The 1H NMR spectrum of the allopurinol hapten is as follows: 1 H NMR (600 MHz, DMSO- d 6) δ 12.35 (s,1H), 7.89 (s, 1H), 4.35 (t, J= 7.2 Hz, 2H), 3.46 (s, 3H), 3.38 (s, 3H), 2.79(t, J = 7.2 Hz, 2H).

[0085] The mass spectrometry results for the allopurinol hapten are as follows: + ESI-MS + [MH] + 250.07.

[0086] The mass spectrometry and NMR results show that the mass spectrometry results correspond to the molecular weight of the allopurinol hapten, and the number of protons in the NMR spectrum corresponds to the number of protons in the allopurinol hapten backbone structure. This indicates that the allopurinol hapten, denoted as AP-2, was successfully prepared, and its structural formula is shown in formula (II).

[0087]

[0088] Example 2

[0089] This embodiment provides an allopurinol artificial antigen and its synthesis method, and identifies the synthesized artificial antigen.

[0090] 1. Synthesis of allopurinol artificial antigens AP-1-KLH and AP-2-OVA

[0091] (1) The allopurinol hapten AP-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 solution was dissolved in 100 μL DMF and stirred at room temperature for 3 h to obtain the hapten activation solution.

[0092] (2) Dissolve hemocyanin (KLH, 10 mg, 0.00015 mol) in 1 mL PBS buffer (0.01 mol / L, pH=7.4) to obtain KLH solution. Stir the KLH solution and hapten activation solution at 4 °C overnight.

[0093] (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;

[0094] (4) Dialyze at 4 ℃ for 3 days, and change the solution with pre-cooled 0.01M PBS buffer every 8 hours;

[0095] (5) After dialysis purification, the allopurinol artificial antigen, denoted as AP-1-KLH, was obtained and stored in a -20 ℃ refrigerator after concentration determination and ultraviolet spectroscopy scanning identification.

[0096] AP-2-OVA was prepared according to the above method, with the only difference being that OVA (chicken egg albumin) was used instead of KLH; and the allopurinol hapten AP-2 obtained in Example 1 was used to replace AP-1 to prepare the allopurinol artificial antigen, which was denoted as AP-2-OVA.

[0097] 2. Identification of allopurinol artificial antigen

[0098] KLH, OVA, AP-1, AP-2, AP-1-KLH and AP-2-OVA were identified by scanning using the ultraviolet full-wavelength method (200-350nm).

[0099] Figure 1 The images show the UV full-wavelength scan identification of allopurinol hapten AP-1, allopurinol artificial antigen AP-1-KLH, and hemocyanin KLH, as follows. 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 AP-1-KLH is significantly different from that of the carrier protein KLH. AP-1 has a strong absorption peak above 343 nm, while after conjugation with KLH, the absorption peak of AP-1-KLH is similar to that of KLH at 230 nm, but significantly higher than that of KLH at 280 nm, and the curve relative to the hapten AP-1 shows a significant shift. Since all unreacted components have been removed by dialysis during the post-conjugation dialysis process, the characteristic peaks of the conjugation product are contributed by the protein-bound drug molecules, indicating that the reaction product is a complex of the carrier protein KLH and AP-1.

[0100] Figure 2 The images show the UV full-wavelength scan identification of allopurinol hapten AP-2, allopurinol artificial antigen AP-2-OVA, and chicken ovalbumin OVA. Similarly, the absorption curve of AP-2-OVA shows characteristic peaks that are different from those of OVA and AP-2, indicating that the reaction product is a complex of carrier protein OVA and AP-2.

[0101] The above results indicate that the present invention successfully prepared allopurinol artificial antigens AP-1-KLH and AP-2-OVA. The structural formula of allopurinol artificial antigen AP-1-KLH is shown in formula (III) and the carrier protein is KLH. The structural formula of allopurinol artificial antigen AP-2-OVA is shown in formula (IV) and the carrier protein is OVA.

[0102]

[0103] Example 3

[0104] Example 3 provides an allopurinol monoclonal antibody and its preparation method.

[0105] 1. Animal immunization

[0106] Using AP-1-KLH prepared in Example 2 as the immunogen, AP-1-KLH was diluted to 5 mg / mL with 0.01 mol / L PBS, then mixed with an equal volume of Freund's complete adjuvant and thoroughly emulsified. This mixture was then used to immunize 8-week-old female BALB / c mice. For the first immunization, three female BALB / c mice were subcutaneously inoculated at multiple sites in the abdomen, with an antigen dose of 100 μg / mouse and 0.1 ml per mouse. A second immunization was performed 14 days later, emulsified with an equal volume of Freund's incomplete adjuvant and the immunogen, using the same dose as the first immunization. Three booster immunizations were administered. After immunization, tail serum was collected. Its titer and inhibition rate were determined. Mice with the best results were selected for a pulse immunization, with an antigen dose of 100 μg / mouse.

[0107] 2. Cell fusion

[0108] 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:

[0109] 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 and centrifuge (1000 rpm, 7 min). Wash the spleen cells three times with RPMI-1640 medium. After the last centrifugation, dilute the spleen cells to a certain volume, count them, and set them aside for later use.

[0110] 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.

[0111] c. Mix the two cells together, bring the volume to 15 mL, centrifuge (1000 rpm / min, 7 min), and discard the supernatant.

[0112] Remove the centrifuge tubes, rotate them in a circular motion at a medium speed of 60°, and then begin the fusion process. The fusion time should be controlled as follows:

[0113] First minute: Slowly add PEG dropwise;

[0114] Second minute: Continue rotating (1 minute);

[0115] Third minute: Add 1 mL of basal culture medium;

[0116] Fourth minute: Add 3 mL of basal culture medium;

[0117] Fifth minute: Add 8 mL of basal culture medium;

[0118] Sixth minute: Add 8 mL of basal culture medium;

[0119] After fusion, the cell culture was centrifuged (1000 rpm / min, 7 min), and the 200 mL HAT culture medium was removed and placed in a clean bench for later use.

[0120] 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.

[0121] 3. Cell selection and cell line establishment:

[0122] 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.

[0123] 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 subcloning was performed using the limiting dilution method. The cells were tested using the same method after seven days. Subcloning was performed three times using the above method to finally obtain the allopurinol monoclonal antibody cell line AP-1.

[0124] 4. Preparation and identification of allopurinol monoclonal antibodies

[0125] 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 Allopurinol hybridoma cells were used, and ascites fluid was collected starting on day 7. The ascites fluid was then purified for antibody removal via immunoaffinity chromatography. Under neutral conditions, Protein G bound to IgG immunoglobulins in the ascites fluid, causing other impurities to efflux 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 allopurinol monoclonal antibodies. The purified monoclonal antibodies were stored at -20 °C.

[0126] 5. Sensitivity evaluation of allopurinol monoclonal antibody (indirect competitive ELISA method)

[0127] Using the allopurinol artificial antigen AP-2-OVA prepared in Example 2 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, and 100 μL / well was used to coat a 96-well microplate. The plate was incubated overnight (12 h) at 4°C.

[0128] 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 store in a resealable bag for later use.

[0129] Allopurinol monoclonal antibody prepared using AP-1-KLH as an immunogen was diluted with PBST at a volume ratio of 1:128000. Allopurinol standard was diluted with PBST to different concentrations, resulting in allopurinol standard dilutions with concentrations of 1000 ng / mL, 500 ng / mL, 60 ng / mL, 25 ng / mL, 8 ng / mL, 5 ng / mL, 1.6 ng / mL, 0.8 ng / mL, 0.4 ng / mL, 0.15 ng / mL, 0.015 ng / mL, and 0 ng / mL.

[0130] Add 50 μL / well of allopurinol standard dilution buffer at different concentrations (three sets in parallel), then add 50 μL / well of diluted allopurinol monoclonal antibody, incubate at 37 ℃ for 40 min, wash five times, and pat dry;

[0131] 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;

[0132] Add 100 μL / well of colorimetric reagent and develop for 10 min;

[0133] The reaction was terminated by adding 50 μL of 10% H2SO4 solution, and the OD value was read at 450 nm.

[0134] ELISA standard curve plotting: Plot B / B0 as the ordinate (B represents the absorbance OD of standards at different concentrations of allopurinol). 450 B0 is the absorbance value (OD) of the blank control well. 450 A standard curve was prepared by fitting the logarithm of the allopurinol standard concentration to the logarithm of the concentration on the x-axis using the Logistic function, as shown below. Figure 3As shown. The limit of detection (LOD) of the indirect competitive ELISA method based on the above-mentioned allopurinol monoclonal antibody and AP-2-OVA is 0.57 ng / mL, the half-maximum inhibitory concentration (IC50) is 5.90 ng / mL, and the quantitative detection range is 1.35~25.67 ng / mL.

[0135] 6. Specificity evaluation of allopurinol monoclonal antibody

[0136] Benzbromarone, febuxostat, and probenecid are allopurinol gout analogs. The specificity of the allopurinol monoclonal antibody prepared in this example was evaluated by cross-reactivity experiments.

[0137] Following the sensitivity evaluation method described above, the only difference is that allopurinol standards are replaced with standards for benzbromarone, febuxostat, and probenecid, and the same dilution factors are used for detection to obtain the IC50 values ​​for each structural analog. 50 value.

[0138] The cross-reactivity rate (CR) of allopurinol is calculated using the following formula: CR (%) = IC 50 (Allopurinol) / IC50 (similar drug) × 100%. The lower the cross-reactivity rate, the stronger the specificity.

[0139] Table 1. Cross-reactivity results of allopurinol monoclonal antibodies with allopurinol and its analogues.

[0140] ;

[0141] Note: NR indicates no reaction, meaning the antibody does not recognize the analogue.

[0142] As shown in Table 1, the cross-reactivity of allopurinol monoclonal antibody to allopurinol was 100%, and the IC50 was [missing information]. 50 The concentration was 5.90 ng / mL, with no cross-contamination with benzbromarone, febuxostat, or probenecid. This indicates that the antibody used to detect allopurinol has high recognition ability and specificity for allopurinol, effectively eliminating interference from the detection of gout analogs such as benzbromarone, febuxostat, and probenecid, and can be specifically used for the detection of allopurinol.

[0143] The above results indicate that the allopurinol monoclonal antibody prepared in this invention exhibits excellent detection performance, high sensitivity, and strong specificity for allopurinol.

[0144] Example 4

[0145] Example 4 provides a combination of allopurinol immunogen and coating antigen.

[0146] Using the allopurinol artificial antigen AP-2-OVA prepared in Example 2 as the coating antigen, and the allopurinol monoclonal antibody prepared in Example 3 using AP-1-KLH 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:

[0147] 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)).

[0148] 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;

[0149] 3. Dilute the allopurinol 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 allopurinol standard 1000 times with PBST to obtain a 1 μg / mL allopurinol standard dilution solution;

[0150] 4. Titer column setup: First, add 50 μL of PBST to each well, then add 50 μL of allopurinol monoclonal antibody at different dilution ratios to each well, and finally add 50 μL of PBST to the last well instead of the antibody.

[0151] 5. Inhibition column setup: First, add 50 μL of allopurinol standard dilution buffer to each well, then add 50 μL of allopurinol monoclonal antibody at different dilution ratios to each well, and finally add 50 μL of PBST to the last well instead of antibody.

[0152] 6. Incubate at 37℃ for 40 min, wash 5 times, and then plate.

[0153] 7. Add goat anti-mouse secondary antibody-HRP (diluted 5000 times with PBST), incubate at 37 ℃ for 30 min, wash 5 times, and plate.

[0154] 8. Add the color developing solution and incubate at 37 ℃ for 10 min;

[0155] 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. 450The antibody dilution factor corresponding to approximately 1.0 is: inhibition rate = (OD value of titer - OD value of inhibition) / OD value of inhibition × 100%.

[0156] The present invention also uses AP-1 conjugated with BSA, OVA and LF to prepare artificial antigens AP-1-BSA, AP-1-OVA and AP-1-LF, and uses the same method as above to evaluate different combinations of immunogens and coating antigens. The results are shown in Table 2.

[0157] Table 2. ELISA detection results of immunogens and coating agents.

[0158] ;

[0159] As shown in Table 2, the monoclonal antibody prepared using the allopurinol artificial antigen AP-1-KLH as an immunogen and AP-2-OVA is the optimal combination of immunogen and coating agent. Under this combination, the monoclonal antibody can not only specifically recognize the target analyte allopurinol, but also has good antibody sensitivity and can be used to specifically recognize the target analyte allopurinol.

[0160] Example 5

[0161] Example 5 provides an ELISA kit for detecting allopurinol.

[0162] 1. Composition

[0163] (1) The enzyme-labeled plate coated with the coating agent is prepared by the following method:

[0164] AP-2-OVA prepared in Example 2 was used as the coating agent and diluted to 500 ng / mL with the coating stock solution (0.05 M carbonate buffer, pH 9.6). 96-well microplates were coated with 100 μL / well 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, then patted dry. Blocking buffer from this kit was then added at 120 μL / well, and the plates were incubated at 37°C in the dark for 1 h. The liquid in the wells was discarded, and the plates were patted dry and then vacuum-sealed with an aluminum foil for storage.

[0165] (2) Standards: 12 allopurinol standards of different concentrations, and allopurinol standard dilutions of 1000 ng / mL, 500 ng / mL, 60 ng / mL, 25 ng / mL, 8 ng / mL, 5 ng / mL, 1.6 ng / mL, 0.8 ng / mL, 0.4 ng / mL, 0.15 ng / mL, 0.015 ng / mL and 0 ng / mL.

[0166] (3) Antibody: Allopurinol monoclonal antibody prepared in Example 3;

[0167] (4) Enzyme-labeled secondary antibody: Horseradish peroxidase-labeled goat anti-mouse secondary antibody;

[0168] (5) Substrate colorimetric solution: composed of solution A and solution B, where solution A is urea peroxide and solution B is tetramethylbenzidine;

[0169] (6) Termination solution: 2 mol / L H2SO4;

[0170] (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;

[0171] (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;

[0172] (9) Blocking solution: pH 7.3, 0.2 mol / L phosphate buffer containing 2% casein.

[0173] 2. Instructions for use

[0174] (1) Sample testing

[0175] 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.

[0176] 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.

[0177] 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).

[0178] 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.

[0179] 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).

[0180] 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.

[0181] 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.

[0182] (2) Drawing the standard curve

[0183] 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.

[0184] (3) Calculation of sample concentration

[0185] 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 allopurinol in the sample.

[0186] In summary, this invention provides two allopurinol haptens with appropriately long spacer arms, and based on these, prepares allopurinol artificial antigens and monoclonal antibodies. The obtained monoclonal monomers exhibit high titers, strong specificity, and high affinity, providing core raw materials for establishing a specific immunoassay method for allopurinol. The immunoassay method established based on the allopurinol artificial antigens and monoclonal antibodies provided by this invention demonstrates excellent specificity and high sensitivity, with a limit of detection (LOD) of 0.57 ng / mL and a half-inhibitory concentration (IC50). 50 The concentration is 5.90 ng / mL, and the quantitative detection range is 1.35~25.67 ng / mL. It has no cross-reactivity with allopurinol analogues and can perform rapid qualitative and quantitative detection of allopurinol in samples. The operation is simple and the detection results are accurate and reliable.

[0187] 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.

[0188] 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.

[0189] 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 substantially equivalents can be substituted for elements of the described embodiments. Furthermore, many modifications can be made without departing from the scope of the invention to adapt particular situations or materials to the teachings of the invention. Therefore, this invention is not intended to be limited to performing the specific embodiments disclosed, but rather to encompass all embodiments falling within the scope of the appended claims. Moreover, unless specifically stated otherwise, any use of the terms first, second, etc., does not indicate any order or importance, but is used to distinguish one element from another.

Claims

1. A combination of allopurinol artificial antigens, characterized in that, It includes an immunogen and a coating antigen, wherein the structure of the immunogen is shown in formula (III) and the carrier protein is hemocyanin, and the structure of the coating antigen is shown in formula (IV) and its carrier protein is chicken ovalbumin; 。 2. The application of the allopurinol artificial antigen combination according to claim 1 in the preparation of allopurinol detection products.

3. The use of the allopurinol artificial antigen combination according to claim 1 in the detection of allopurinol for the purpose of non-disease treatment diagnosis.

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