Xanthine derivative, immunogen and specific antibody thereof, and preparation method of xanthine detection kit

Abstract

The invention relates to preparation of a xanthine (Xanthine) detection kit by using a chemiluminescence immunoassay technology. Xanthine is purine widely existing in body fluid, tissues and plant cells and is small in molecular weight and poor in immunogenicity, and when a corresponding antibody is prepared, a hapten needs to be coupled with a specific macromolecular carrier to prepare a complete antigen. According to the invention, the antibody with good specificity and strong affinity is successfully prepared by designing and transforming the hapten. The invention mainly relates to design and synthesis of a xanthine hapten, preparation of a xanthine complete antigen and an anti-xanthine antibody, a method for measuring the concentration of xanthine, and composition and components of a reagent. The design and synthesis of the hapten mainly comprise the design and chemical synthesis of xanthine derivatives. The xanthine derivative disclosed by the invention has a structure as shown in a formula (I): # imgabs0 #, wherein R is-(CH2) n-COOH, and n is any integer between 1 and 10.

Description

Technical Field

[0001] The present invention relates to the field of biomedical immunoassays, and in particular to a xanthine derivative, a xanthine immunogen, a specific antibody thereof, and a xanthine detection kit. Background Art

[0002] Xanthine (Xanthine), its structural formula is shown in formula (III):

[0003]

[0004] Xanthine, also known as 2,6-dihydroxypurine, has a molecular formula of C5H4N4O2 and a molecular weight of 152.11. It is a purine widely found in body fluids, tissues, and plant cells. Xanthines and their derivatives are a group of alkaloids commonly used as mild stimulants and bronchodilators, particularly for treating asthma or flu symptoms. Compared to more potent stimulants such as sympathomimetics, xanthines primarily act to antagonize adenosine and enhance central nervous system alertness. Normally, hypoxanthine is metabolized to xanthine by the enzyme xanthine oxidase, which further metabolizes it into uric acid and superoxide, which are then excreted in the urine. A rare hereditary condition called xanthinuria results in xanthine accumulation in the urine and blood, ultimately leading to renal failure. Recent studies have shown that elevated xanthine levels can cause ischemic damage, making xanthine an effective marker for detecting tissue hypoxia. Early detection of xanthine changes in biological fluids is crucial for metabolic studies, diagnosis, and treatment monitoring. Furthermore, xanthine has been found in patients with urinary stones. When adenosine monophosphate is degraded to uric acid, hypoxanthine is oxidized to form xanthine. Measurements of xanthine and hypoxanthine in plasma and urine have begun to be used to monitor the efficacy of allopurinol therapy. During the treatment of hydrocephalus, cerebrospinal fluid (CSF) xanthine and hypoxanthine levels have been used as treatment guidance and markers of disease progression.

[0005] Currently, xanthine detection mainly includes colorimetry, which is cumbersome to operate, time-consuming and labor-intensive, with poor accuracy and easily interfered with. In clinical testing, it is impossible to take into account both accuracy and timeliness at the same time. The present invention starts with antigen design and antibody preparation, and adopts an advanced fully automatic chemiluminescence detection platform, which completely solves the pain points of xanthine clinical testing and has broad application prospects. This detection reagent can be used for high-throughput, automated, multi-type sample determination, and has the outstanding advantages of high sensitivity, specificity and good stability in reagent performance. Summary of the Invention

[0006] The purpose of the present invention is to provide a xanthine derivative, a xanthine immunogen and a specific antibody thereof, and a xanthine detection kit to improve the defects of the existing xanthine determination method in terms of poor specificity, low sensitivity, and inability to perform automated analysis.

[0007] According to one aspect of the present invention, there is provided a xanthine derivative having the structural formula shown in formula (I):

[0008]

[0009] Wherein, R is a connecting group -(CH2)n-COOH, and n is any integer between 1 and 10. The xanthine derivative of the present invention has a basic structure for preparing a xanthine immunogen with immunogenicity, and provides a structural basis for preparing a new xanthine detection reagent.

[0010] According to another aspect of the present invention, there is also provided a xanthine immunogen having the structural formula shown in formula (II):

[0011]

[0012] Wherein, R is a linking group -(CH2)n-COOH, n is any integer between 1 and 10, and the carrier is an immunogenic protein or polypeptide. The xanthine immunogen of the present invention has high immunogenicity, can stimulate an immune response in an animal body, and produces high-titer anti-xanthine-specific antibodies with strong antibody affinity, making it suitable for preparing a highly sensitive and specific xanthine competitive detection reagent.

[0013] When n=1, R in the xanthine immunogen is -CH2-COOH. Suitable carriers for the xanthine immunogen are preferably protein carriers, although other immunogenic substances with sufficiently large molecular weight and sufficient reactive groups can also serve as carriers. The most commonly used immunogenic carriers include serum proteins, keyhole limpet hemocyanin (KLH), thyroglobulin, and polylysine. The carrier in the present invention is preferably keyhole limpet hemocyanin.

[0014] According to another aspect of the present invention, there is also provided an anti-xanthine-specific antibody produced by immunizing an animal with an immunogen. The antibody is produced by immunizing an animal with any of the aforementioned xanthine immunogens. The "antibody" referred to in the present invention refers not only to antibodies against intact protein molecules, but also includes antibodies against polypeptide fragments or derivatives of polypeptide fragment antibodies that retain the specific binding ability of intact antibodies. The antibodies of the present invention may be polyclonal antibodies, monoclonal antibodies, or recombinant antibodies, preferably monoclonal antibodies.

[0015] The antibodies of the present invention can be prepared using existing techniques. A typical method for obtaining polyclonal antibodies is to use a single immunogen, with or without an adjuvant, to immunize one or more sites in an animal, such as rabbits, goats, mice, sheep, guinea pigs, horses, alpacas, or camels. Immunizations are repeated 5-7 times until the antibody titer reaches a maximum. Blood is collected from the animal at regular intervals to obtain an appropriate amount of specific antiserum. Monoclonal antibodies can be produced using hybridoma cell technology. Recombinant antibodies can be produced using genetic engineering expression techniques.

[0016] According to another aspect of the present invention, a method for preparing a xanthine immunogen is provided, comprising the steps of preparing the aforementioned xanthine derivative and linking the aforementioned xanthine derivative to a carrier to obtain the xanthine immunogen. The xanthine derivative prepared by the aforementioned method is linked to an immunogenic protein or polypeptide to obtain the xanthine immunogen of the present invention having strong immunogenicity. This method is simple to operate.

[0017] In the method for preparing the xanthine immunogen of the present invention, a method for preparing the xanthine derivative is also provided, which comprises the following steps: step 1, subjecting xanthine to a substitution reaction with a halogenated -(CH2)n-COO-tert-butyl ester in an N,H-dimethylformamide environment to obtain 2-(CH2)n-COO-tert-butyl ester xanthine; step 2, dissolving the 2-(CH2)n-COO-tert-butyl ester xanthine in a strong acid solution to obtain a xanthine derivative. This preparation method replaces the hydrogen at position 2 of xanthine with a halogenated -(CH2)n-COO-tert-butyl ester to generate 2-(CH2)n-COO-tert-butyl ester xanthine, and then, in an acidic environment, opens the 2-(CH2)n-COO-tert-butyl ester bond on the 2-(CH2)n-COO-tert-butyl ester xanthine to form a xanthine derivative having -(CH2)n-COOH at position 2 of xanthine. The preparation method is simple to operate, has mild reaction conditions, high stability and good repeatability.

[0018] In the above-mentioned preparation step 1 of the xanthine derivative, the following steps are further included: Step 1-1, dissolving xanthine and halo-(CH2)n-COO-tert-butyl ester in N,N-dimethylformamide (DMF) to obtain a mixed solution; Step 1-2, adding ethyl acrylate to the mixed solution to obtain a solid-liquid mixture; Step 1-3, filtering the solid-liquid mixture to obtain 2-(CH2)n-COO-tert-butyl ester xanthine. Xanthine and halo-(CH2)n-COO-tert-butyl ester have good solubility in the organic solvent DMF. Ethyl acrylate is added to precipitate 2-(CH2)n-COO-tert-butyl ester xanthine, and filtering to obtain the reactant.

[0019] In step 1-1 above, after obtaining the mixed solution, the mixed solution is further heated to a constant temperature, preferably at a temperature of 70 to 100° C. for at least 32 hours. Heating the mixed solution to a constant temperature for at least 32 hours can ensure sufficient dissolution and reaction.

[0020] In the present invention, in the above steps 1-2, the reaction mixture is stirred and solidified at a low temperature of 0 to 8° C. to increase the yield.

[0021] In the above-mentioned method for preparing xanthine derivatives of the present invention, there are no special requirements for the specific operation of filtering the solid-liquid mixture to obtain 2-(CH2)-COO-tert-butyl xanthine, as long as the desired target product can be separated from the solid-liquid mixture. In the present invention, the above-mentioned steps 1-3 include filtering the solid-liquid mixture to obtain a solid substance; washing and purifying the solid substance with acetone to obtain a purified product; and vacuum drying the purified product to obtain 2-(CH2)n-COO-tert-butyl xanthine. After the steps of filtering, washing, purifying, and vacuum drying, the obtained 2-(CH2)n-COO-tert-butyl xanthine has a relatively high purity and yield.

[0022] In the above-mentioned method for preparing xanthine derivatives of the present invention, the specific operation process of the step of dissolving 2-(CH2)n-COO-tert-butyl xanthine in a strong acid solution to obtain a xanthine derivative can be appropriately adjusted according to the difference between 2-(CH2)n-COO-tert-butyl xanthine and the acid solution. In the present invention, preferably, the above-mentioned step 2 includes: dissolving 2-(CH2)n-COO-tert-butyl xanthine in a strong acid solution to obtain a reaction solution; stirring the reaction solution at 40-60°C for 2.5-6.5 hours to obtain a stirred liquid; drying the stirred liquid to obtain a dried product; washing and purifying the dried product with acetone to obtain a xanthine derivative. Stirring at a high temperature of about 60°C can promote the formation of the target xanthine derivative. The dried product containing the target xanthine derivative is obtained by evaporation and drying. After washing with acetone, the organic residue on the dried product is removed to obtain the target xanthine derivative with higher purity.

[0023] In the preparation method of the xanthine derivative of the present invention, when n=1, the preparation steps of the xanthine derivative are as follows:

[0024]

[0025] When n=1, the reaction steps are the same as above, except that tert-butyl bromoacetate is used as the synthetic raw material. Therefore, the linker group R of the final product xanthine derivative is -CH2-COO-.

[0026] In the above-mentioned method for preparing xanthine immunogens of the present invention, the step of connecting the carrier and the xanthine derivative can be reasonably improved according to the different carriers in actual operation. In the present invention, the above-mentioned connection step includes: step 2-1, preparing a carrier solution and a xanthine derivative solution; wherein the mass ratio of the carrier to the xanthine derivative is 1 to 8:1; preferably, the carrier is serum protein, keyhole limpet hemocyanin, thyroglobulin or polylysine; step 2-2, adding the activated xanthine derivative solution dropwise to the carrier solution to obtain a crude xanthine immunogen; step 2-3, stirring the added mixture at 2 to 10°C overnight or reacting at room temperature for 2 hours to obtain a crude xanthine immunogen; step 2-4, purifying the crude xanthine immunogen to obtain a xanthine immunogen. The preparation steps of the present invention can obtain the target product through simple activation, dropwise addition, and purification steps. The preparation method is simple, the process stability is high, and the reproducibility is good.

[0027] In the above-mentioned method for preparing the xanthine immunogen of the present invention, during the actual operation of preparing the carrier solution and the xanthine derivative solution, appropriate solvent concentrations and pH values are rationally selected based on the type of carrier. In the above-mentioned step 2-1 of the present invention, the carrier solution is prepared by dissolving the carrier in a 0.05-0.20 M phosphate buffer solution at a pH of 8.0-9.5 to obtain a carrier solution; the xanthine derivative solution is prepared by placing the xanthine derivative, 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide, and N-hydroxysulfosuccinimide in N,N-dimethylformamide, methanol, and a 5-20 mM potassium phosphate buffer solution at a pH of 4.0-6.0, stirring at room temperature to obtain an activated xanthine derivative solution. Dissolving the carrier in a phosphate buffer solution at a concentration of 0.10-0.25 M and a pH of 8.0-9.5 allows the amino groups on the carrier and the carboxyl groups of the activated xanthine derivative to fully react and bind in a slightly alkaline environment. The carboxyl group of the xanthine derivative can be fully activated in a slightly acidic solution by using a phosphate buffer solution with a concentration of 5 to 20 mM and a pH of 4.0 to 6.0.

[0028] In the method for preparing the xanthine immunogen of the present invention, in the step of dropwise addition to obtain a crude xanthine immunogen, in order to further increase the xanthine immunogen content in the crude xanthine immunogen, the dropwise addition in steps 2-2 and 2-3 of the present invention allows the xanthine derivative to react more fully with the carrier; and stirring overnight at 2-10° C. further promotes the formation of the xanthine immunogen, thereby increasing the xanthine immunogen content in the crude product.

[0029] In the above-mentioned method for preparing a xanthine immunogen of the present invention, any operation capable of purifying a crude xanthine immunogen to obtain the xanthine immunogen is applicable to the present invention. Preferably, in steps 2-4 above, the crude xanthine immunogen is purified by dialysis to obtain the xanthine immunogen. Dialysis is a simple method and has a good purification effect.

[0030] According to another aspect of the present invention, a xanthine detection reagent is provided, comprising an anti-xanthine-specific antibody, a xanthine enzyme-labeled conjugate, and an enzyme substrate, wherein the anti-xanthine-specific antibody is any of the aforementioned anti-xanthine-specific antibodies; and the xanthine enzyme-labeled conjugate contains the aforementioned xanthine derivative. The xanthine enzyme-labeled conjugate is formed by coupling an enzyme and a hapten, wherein the hapten is the aforementioned xanthine derivative.

[0031] The xanthine detection reagent of the present invention has a detection sensitivity far superior to that of corresponding products in the prior art due to the high specificity of the anti-xanthine-specific antibody and its strong binding affinity with xanthine. Preferably, the enzyme-labeled conjugate is an alkaline phosphatase-hapten enzyme-labeled conjugate; and the substrate of the enzyme is AMPPD or APS-5. Detection reagents using the alkaline phosphatase-hapten enzyme-labeled conjugate and AMPPD or APS-5 as enzyme substrates can conveniently and accurately determine the xanthine content in a sample and are suitable for high-throughput automated detection.

[0032] According to another aspect of the present invention, a xanthine detection kit is also provided, comprising the above-mentioned anti-xanthine-specific antibody and an indicator reagent for detecting the anti-xanthine-specific antibody and the xanthine complex. The indicator reagent is selected from an enzyme reagent, a radioisotope reagent, a fluorescent reagent, and a luminescent reagent. Preferably, the indicator reagent comprises a xanthine enzyme-labeled conjugate and an enzyme substrate, wherein the xanthine enzyme-labeled conjugate can be coupled to a xanthine derivative of the present invention, enabling convenient and accurate determination of the xanthine content in a sample, and being suitable for high-throughput automated detection.

[0033] The technical solution of the present invention is applied to a xanthine derivative obtained by replacing hydrogen at a specific site with a specific R group, and the xanthine immunogen formed by connecting it to a specific carrier has high immunogenicity, high specificity of antibodies produced by immune-induced animals, and strong specific binding ability to xanthine. High-throughput and rapid detection of xanthine can be achieved on a fully automatic chemiluminescence immunoassay using alkaline phosphatase chemiluminescence immunoassay technology, and has the advantages of simple operation, high sensitivity, strong specificity, and accurate results. It can also effectively reduce the cost of xanthine detection, which is conducive to clinical promotion and use. BRIEF DESCRIPTION OF THE DRAWINGS

[0034] Figure 1 : Standard curve of xanthine ELISA detection.

[0035] Figure 2 : Calibration curve of xanthine alkaline phosphatase chemiluminescence.

[0036] Figure 3 : The xanthine chemiluminescence detection reagent of the present invention is compared with the xanthine ELISA detection reagent of a well-known foreign manufacturer for sample comparison results. DETAILED DESCRIPTION

[0037] It should be noted that, in the absence of conflict, the embodiments and features of the embodiments of the present invention can be combined with each other. The present invention will be described in detail below with reference to the accompanying drawings and in combination with the embodiments.

[0038] Example 1: Synthesis and structural confirmation of xanthine derivatives

[0039] The chemical structure of the xanthine derivatives used in the following examples is shown in formula (IV):

[0040]

[0041] The specific synthesis steps of the xanthine derivative represented by formula (IV) are as follows:

[0042] 1. Synthesis of Compound 2

[0043]

[0044] 15.0 g of compound 1 and 26 g of tert-butyl bromoacetate were weighed and dissolved in 150 mL of N,N-dimethylformamide (DMF). The solution was then heated to 95°C overnight. 500 mL of ethyl acrylate (EA) was added to this solution, and the reaction mixture was stirred at 0°C for 25 minutes. The solid precipitate was filtered, washed with acetone, and vacuum-dried to obtain 9.0 g of compound 2 as a brown solid, with a yield of 24%.

[0045] 2. Synthesis of xanthine derivatives

[0046]

[0047] (1) 3.00 g of compound 2 was weighed and dissolved in 30 mL of 1.5 M HCl. The solution was stirred at 60°C for 3 hours. The reaction mixture was evaporated to dryness, and the dried residue was washed with acetone to obtain 1.00 g of a xanthine derivative as a reddish-brown solid with a yield of 47%.

[0048] (2) The compound was subjected to nuclear magnetic resonance spectroscopy using a Varian III plus 300 MHz spectrometer with TMS as the internal standard. The result showed that the compound was 2-(CH2)2-COO-tert-butyl ester xanthine.

[0049] Example 2: Preparation steps of derivatives when n=2

[0050] 1. Synthesis of 2-(CH2)2-COO-tert-butyl ester xanthine

[0051] (1) 18.0 g of compound 1 and 27.8 g of tert-butyl bromopropionate were weighed and dissolved in 150 mL of N,N-dimethylformamide (DMF). The solution was then heated to 95°C overnight. 550 mL of ethyl acrylate (EA) was added to the solution, and the reaction mixture was stirred at 4°C for 25 min. The solid precipitate in the solution was filtered, washed with acetone, and vacuum-dried to obtain 4.20 g of the intermediate product as a brown solid with a yield of 13%.

[0052] (2) The compound was subjected to nuclear magnetic resonance spectroscopy using a Varian III plus 300 MHz spectrometer with TMS as the internal standard. The result showed that the compound was 2-(CH2)2-COO-tert-butyl ester xanthine.

[0053] 2. Synthesis of xanthine derivatives

[0054] (1) 3.00 g of the above-mentioned 2-(CH2)2-COO-tert-butyl ester xanthine was weighed and dissolved in 30 mL of 1.5 M HCl. The solution was stirred at 60°C for 2 hours. The reaction mixture was evaporated to dryness, and the dried residue was washed with acetone to obtain 2.0 g of a xanthine derivative as a reddish-brown solid with a yield of 50.2%.

[0055] (2) Structural identification of the purified product obtained above

[0056] a. The above compound was subjected to NMR spectroscopy scanning using Varian III plus 300 MHz, using TMS as the internal standard, and the results were characterized as 2-(CH2)2-COO-xanthine.

[0057] b. The obtained derivative was analyzed and identified by chromatography / mass spectrometry (LC / MS), and it was determined that the final compound was 2-(CH2)2-COO-xanthine.

[0058] Example 3: When n=3, the preparation steps of xanthine derivatives:

[0059] 1. Synthesis of 2-(CH2)3-COO-tert-butyl ester xanthine

[0060] (1) 18.0 g of compound 1 and 29.5 g of tert-butyl bromobutyrate were weighed and dissolved in 180 mL of N,N-dimethylformamide (DMF). The solution was then heated to 95°C overnight. 525 mL of ethyl acrylate (EA) was added to the solution, and the reaction mixture was stirred at 5°C for 30 min. The solid precipitate in the solution was filtered, washed with acetone, and vacuum-dried to obtain 3.70 g of compound 2 as a white solid with a yield of 11.2%.

[0061] (2) The compound was subjected to nuclear magnetic resonance spectroscopy using a Varian III plus 300 MHz spectrometer with TMS as the internal standard. The result showed that the compound was 2-(CH2)3-COO-tert-butyl ester xanthine.

[0062] 2. Synthesis of xanthine derivatives

[0063] (1) 3.00 g of compound 2 was weighed and dissolved in 30 mL of 1.5 M HCl. The solution was stirred at 55°C for 4.5 hours. The reaction mixture was evaporated to dryness, and the dried residue was washed with acetone to obtain 1.1 g of a xanthine derivative as a reddish-brown solid with a yield of 47%.

[0064] (2) Structural identification of the purified product obtained above

[0065] a. The compound was subjected to nuclear magnetic resonance spectroscopy using a Varian III plus 300 MHz spectrometer with TMS as the internal standard. The result showed that the compound was 2-(CH2)3-COO-xanthine.

[0066] b. The obtained derivative was analyzed and identified by chromatography / mass spectrometry (LC / MS), and it was determined that the final compound was 2-(CH2)3-COO-xanthine.

[0067] In the above embodiment, when n=1, 2 and 3, tert-butyl bromoacetate, tert-butyl bromopropionate and tert-butyl bromobutyrate were selected as the synthetic raw materials in the synthesis of the intermediate compounds in the preparation of the xanthine derivatives, so the linking groups R1 of the final products of the xanthine derivatives were -CH2-COO-, -(CH2)2-COO- and -(CH2)3-COO-. When n is another integer between 1 and 10, when other tert-butyl brominated organic acid esters similar to tert-butyl bromoacetate are used for experiments, the synthesis method is exactly the same except for the different value of n.

[0068] Example 4: Synthesis of BSA-xanthine derivative immunogens

[0069] The BSA-xanthine immunogen is formed by linking bovine serum albumin (BSA) with the -(CH2)n-COO- group of a xanthine derivative represented by formula (I). In this embodiment, the synthesis method of the immunogen is described in detail using n=1 as an example. The specific steps are as follows:

[0070] 1. Dissolve bovine serum albumin (20 mg) in 5 ml of 0.2 M phosphate buffer, pH 8.5;

[0071] 2. Add the following chemicals to a small beaker and stir to dissolve: 20 mg of the synthesized xanthine derivative, 0.3 ml of D,D-dimethylformamide (DMF), 0.3 ml of methanol, 1.0 ml of 10 mM, pH 5.0 phosphate buffer, 20 mg of 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide (EDAc), and 2.5 mg of N-hydroxysulfosuccinimide (Sulfo-NHS). Stir and dissolve these chemicals at room temperature for 10 minutes.

[0072] 3. Add the activated solution dropwise to the BSA solution and stir overnight at 2-10°C to obtain a crude antigen product; purify the synthesized antigen by dialysis to obtain 2-acetoxyxanthine immunogen.

[0073] Example 5: Synthesis of KLH-xanthine derivative immunogens

[0074] The KLH-acetylxanthine immunogen is formed by linking hemocyanin (KLH) with the -(CH2)n-COO- group of an acetylxanthine derivative represented by formula (I). In this embodiment, the synthesis method of the immunogen is described in detail using n=2 as an example. The specific steps are as follows:

[0075] 1. Dissolve hemocyanin (20 mg) in 5 ml of 0.18 M phosphate buffer, pH 8.5;

[0076] 2. Add the following chemicals to a small beaker and stir to dissolve: 20 mg of the synthesized xanthine derivative, 0.3 ml of D, D-dimethylformamide (DMF), 0.3 ml of methanol, 1.0 ml of 10 mM, pH 5.0 phosphate buffer, 20 mg of 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide (EDAc), and 2 mg of N-hydroxysulfosuccinimide (Sulfo-NHS). Stir and dissolve these chemicals at room temperature for 10 min.

[0077] 3. Add the activated solution dropwise to the KLH solution and stir overnight at 2-10°C to obtain a crude xanthine immunogen. Purify the synthesized antigen by dialysis to obtain 2-propionic acid xanthine immunogen.

[0078] Example 6: Synthesis of thyroglobulin-xanthine derivative immunogen

[0079] The thyroglobulin-xanthine immunogen is formed by linking thyroglobulin with the -(CH2)n-COO- group of a xanthine derivative represented by formula (I). In this embodiment, the synthesis method of the immunogen is described in detail using n=3 as an example. The specific steps are as follows:

[0080] 1. Dissolve thyroglobulin (20 mg) in 5 ml of 0.20 M phosphate buffer, pH 9.0;

[0081] 2. Add the following chemicals to a small beaker and stir to dissolve: 10 mg of the synthesized xanthine derivative, 0.3 ml of N,N-dimethylformamide (DMF), 0.3 ml of methanol, 1.0 ml of 20 mM, pH 5.0 potassium phosphate buffer, 20 mg of 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide (EDAc), and 2.0 mg of N-hydroxysulfosuccinimide (Sulfo-NHS). Stir and dissolve these chemicals at room temperature for 30 min.

[0082] 3. Add the activated solution dropwise to the thyroglobulin solution and stir overnight at 2-10°C to obtain the complete antigen; purify the synthesized antigen by dialysis to obtain 2-butyryl xanthine immunogen.

[0083] Example 7: Synthesis of polylysine-xanthine derivative immunogens

[0084] The polylysine-xanthine immunogen is formed by linking polylysine with the -(CH2)n-COO- group of a xanthine derivative represented by formula (I). In this embodiment, the synthesis method of the immunogen is described in detail using n=3 as an example. The specific steps are as follows:

[0085] 1. Dissolve poly-lysine (20 mg) in 5 ml of 0.20 M, pH 9.0 phosphate buffer;

[0086] 2. Add the following chemicals to a small beaker and stir to dissolve: 10 mg of the synthesized xanthine derivative, 0.3 ml of N,N-dimethylformamide (DMF), 0.3 ml of methanol, 1.0 ml of 20 mM, pH 5.0 potassium phosphate buffer, 20 mg of 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide (EDAc), and 2.0 mg of N-hydroxysulfosuccinimide (Sulfo-NHS). Stir and dissolve these chemicals at room temperature for 30 min.

[0087] 3. Add the activated solution dropwise to the poly-lysine solution and stir overnight at 2-10°C to obtain the complete antigen; centrifuge the synthesized antigen to obtain the supernatant, and purify the supernatant by dialysis to obtain 2-butyryl xanthine immunogen.

[0088] Similarly, when n is other integers ranging from 1 to 10, xanthine immunogens such as those shown in formula (II) can be prepared using the same method, and the experimental results show no significant differences. Xanthine immunogens prepared using xanthine derivatives with different n values all exhibit strong immunogenicity, and the corresponding specific antibodies all have excellent performance. Of course, the carrier is still an immunogenic protein, which can be serum protein, keyhole limpet hemocyanin (KLH), thyroglobulin, and polylysine. Preferably, the carrier is keyhole limpet hemocyanin.

[0089] Example 8: Preparation of anti-xanthine specific monoclonal antibodies

[0090] This example provides a method for preparing a xanthine monoclonal antibody, which comprises the following steps:

[0091] 1. Animal immunization

[0092] Two eight-week-old female Balb / C mice were immunized with the artificial antigen prepared in Example 5, using multiple subcutaneous injections at the nape of the neck. The primary immunization used the immunogen emulsified in Freund's complete adjuvant (CFA). Equal volumes of the immunogen and CFA were mixed and emulsified, with a single mouse dose of 0.1 mg. After the primary immunization, booster immunizations were administered every 14 days at a dose of 0.1 mg using the same emulsification method. Five immunizations were performed, starting with the third booster immunization. Seven days after each immunization, 30 μL of blood was collected from the tail of the mice, centrifuged, and stored at -20°C for titer and specificity determination.

[0093] 2. ELISA indirect enzyme-linked immunosorbent assay to analyze the effect of antiserum

[0094] Using a conventional antibody titer determination method, blank serum without antibody was used as a control, and the antiserum was diluted a certain multiple and then subjected to ELISA detection. The final detection showed that the titer of the anti-xanthine specific antibody of the present invention was 1:30,000-1:50,000, indicating that the antibody prepared by the present invention has strong specificity and high sensitivity.

[0095] 3. Cell fusion and screening of positive hybridomas

[0096] (1) Resuscitation of myeloma cells: Take the myeloma cells out of liquid nitrogen and quickly thaw them in a 37°C water bath. Centrifuge at 1000 rpm for 5 minutes after thawing. Pour off the supernatant in a clean bench and add about 1 mL of complete culture medium to the cell pellet. Blow away the cells. Use a pipette to take out the cells and mix them with complete culture medium. Place them in a 10 cm diameter culture dish and expand to 4 to 6 dishes. Change the medium several times during this period. When the cells cover the bottom of each culture dish, they can be used for cell fusion.

[0097] (2) Cell preparation: Take out two small culture dishes, pour part of the culture medium into one to cool the dissection tools, and absorb a small amount of culture medium into the other and put it into a cell sieve for grinding the spleen. Blow down the revived myeloma cells and transfer them to a 50mL centrifuge tube, seal it, and centrifuge it at 1200r / min for 5 minutes. Kill the mice after five immunizations, soak them in 75% alcohol for about 1 minute, put them in a clean bench, take blood from the heart, warm them at 37℃ for 30 minutes, centrifuge them for 15 minutes, and take the serum and store it at -20℃. After taking the mouse spleen cells, grind them thoroughly in a cell sieve, wash them with the pre-packaged basic solution, and transfer them to a 50mL centrifuge tube, seal it, discard the supernatant of the centrifuged myeloma cells, add the basic solution to wash them again, and centrifuge them together with the spleen cells at 1200r / min for 5 minutes. After the second centrifugation, discard the supernatant of the myeloma cells, add 2mL of basic solution, and blow them evenly. Remove the supernatant from the spleen cells, pass them through a cell sieve, and add them to the myeloma cells. After pipetting evenly, add basal medium to 20 mL and centrifuge at 1200 rpm for 5 minutes. Remove the centrifuged mixed cells and discard the supernatant. Aspirate the excess medium with a pipette and shake the precipitated cells to disperse. Then, incubate the mixture in a 37°C cell culture incubator for 5 minutes.

[0098] (3) Cell fusion: After the incubation, place the centrifuge tube in 37°C warm water and keep rotating. Use the pipette tip to absorb 1mL of PEG preheated to 37°C. Slowly add PEG to the precipitated cells within the first minute. Let it stand for 1 minute. Preheat the basal culture medium. Add 1mL within the third minute, 3mL within the fourth minute, and 16mL within the fifth to sixth minutes. Stir gently and add along the wall to separate the PEG. Seal the centrifuge tube and centrifuge at 900r / min for 8 minutes. Pour off the supernatant and add the fused cells to HAT complete culture medium. Stir gently and evenly add to four 24-well culture plates. Keep the volume of HAT culture medium containing fused cells the same in each well.

[0099] (4) Screening of positive hybridomas: within 4 days after fusion, half of the medium was replaced with HT medium. After 8 days, the medium was replaced with HT medium in each well. On the 10th day, the supernatant in the multi-well culture plate was extracted and the specific antibodies in the culture medium were detected by indirect ELISA. The positive hybridoma cells with high titer and strong affinity were selected. The positive wells with the best fusion effect were screened and marked. Under sterile conditions, the cells were transferred to a new 96-well culture plate. Each original well was cloned into two 96-well plates. After the cells adhered to the wall and covered 1 / 4 of the bottom of the well, the supernatant was taken and tested by ELISA. The titer and inhibition rate were also used as measurement indicators. The strong positive cells were subcloned by limiting dilution method. This was repeated 3 to 4 times (note that the positive well cells selected in each round need to be expanded and cultured and then frozen for future use). Until each well on each plate is positive and the titer and inhibition are similar after detection, the hybridoma cell line was successfully established and a hybridoma cell line that can stably secrete uniform antibodies was obtained. Single cell clones were picked and those that tested positive were transferred to 24-well cell culture plates, 6-well cell culture plates, and 10 cm cell culture dishes for expansion and freezing.

[0100] 4. Large-scale preparation of monoclonal antibodies

[0101] After obtaining hybridoma cell clones that secrete specific monoclonal antibodies, large-scale monoclonal antibodies are typically produced using in vitro culture and in vivo monoclonal antibody induction methods. Ten or more Balb / c mice aged 8 weeks or older are injected intraperitoneally with liquid slurry at a dose of 0.5 mL per mouse. One to two weeks later, hybridoma cells are injected into the peritoneal cavity of the mice. The mice are observed daily after cell inoculation, especially starting on day 7, when the abdominal cavity will swell. Before the mice die, ascites is collected using a sterile disposable syringe. The collected ascites is centrifuged at 12,000 rpm for 10 minutes to remove the upper fat layer and the lower fibrin layer. The middle layer is collected and the titer and inhibition rate are determined by ELISA. After purification, the xanthine monoclonal antibody is stored at -20°C until further use.

[0102] Example 9: Xanthine ELISA test

[0103] The titer and specificity of the antiserum were determined by ELISA indirect enzyme-linked immunosorbent assay, and the steps are as follows:

[0104] 1. ELISA test for xanthine using the antibody prepared in Example 8

[0105] This test uses a competitive immunoassay to measure the xanthine content in a liquid sample. The principle is that xanthine in the sample competes with a conjugated xanthine derivative (HRP-xanthine derivative enzyme conjugate) for binding to the limited sites on the antibody coated on the ELISA plate. If there is little or no xanthine in the liquid sample, the xanthine derivative conjugated to the HRP enzyme will bind to the antibody in the ELISA plate. Conversely, if the liquid sample contains a large amount or a certain amount of xanthine, the enzyme-xanthine derivative conjugate will reduce its binding to the antibody, thereby weakening the color signal. Therefore, the absorbance generated by the test is inversely proportional to the xanthine content in the liquid sample.

[0106] 2. The specific steps for establishing the standard curve of xanthine ELISA are as follows:

[0107] (1) Preparation of standard products

[0108] Xanthine powder (purchased from Merck) was dissolved in methanol to prepare a 1 mg / mL stock solution. The stock solution was diluted sequentially with ELISA buffer containing 50.0 mM Tris, 1000 mM NaCl, 333.00 ng / mL, 111.00 ng / mL, 37.00 ng / mL, 12.30 ng / mL, 4.11 ng / mL, and 0.00 ng / mL standard solutions.

[0109] (2) Preparation of standard curve using xanthine ELISA test method

[0110] Use PBS to dilute the anti-xanthine antibody prepared in Example 7 to a final concentration of 1:10000, and coat 100 μL / well on a 96-well enzyme-labeled plate and place it at 4°C for 12-24 hours; wash the 96-well enzyme-labeled plate coated with the anti-xanthine antibody three times with PBS, add 200 μL / well of 0.5% BSA solution, block it at 4°C and place it for 8-16 hours. Then wash it three times with PBS and add 20 μL / well of standard. Then add 100 μL / well of HRP-xanthine conjugate at a working concentration; incubate it at room temperature for 30 minutes and then wash the plate 5 times with PBS; then add 100 μL of TMB substrate to each well and incubate it at room temperature for 30 minutes. Then add 100 μL of stop solution (2M sulfuric acid) to each well. Determine the absorbance at 450 nm. Calibrate according to the absorbance at 450 nm corresponding to each standard to prepare a standard curve. The results are shown in the attached figure. Figure 1 shown.

[0111] Example 10: Preparation of Xanthine Alkaline Phosphatase Chemiluminescent Detection Reagent

[0112] The antibody prepared in Example 8 was used to prepare a xanthine alkaline phosphatase chemiluminescence detection reagent.

[0113] 1. A xanthine detection kit. This experiment adopts the principle of competitive chemiluminescent immunoassay reagent. The analyte in the sample and the antigen labeled with a luminescent marker competitively bind to a certain number of antibody binding sites. The amount of antigen bound to the antibody is inversely proportional to the concentration of the analyte in the sample. When the reaction system reaches equilibrium, a substrate solution is added to detect the amount of antigen labeled with a luminescent marker bound to the antibody. A standard curve is made using a known standard substance, and the luminescence value of the unknown sample is calibrated on the standard curve to obtain the concentration of xanthine in the unknown sample. It is characterized in that it includes xanthine labeled with a chemiluminescent marker and a xanthine monoclonal antibody coated on a fixed carrier.

[0114] 2. The chemiluminescent marker in the xanthine labeled with the chemiluminescent marker is alkaline phosphatase.

[0115] 3. The antibody coated on the fixed carrier is an anti-xanthine antibody coated on magnetic beads.

[0116] (1) Obtaining the standard curve: Set the reaction parameters of the alkaline phosphatase chemiluminescence analyzer (see Table 1). The on-machine detection process is as follows: add the sample (or standard), reagent 1 and reagent 2 at the same time, incubate at 37°C for 10 minutes; add the luminescent substrate solution, measure the luminescence value, and the machine automatically calculates the calibration curve and calculates the sample concentration. The calibration curve is as shown in the attached figure. Figure 2 shown.

[0117] Table 1: Alkaline phosphatase chemiluminescence analyzer reaction parameters

[0118]

[0119] (2) Sample comparison was performed using the xanthine alkaline phosphatase chemiluminescence detection reagent of the present invention and the xanthine ELISA detection reagent of a well-known foreign manufacturer. The detection data and data analysis are shown in the attached Figure 3 .

[0120] Example 11: Analog cross-reaction test

[0121] Nine common xanthine structural analogs were selected for cross-reaction testing and measured using alkaline phosphatase chemiluminescence detection reagent. The nine common xanthine structural analogs and their cross-reaction rates are shown in Table 2.

[0122] Table 2: Cross-reactivity results of common xanthine structural analogs

[0123] Xanthine Cross-reaction (%) Xanthine 100％ Hypoxanthine 0.05％ uric acid 0.02％ Theophylline 0.00％ Theobromine 0.00％ caffeine 0.00％ Purine 0.01％ Adenine 0.01％ Guanine 0.02％

[0124] Determination results: The cross-reaction results of the above 9 common xanthine structural analogs show that the antibody prepared by the present invention has good specificity.

[0125] From the above results, it can be seen that the complete xanthine immunogen prepared from the xanthine derivative provided by the present invention has strong immunogenicity, the antibodies generated are highly specific, and have good affinity for xanthine. The alkaline phosphatase chemiluminescent reagent prepared using the above antibodies has good stability and high sensitivity, and can achieve high-throughput and rapid detection of xanthine on a fully automatic chemiluminescence analyzer. It has the advantages of simple operation, high sensitivity, strong specificity, and accurate results. It can also effectively reduce the cost of xanthine detection, which is conducive to clinical promotion and use.

[0126] It should be noted that the above are only embodiments of the present invention and do not limit the patent scope of the present invention. Any equivalent structure or equivalent process transformation made using the contents of the present invention description and drawings, or directly or indirectly applied in other related technical fields, are also included in the patent protection scope of the present invention.

Claims

1. A xanthine derivative, characterized in that It has the structure shown in formula (I): Wherein, R is a connecting group R is -(CH2) n —COOH, n is any integer between 1 and 10.

2. A xanthine immunogen, characterized in that It has the structural formula shown in formula (II): Where R is —(CH2) n —COOH, n is any integer between 1 and 10, and the carrier is an immunogenic protein or polypeptide; preferably, R is —(CH2) n —COOH, the carrier is serum protein, keyhole limpet hemocyanin, thyroglobulin or polylysine.

3. An anti-xanthine specific antibody produced by immunizing an animal with an immunogen, characterized in that: The anti-xanthine specific antibody is obtained by immunizing an animal with the xanthine immunogen according to claim 2.

4. A method for preparing a xanthine immunogen, characterized in that: The preparation method comprises: preparing the xanthine derivative according to claim 1; and connecting the xanthine derivative with a carrier to obtain the xanthine immunogen; wherein the carrier is an immunogenic protein or polypeptide; the steps of preparing the xanthine derivative are as follows: A1. Substitution reaction of xanthine with halo-(CH2)n-COO-tert-butyl ester in N,N-dimethylformamide (DMF) to obtain 2-(CH2)n-COO-tert-butyl ester xanthine; A2. Dissolve the 2-(CH2)n-COO-tert-butyl ester xanthine in a strong acid solution to obtain a reaction solution, and stir the reaction solution at 40-60°C for 2.5-6.5h to obtain the xanthine derivative.

5. The preparation method according to claim 4, characterized in that The step A1 comprises the following steps: a. dissolving xanthine and halo-(CH2)n-COO-tert-butyl ester in di-N,N-dimethylformamide (DMF) to obtain a mixed solution; wherein the molar ratio of xanthine to halo-(CH2)n-COO-tert-butyl ester is 0.8 to 1.6:1; preferably, after obtaining the mixed solution, the method further comprises heating the mixed solution to a constant temperature, more preferably, the temperature of heating to the constant temperature is 70 to 100° C., and the constant temperature time is greater than or equal to 32 hours; b. adding ethyl acrylate to the mixed solution to obtain a reaction mixture, and stirring the reaction mixture at a low temperature of 0 to 8° C. to obtain a solid-liquid mixture; c. Filtering, washing, purifying and vacuum drying the reaction mixture to obtain the 2-(CH2)n-COO-tert-butyl ester xanthine.

6. The preparation method according to claim 4 or 5, characterized in that When n=1, the preparation steps of the xanthine derivative are as follows:

7. The preparation method according to claim 4, characterized in that The step of coupling the carrier with the xanthine derivative comprises: a. preparing a carrier solution and a xanthine derivative solution; preferably, the carrier is serum protein, keyhole limpet hemocyanin, thyroglobulin or polylysine; b. adding the xanthine derivative solution to the carrier solution to obtain a mixed solution of activated xanthine derivative and carrier; c. stirring the mixed solution at 2-10° C. overnight (or reacting at room temperature for 2 hours) to obtain the crude xanthine immunogen; d. Purify the crude xanthine immunogen product to obtain the pure xanthine immunogen; preferably, purify by dialysis using a dialysis bag.

8. The preparation method according to claim 7, characterized in that In step a, the step of preparing the carrier solution includes dissolving the carrier in a 0.05-0.2M phosphate buffer solution with a pH of 8.0-9.5 to obtain the carrier solution; and the step of preparing the xanthine derivative solution includes placing the xanthine derivative, 1-ethyl-3-(-3-dimethylaminopropyl)carbodiimide (EDAc) and N-hydroxysulfosuccinimide (Sulfo-NHS) in N,N-dimethylformamide, methanol and 5-20mM phosphate buffer solution with a pH of 4.0-6.0 and stirring at room temperature to obtain a solution of the xanthine derivative in an activated state; wherein the mass ratio of the carrier to the xanthine derivative is 1-8:

1.

9. A xanthine detection kit, characterized in that, The kit contains the xanthine derivative according to claim 1 and or the antibody according to any one of claim 3.

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