Indoxyl sulfate derivative, immunogen, antibody and detection reagent preparation and application

Abstract

The invention relates to a preparation method of an indol sulfate (IS) detection kit by using a chemiluminescence immunoassay technology. Indoxyl sulfate is a protein-bound urotoxin in a human body, has small molecular weight and poor 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 high affinity is successfully prepared by designing and transforming the hapten. The invention mainly relates to design and synthesis of an indol sulfate hapten, preparation of an indol sulfate complete antigen and an anti-indol sulfate antibody, a method for determining the concentration of indol sulfate, and composition and components of a reagent. The design and synthesis of the hapten mainly comprise design and chemical synthesis of an indol sulfate derivative. The indoxyl sulfate derivative disclosed by the invention has a structure as shown in a formula (I): # imgabs0 #, wherein R is-(CH2) n-COOH, and n is an integer between 1 and 10.

Description

Technical Field

[0001] The present invention relates to the field of biomedical detection, and particularly to an indoxyl sulfate derivative, an indoxyl sulfate immunogen, its specific antibody, and an indoxyl sulfate detection kit. Background Art

[0002] Indoxyl sulfate (IS), whose structural formula is shown as formula (Ⅲ):

[0003]

[0004] Indoxyl sulfate is the most abundant indole compound accumulated in the body of uremic patients. It mainly comes from tryptophan in food, is generated into indole by the action of Escherichia coli in the intestine and then enters the blood, and is formed in the liver through oxidation and sulfation after being transported. The kidney is its main excretion route. Due to the decline of kidney function in uremic patients, it cannot be effectively excreted from the body. Coupled with the competition of various uremic toxins for excretion pathways, indoxyl sulfate is easily accumulated in the body and is one of the most critical toxins leading to renal fibrosis. Clinical observations show that indoxyl sulfate can also cause damage to renal tubules and glomeruli, degeneration of myocardial cells, hyperplasia of vascular smooth muscle cells, arterial calcification, and affect bone metabolism.

[0005] Currently, the main methods for detecting indoxyl sulfate are enzyme-linked immunosorbent assay and high-performance liquid chromatography. The above methods are cumbersome to operate, time-consuming and laborious, and in clinical tests, they cannot take into account both accuracy and timeliness. The present invention starts from antigen design and antibody preparation, and uses an advanced fully automated chemiluminescence detection platform to completely solve the pain points of indoxyl sulfate clinical tests and has broad application prospects. This detection reagent can perform high-throughput, automated, and multi-type sample determination, and has outstanding advantages of high sensitivity, good specificity and stability in reagent performance. Summary of the Invention

[0006] The purpose of the present invention is to provide an indoxyl sulfate derivative, an indoxyl sulfate immunogen, its specific antibody, and an indoxyl sulfate detection kit to improve the defects of poor specificity, low sensitivity, and inability to perform automated analysis in the existing indoxyl sulfate determination methods.

[0007] According to one aspect of the present invention, an indoxyl sulfate derivative is provided, which has the structural formula shown as formula (Ⅰ):

[0008]

[0009] Among them, R is a linking group -(CH2)n-COOH, where n is an integer between 1 and 10. The indoxyl sulfate derivative of the present invention has the basic structure for preparing an indoxyl sulfate immunogen with immunogenicity, providing a structural basis for preparing a new indoxyl sulfate detection reagent.

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

[0011]

[0012] Among them, R is a linking group -(CH2)n-COOH, where n is an integer between 1 and 10, and the carrier is a protein or polypeptide with immunogenicity. The indoxyl sulfate immunogen of the present invention has high immunogenicity, can stimulate the animal body to produce an immune response, produce high-titer anti-indoxyl sulfate specific antibodies, and has high affinity, and is suitable for preparing a highly sensitive and highly specific indoxyl sulfate competitive assay detection reagent.

[0013] When n = 1, R in the above indoxyl sulfate immunogen is -CH2-COOH. The carrier suitable for the above indoxyl sulfate immunogen is preferably a protein carrier, but other types of immunogenic substances with a sufficiently large molecular weight and a sufficient number of active groups can also be used as carriers. The most commonly used immunogenic carriers include serum albumin, keyhole limpet hemocyanin (KLH), thyroglobulin, and polylysine. The carrier in the present invention is preferably keyhole limpet hemocyanin.

[0014] According to yet another aspect of the present invention, there is also provided an anti-indoxyl sulfate specific antibody produced by immunizing an animal with an immunogen, and the antibody is produced by immunizing an animal with any one of the above indoxyl sulfate immunogens. The "antibody" referred to in the present invention not only refers to a complete protein molecule antibody, but also includes a polypeptide fragment antibody or a derivative of a polypeptide fragment antibody that retains the specific binding ability of the complete antibody. The antibody in the present invention is a polyclonal antibody, a monoclonal antibody, or a recombinant antibody, and is preferably a monoclonal antibody.

[0015] The antibody of the present invention can be prepared by existing technologies. A typical method for obtaining a polyclonal antibody is to use a single immunogen, with or without adjuvant, to immunize an animal at one or more sites. Host animals include rabbits, goats, mice, sheep, guinea pigs, etc. Immunize continuously 5 - 7 times until the antibody titer reaches the highest, and the animal is bled regularly to obtain an appropriate amount of specific antiserum. Monoclonal antibodies can be prepared by hybridoma cell technology. Recombinant antibodies can be prepared by genetic engineering expression technology.

[0016] According to another aspect of the present invention, a method for preparing an indoxyl sulfate immunogen is also provided. The preparation method includes the steps of preparing the above indoxyl sulfate derivative and connecting the above indoxyl sulfate derivative with a carrier to obtain the indoxyl sulfate immunogen. By connecting the indoxyl sulfate derivative prepared by the above method with an immunogenic protein or polypeptide, the indoxyl sulfate immunogen with strong immunogenicity of the present invention can be obtained, and the preparation method is simple to operate.

[0017] In the method for preparing the indoxyl sulfate immunogen of the present invention, a method for preparing the above indoxyl sulfate derivative is also provided. The preparation method includes the following steps: (1) Substituting indoxyl sulfate with halo-(CH2)n-COO-tert-butyl ester in an N,H-dimethylformamide environment to obtain 2-(CH2)n-COO-tert-butyl ester indoxyl sulfate; (2) Dissolving 2-(CH2)n-COO-tert-butyl ester indoxyl sulfate in a strong acid solution to obtain an indoxyl sulfate derivative. This preparation method substitutes the hydrogen at the 2-position of indoxyl sulfate with halo-(CH2)n-COO-tert-butyl ester to generate 2-(CH2)n-COO-tert-butyl ester indoxyl sulfate, and then in an acidic environment, the 2-(CH2)n-COO-tert-butyl ester bond on 2-(CH2)n-COO-tert-butyl ester indoxyl sulfate is opened to form an indoxyl sulfate derivative with -(CH2)n-COOH at the 2-position of indoxyl sulfate. This preparation method is simple to operate, and the reaction conditions are mild, with high stability and good repeatability.

[0018] In the above step (1) of preparing the indoxyl sulfate derivative, the following steps are also included: 1-1, dissolving indoxyl sulfate and halo-(CH2)n-COO-tert-butyl ester in N,N-dimethylformamide (DMF) to obtain a mixed solution; 1-2, adding ethyl acrylate to the mixed solution to obtain a solid-liquid mixture; 1-3, filtering the solid-liquid mixture to obtain 2-(CH2)n-COO-tert-butyl ester indoxyl sulfate. Indoxyl sulfate and halo-(CH2)n-COO-tert-butyl ester have good solubility in DMF organic solvent. Adding ethyl acrylate precipitates 2-(CH2)n-COO-tert-butyl ester indoxyl sulfate, and filtering to obtain the reactant.

[0019] In the above step 1-1, after obtaining the mixed solution, the step of heating the mixed solution to a constant temperature is also included. Preferably, the temperature for heating to a constant temperature is 70-100 °C, and the time for maintaining the constant temperature is greater than or equal to 32 h. Heating the mixed solution to a constant temperature and maintaining it for more than 32 h can ensure sufficient dissolution and reaction.

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

[0021] In the above-mentioned method for preparing the indoxyl sulfate derivative of the present invention, there are no special requirements for the specific operation of filtering the solid-liquid mixture to obtain indoxyl sulfate 2-(CH2)-COO-tert-butyl ester, as long as the desired target product can be separated from the solid-liquid mixture. In the present invention, the above-mentioned step 1-3 includes 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 indoxyl sulfate 2-(CH2)n-COO-tert-butyl ester. After the steps of filtration, washing and purification, and vacuum drying, the obtained indoxyl sulfate 2-(CH2)n-COO-tert-butyl ester has relatively high purity and yield.

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

[0023] In the above-mentioned method for preparing the indoxyl sulfate derivative of the present invention, when n = 1, the preparation steps of the above-mentioned indoxyl sulfate derivative are as follows:

[0024]

[0025] When n = 1, it is the same as the above reaction steps, except that tert-butyl bromoacetate is used as the synthetic raw material, so the linking group R of the obtained final product indoxyl sulfate derivative is -CH2-COO-.

[0026] In the above-mentioned method for preparing the indoxyl sulfate immunogen of the present invention, for the step of connecting the carrier and the indoxyl sulfate derivative, in actual operation, it can be reasonably improved according to the differences of the carriers. In the present invention, the above-mentioned connecting step (2) includes: 2-1. Prepare a carrier solution and an indoxyl sulfate derivative solution; wherein, the mass ratio of the carrier to the indoxyl sulfate derivative is 1-8:1; preferably, the carrier is serum albumin, keyhole limpet hemocyanin, thyroglobulin or polylysine; 2-2. Drop the activated indoxyl sulfate derivative solution into the carrier solution to obtain a crude indoxyl sulfate immunogen; 2-3. Stir the dropped mixture overnight at 2-10°C or react at room temperature for 2 hours to obtain a crude indoxyl sulfate immunogen; Step 2-4. Purify the crude indoxyl sulfate immunogen to obtain the indoxyl sulfate immunogen. The preparation steps of the present invention can obtain the target product through simple activation, dropping, and purification steps. The preparation method is simple, has high process stability, and good reproducibility.

[0027] In the above preparation method of the indoxyl sulfate immunogen of the present invention, in the actual operation of the steps of preparing the carrier solution and the indoxyl sulfate derivative solution, a suitable solvent concentration and pH value are reasonably selected according to the type of the carrier. In step 2-1 of the present invention, the step of preparing the carrier solution is to dissolve the carrier in a phosphate buffer solution with a concentration of 0.05-0.20M and a pH of 8.0-9.5 to obtain a carrier solution; the step of preparing the indoxyl sulfate derivative solution is to place the indoxyl sulfate derivative, 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide and N-hydroxysulfosuccinimide in N,N-dimethylformamide, methanol, and a phosphate buffer solution with a concentration of 5-20mM and a pH of 4.0-6.0, and stir at room temperature to obtain an activated indoxyl sulfate derivative solution. Using a phosphate buffer solution with a concentration of 0.10-0.25M and a pH of 8.0-9.5 to dissolve the carrier can enable the amino group on the carrier and the carboxyl group of the activated indoxyl sulfate derivative to react and combine fully in a slightly alkaline environment. Using a phosphate buffer solution with a concentration in the range of 5-20mM and a pH in the range of 4.0-6.0 can activate the carboxyl group of the indoxyl sulfate derivative sufficiently in a slightly acidic solution.

[0028] In the above preparation method of the indoxyl sulfate immunogen of the present invention, in the step of dropping to obtain the crude indoxyl sulfate immunogen, in order to further increase the content of indoxyl sulfate immunogen in the crude indoxyl sulfate immunogen. In steps 2-2 and 2-3 of the present invention, through the dropping step, the indoxyl sulfate derivative can react with the carrier more fully; stirring overnight at 2-10°C can further promote the formation of indoxyl sulfate immunogen, thereby increasing the content of indoxyl sulfate immunogen in the crude product.

[0029] In the method for preparing the indoxyl sulfate immunogen of the present invention, any operation capable of purifying the indoxyl sulfate immunogen from the crude indoxyl sulfate immunogen is applicable to the present invention. Preferably, in the above steps 2-4, dialysis is used to purify the crude indoxyl sulfate immunogen to obtain the indoxyl sulfate immunogen. The dialysis method is simple and has good purification effect.

[0030] According to another aspect of the present invention, there is also provided an indoxyl sulfate detection reagent, comprising a specific antibody against indoxyl sulfate, an indoxyl sulfate enzyme-labeled conjugate, and a substrate of the enzyme, wherein the specific antibody against indoxyl sulfate is any one of the above specific antibodies against indoxyl sulfate; the indoxyl sulfate enzyme-labeled conjugate contains the above indoxyl sulfate derivative; the indoxyl sulfate enzyme-labeled conjugate is formed by conjugating an enzyme and a hapten, wherein the hapten is the above indoxyl sulfate derivative.

[0031] In the above indoxyl sulfate detection reagent of the present invention, due to the strong specificity of the specific antibody against indoxyl sulfate and the strong binding ability to indoxyl sulfate, the detection sensitivity is much higher than that of the corresponding products in the prior art. Preferably, the above enzyme-labeled conjugate is an alkaline phosphatase-hapten enzyme-labeled conjugate; the above substrate of the enzyme is AMPPD or APS-5. The detection reagent using the alkaline phosphatase-hapten enzyme-labeled conjugate and AMPPD or APS-5 as the substrate of the enzyme can conveniently and accurately determine the content of indoxyl sulfate in the sample and is suitable for high-throughput automated detection.

[0032] According to another aspect of the present invention, there is also provided an indoxyl sulfate detection kit, containing the above specific antibody against indoxyl sulfate and / or an indicator reagent for detecting the specific antibody against indoxyl sulfate and indoxyl sulfate complex. The indicator reagent is selected from enzyme reagents, radioisotope reagents, fluorescent reagents, and luminescent reagents. Preferably, the indicator reagent is composed of an indoxyl sulfate enzyme-labeled conjugate and a substrate of the enzyme. Among them, the indoxyl sulfate enzyme-labeled conjugate can be conjugated with the indoxyl sulfate derivative of the present invention, which can conveniently and accurately determine the content of indoxyl sulfate in the sample and is suitable for high-throughput automated detection.

[0033] Applying the technical solution of the present invention, the indoxyl sulfate derivative obtained by substituting the hydrogen at a specific site with a specific R group and the indoxyl sulfate immunogen formed by connecting with a specific carrier have high immunogenicity. The antibody produced by immunizing animals has high specificity and strong specific binding ability to indoxyl sulfate. The high-throughput and rapid detection of indoxyl sulfate can be realized on a fully automatic chemiluminescence immunoassay analyzer by using the alkaline phosphatase chemiluminescence immunoassay technology, and it has the advantages of simple operation, high sensitivity, strong specificity, accurate results, etc. It can also effectively reduce the detection cost of indoxyl sulfate and is beneficial to clinical promotion and use. Brief Description of the Drawings

[0034] Appendix Figure 1 : Standard curve for the ELISA detection of indoxyl sulfate.

[0035] Appendix Figure 2 : Chemiluminescence calibration curve of indoxyl sulfate alkaline phosphatase.

[0036] Appendix Figure 3 : Comparison results of samples between the chemiluminescence detection reagent of indoxyl sulfate alkaline phosphatase of the present invention and the ELISA detection reagent of well-known foreign manufacturers. Detailed implementation manners

[0037] It should be noted that, without conflict, the embodiments in this application and the features in the embodiments can be combined with each other. The present invention will be described in detail below with reference to the drawings and in combination with the embodiments.

[0038] Example 1: Synthesis and structure confirmation of indoxyl sulfate derivatives

[0039] The chemical structure of the indoxyl sulfate derivative used in the following examples is shown in formula (IV):

[0040]

[0041] The specific synthesis steps of the indoxyl sulfate derivative shown in formula (IV) are as follows:

[0042] (1) Synthesis of compound 2

[0043] The specific synthesis steps of compound 2 are as follows:

[0044]

[0044] Weigh 15.0 g of compound 1 and 26 g of tert-butyl bromoacetate, dissolve them together in 150 mL of N,N-dimethylformamide (DMF), and then heat the solution to 95 °C overnight. Add 500 mL of ethyl acrylate (EA) to this solution, and stir the reaction mixture at 0 °C for 25 min. Filter out the solid precipitate in the solution, wash it with acetone, and then perform vacuum drying to finally obtain 11.0 g of compound 2 in the form of a brown solid, with a yield of 29%.

[0045] (2) Synthesis of indoxyl sulfate derivative

[0046]

[0047] Weigh 3.00 g of compound 2, dissolve it in 30 mL of HCl (1.5 M), and stir this solution at 60 °C for 3 hours. Evaporate and dry the reaction mixture, wash the dried residue with acetone, and finally obtain 1.90 g of the indoxyl sulfate derivative in the form of a red-brown solid, with a yield of 89%.

[0048] The above compound was scanned by nuclear magnetic resonance spectroscopy using Varian III plus 300 MHz, with TMS as the internal standard. The result was characterized as indoxyl sulfate 2-(CH2)2-COO-tert-butyl ester.

[0049] Example 2: When n = 2, the preparation steps of the derivative are as follows;

[0050] (1) Synthesis of indoxyl sulfate 2-(CH2)2-COO-tert-butyl ester

[0051] Weigh 18.0 g of Compound 1 and 27.8 g of tert-butyl bromopropionate, dissolve them together in 150 mL of N,N-dimethylformamide (DMF), then heat the solution to 95 °C overnight. Add 550 mL of ethyl acrylate (EA) to this solution, and stir the reaction mixture at 4 °C for 25 min. Filter out the solid precipitate in the solution, wash it with acetone and then dry it under vacuum. Finally, 7.20 g of a brown solid intermediate product is obtained, with a yield of 22%.

[0052] The above compound was scanned by nuclear magnetic resonance spectroscopy using Varian III plus 300 MHz, with TMS as the internal standard. The result was characterized as indoxyl sulfate 2-(CH2)2-COO-tert-butyl ester.

[0053] (2) Synthesis of indoxyl sulfate derivative

[0054] Weigh 3.00 g of the above indoxyl sulfate 2-(CH2)2-COO-tert-butyl ester, dissolve it in 30 mL of HCl (1.5 M), and stir this solution at 60 °C for 2 hours. Evaporate and dry the reaction mixture, wash the dried residue with acetone. Finally, 2.0 g of a red-brown solid indoxyl sulfate derivative is obtained, with a yield of 91.2%.

[0055] Perform structural identification on the above obtained purified product

[0056] 1. The above compound was scanned by nuclear magnetic resonance spectroscopy using Varian III plus 300 MHz, with TMS as the internal standard. The result was characterized as indoxyl sulfate 2-(CH2)2-COO-.

[0057] 2. Analyze and identify the obtained derivative using chromatography / mass spectrometry (LC / MS), and it can be determined that the finally obtained compound is indoxyl sulfate 2-(CH2)2-COO-.

[0058] Example 3: When n = 3, the preparation steps of the indoxyl sulfate derivative are as follows;

[0059] (1) Synthesis of Indoxyl Sulfate 2-(CH2)3-COO-tert-butyl Ester

[0060] Weigh 18.0 g of Compound 1 and 29.5 g of tert-butyl bromobutyrate, dissolve them together in 180 mL of N,N-dimethylformamide (DMF), and then heat the solution to 95 °C overnight. Add 525 mL of ethyl acrylate (EA) to this solution and stir the reaction mixture at 5 °C for 30 min. Filter out the solid precipitate in the solution, wash it with acetone, and then dry it under vacuum to finally obtain 6.90 g of white solid Compound 2 with a yield of 21%.

[0061] Perform nuclear magnetic resonance spectroscopy scanning on the above compound using Varian III plus 300 MHz with TMS as the internal standard. The result is characterized as the compound shown by Indoxyl Sulfate 2-(CH2)3-COO-tert-butyl Ester.

[0062] (2) Synthesis of Indoxyl Sulfate Derivatives

[0063] Weigh 3.00 g of Compound 2, dissolve it in 30 mL of HCl (1.5 M), and stir this solution at 55 °C for 4.5 hours. Evaporate and dry the reaction mixture, wash the dried residue with acetone, and finally obtain 2.1 g of red-brown solid Indoxyl Sulfate derivative with a yield of 90%.

[0064] Conduct structural identification on the above-obtained purified product;

[0065] 1. Perform nuclear magnetic resonance spectroscopy scanning on the above compound using Varian III plus 300 MHz with TMS as the internal standard. The result is characterized as Indoxyl Sulfate 2-(CH2)3-COO-.

[0066] 2. Analyze and identify the obtained derivative using chromatography / mass spectrometry (LC / MS) technology, and it can be determined that the finally obtained compound is Indoxyl Sulfate 2-(CH2)3-COO-.

[0067] In the above examples, when n = 1, 2, and 3, tert-butyl bromoacetate, tert-butyl bromopropionate, and tert-butyl bromobutyrate were respectively selected as the synthesis raw materials for the synthesis of intermediate compounds in the preparation of Indoxyl Sulfate derivatives. Therefore, the linking group R of the finally obtained Indoxyl Sulfate derivative 1 are -CH2-COO-, -(CH2)2-COO-, and -(CH2)3-COO- respectively. When n is other integers from 1 to 10, when using other tert-butyl bromoorganic acids similar to tert-butyl bromoacetate for experiments, except for the different values of n, the synthesis methods are exactly the same.

[0068] Example 4: Synthesis of BSA-Indoxyl Sulfate Derivative Immunogen

[0069] The BSA-indoxyl sulfate immunogen is formed by connecting Bovine Serum Albumin (BSA) with the -(CH2)n-COO- group of the indoxyl sulfate derivative shown in formula (I). In this example, taking n = 1 as an example, the synthesis method of this immunogen is described in detail as follows:

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

[0071] (2) Add the following chemicals into a small beaker and stir to dissolve: 20 mg of the synthesized indoxyl sulfate derivative, 0.3 ml of D,D-dimethylformamide (DMF), 0.3 ml of methanol, 1.0 ml of 10 mM phosphate buffer with pH 5.0, 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 min;

[0072] (3) Drop the activated solution into the BSA solution and stir overnight at 2 - 10 °C to obtain the crude antigen; Purify the synthesized antigen by dialysis to obtain 2-acetoxysulfate indoxyl immunogen.

[0073] Example 5: Synthesis of KLH-Indoxyl Sulfate Derivative Immunogen

[0074] The KLH-acetyl indoxyl sulfate immunogen is formed by connecting keyhole limpet hemocyanin (KLH) with the -(CH2)n-COO- group of the acetyl indoxyl sulfate derivative shown in formula (I). In this example, taking n = 2 as an example, the synthesis method of this immunogen is described in detail as follows:

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

[0076] (2) Add the following chemicals into a small beaker and stir to dissolve: 20 mg of the synthesized indoxyl sulfate derivative, 0.3 ml of D,D-dimethylformamide (DMF), 0.3 ml of methanol, 1.0 ml of 10 mM phosphate buffer with pH 5.0, 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 indoxyl sulfate immunogen; purify the synthesized antigen by dialysis to obtain 2-propionate indoxyl sulfate immunogen.

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

[0079] The thyroglobulin-indoxyl sulfate immunogen is formed by linking thyroglobulin with the -(CH2)n-COO- group of the indoxyl sulfate derivative shown in formula (I). In this example, taking n = 3 as an example, the synthesis method of this immunogen is described in detail as follows:

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

[0081] (2) Add the following chemicals to a small beaker and stir to dissolve: 10 mg of the synthesized indoxyl sulfate derivative, 0.3 ml of N,N-dimethylformamide (DMF), 0.3 ml of methanol, 1.0 ml of 20 mM potassium phosphate buffer with pH 5.0, 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 a complete antigen; purify the synthesized antigen by dialysis to obtain 2-butyrate indoxyl sulfate immunogen.

[0083] Example 7: Synthesis of polylysine-indoxyl sulfate derivative immunogen

[0084] The polylysine-indoxyl sulfate immunogen is formed by linking polylysine with the -(CH2)n-COO- group of the indoxyl sulfate derivative shown in formula (I). In this example, taking n = 3 as an example, the synthesis method of this immunogen is described in detail as follows:

[0085] (1) Dissolve polylysine (20 mg) in 5 ml of 0.20 M phosphate buffer with pH 9.0.

[0086] (2) Add the following chemicals into a small beaker and stir to dissolve: 10 mg of the synthesized indoxyl sulfate derivative, 0.3 ml of N,N-dimethylformamide (DMF), 0.3 ml of methanol, 1.0 ml of 20 mM potassium phosphate buffer at pH 5.0, 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 a reaction of 30 min.

[0087] (3) Dropwise add the activated solution into the polylysine solution and stir overnight at 2 - 10 °C to obtain the complete antigen; centrifuge the synthesized antigen and take the supernatant, and purify the supernatant by dialysis to obtain the indoxyl sulfate immunogen of 2-butyric acid.

[0088] Similarly, when n takes other integers within the range of 1 - 10, indoxyl sulfate immunogens as shown in formula (II) can be prepared by the same method, and there are no significant differences in the experimental results. The indoxyl sulfate immunogens prepared using indoxyl sulfate derivatives with different n values all have strong immunogenicity, and the corresponding specific antibodies prepared 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-Indoxyl Sulfate Specific Monoclonal Antibody

[0090] This example proposes a method for preparing an indoxyl sulfate monoclonal antibody, which includes the following steps:

[0091] (1) Animal immunization: Immunize two 8-week-old Balb / C female mice with the artificial antigen prepared in Example 5 by the method of multiple subcutaneous injections at the back of the neck. For the primary immunization, use the immunogen emulsified with Freund's complete adjuvant. Mix the immunogen and Freund's complete adjuvant in the same volume for emulsification. The immunogen dose for a single mouse is 0.1 mg; strengthen the immunization every 14 days after the primary immunization, with a dose of 0.1 mg and the same emulsification method; immunize a total of 5 times. Starting from the 3rd booster immunization, 30 μL of blood is collected from the mouse tail 7 days after each immunization, and the antiserum is obtained by centrifugation and stored at -20 °C for serum titer determination and specificity determination.

[0092] (2) Analyze the effect of the antiserum by ELISA indirect enzyme-linked immunosorbent assay Using the conventional antibody titer determination method, with the blank serum without antibody as the control, the antiserum was diluted by a certain multiple and then subjected to ELISA detection. Finally, the titer of the anti-indoxyl sulfate specific antibody of the present invention was detected to be 1:30,000 - 1:50,000, indicating that the antibody prepared by the present invention has strong specificity and high sensitivity.

[0093] (3) Cell fusion and screening of positive hybridomas

[0094] 1. Resuscitation of myeloma cells: Take out the myeloma cells from liquid nitrogen, quickly put them into a 37°C water bath for thawing. After melting, centrifuge at 1000 r / min for 5 minutes. In the laminar flow hood, pour out the supernatant, and add about 1 mL of complete culture medium to the cell pellet. Disperse the cells, take out and mix evenly with the complete culture medium with a pipette, and then put them into a 10-cm diameter culture dish and expand to 4 - 6 dishes. During this period, the medium needs to be changed several times. When the cells in each culture dish cover the bottom, they can be used for cell fusion.

[0095] 2. Cell preparation: Take out 2 small culture dishes. Pour part of the culture medium into one for cooling the dissection tools, and suck a small amount of culture medium into the other and put a cell sieve for grinding the spleen. Blow down the resuscitated myeloma cells and transfer them to a 50-mL centrifuge tube and seal it. Centrifuge at 1200 r / min for 5 minutes. Sacrifice the mice after five immunizations, soak them in 75% alcohol for about 1 minute, put them into the laminar flow hood, take blood from the heart, incubate at 37°C for 30 minutes, centrifuge for 15 minutes, and store the serum at -20°C. After taking the mouse spleen cells, grind them thoroughly in the cell sieve, wash them with the pre-divided basal solution, and transfer them to a 50-mL centrifuge tube and seal it. Discard the supernatant of the centrifuged myeloma cells, add the basal solution and wash them once again, and centrifuge together with the spleen cells at 1200 r / min for 5 minutes. After secondary centrifugation, discard the supernatant of the myeloma cells, add 2 mL of basal solution, and pipette to mix evenly. Remove the supernatant of the spleen cells, pass them through the cell sieve and add them to the myeloma cells, pipette to mix evenly, then add the basal culture medium to 20 mL, and centrifuge at 1200 r / min for 5 minutes. Take out the centrifuged mixed cells, discard the supernatant, suck off the excess culture medium with a pipette, and disperse the precipitated cells, then incubate in a 37°C cell culture incubator for 5 minutes.

[0096] 3. Cell fusion: After the incubation, place the centrifuge tube in 37°C warm water, keep rotating throughout the process, use the gun 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, add 3mL within the fourth minute, and add 16mL within the fifth to sixth minutes, with gentle stirring, add along the wall to separate the PEG. Seal the centrifuge tube and centrifuge at 900r / min for 8 minutes, pour off the supernatant, add the fused cells to the HAT complete culture medium, stir gently, and evenly add to 4 24-well culture plates. Keep the volume of HAT culture medium containing fused cells in each well the same.

[0097] 4. Screening of positive hybridomas: half-change the medium with HT medium within 4 days after fusion, and change the medium with HT medium in each well after 8 days. On the 10th day, extract the supernatant from the multi-well culture plate, detect the specific antibodies in the culture medium with indirect ELISA, select positive hybridoma cells with high titer and strong affinity, screen out the positive wells with the best fusion effect, and mark them. Under sterile conditions, transfer to a new 96-well culture plate, clone each original well into two 96-well plates, and after the cells adhere to the wall and cover 1 / 4 of the bottom of the well, take the supernatant and ELISA test. The titer and inhibition rate are also used as measurement indicators. The strong positive ones are subcloned by limiting dilution method, and this is 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 standby use), until each well of each plate is positive and the titer and inhibition are similar after detection. At this point, the hybridoma cell line is successfully established, and a hybridoma cell line that can stably secrete uniform antibodies is obtained. Single cell clones were picked and those that tested fully positive were transferred to 24-well cell culture plates, 6-well cell culture plates, and 10 cm cell culture dishes for expansion and cryopreservation in time.

[0098] (4) Large-scale preparation of monoclonal antibodies After obtaining hybridoma cell clones that secrete specific monoclonal antibodies, monoclonal antibodies are usually prepared in large quantities by in vitro culture and in vivo monoclonal antibody induction methods. Liquid lysate is injected into the peritoneal cavity of more than ten Balb / c mice over 8 weeks old in advance, with a dose of 0.5 mL / mouse. Hybridoma cells are injected into the peritoneal cavity of mice 1 to 2 weeks later. After inoculation of cells, the state of the mice is observed every day. In particular, from the 7th day onwards, the abdominal cavity of the mice will swell. Before the death of the mice, ascites is collected aseptically with a disposable syringe. The collected ascites is centrifuged at 12000r / min for 10 minutes to remove the upper fat and lower fibrin, collect the middle layer, and determine its titer and inhibition rate by ELISA. After purification, it is stored at -20℃ for standby use to obtain indoxyl sulfate monoclonal antibodies.

[0099] Example 9: Indoxyl sulfate ELISA test The titer and specificity of the antiserum were determined by indirect ELISA method, and the steps were as follows: The antibody prepared in Example 8 was used for the ELISA test of indoxyl sulfate. This test uses a competitive immunoassay to determine the content of indoxyl sulfate in liquid samples. The principle is as follows: Indoxyl sulfate in the sample competes with the conjugated indoxyl sulfate derivative (HRP-indoxyl sulfate derivative enzyme conjugate) for the limited sites on the antibody coated on the enzyme-linked immunosorbent assay (ELISA) plate. If there is little or no indoxyl sulfate in the liquid sample, the HRP enzyme-conjugated indoxyl sulfate derivative will bind to the antibody in the ELISA plate. On the contrary, if the liquid sample contains a large amount or a certain amount of indoxyl sulfate, then the enzyme-indoxyl sulfate derivative conjugate will reduce its binding to the antibody, thereby weakening the color development signal. Therefore, the absorbance generated by the test is inversely proportional to the content of indoxyl sulfate in the liquid sample. The specific steps are as follows:

[0100] (1) Establishment of the standard curve for indoxyl sulfate ELISA detection

[0101] 1. Preparation of standards

[0102] The indoxyl sulfate powder (purchased from Cayman) was dissolved in methanol solution to prepare a stock solution of 1 mg / ml. The stock solution was serially diluted with ELISA buffer to standard solutions of 100.00 ng / mL, 50.00 ng / mL, 25.00 ng / mL, 12.50 ng / mL, 6.25 ng / mL, 3.13 ng / mL, 1.56 ng / mL and 0.00 ng / mL. The ELISA buffer contains 50.0 mM Tris, 100 mM NaCl and 0.2% BSA.

[0103] 2. Preparation of the standard curve using the ELISA test method for indoxyl sulfate

[0104] The anti-indoxyl sulfate antibody prepared in Example 7 was diluted with PBS to a final concentration solution of 1:10000, and 100 μL / well was coated on a 96-well ELISA plate and placed at 4 °C for 12 - 24 h; after washing the 96-well ELISA plate coated with the anti-indoxyl sulfate antibody 3 times with PBS, 200 μL / well of 0.5% BSA solution was added and blocked at 4 °C for 8 - 16 h. Then it was washed 3 times with PBS, and 20 μL / well of the standard was added. Then 100 μL / well of the working concentration of HRP-indoxyl sulfate conjugate was added; after incubating at room temperature for 30 min, the plate was washed 5 times with PBS; then 100 μL of TMB substrate was added to each well and incubated at room temperature for 30 min. Then 100 μL of the stop solution (2 M sulfuric acid) was added to each well. The absorbance at 450 nm was measured. Calibrate according to the absorbance at 450 nm corresponding to each standard to make a standard curve, and the results are as attached Figure 1as shown

[0105] Example 10: Preparation of a Chemiluminescent Detection Reagent for Indoxyl Sulfate Alkaline Phosphatase

[0106] The antibody obtained in Example 8 was used to prepare a chemiluminescent detection reagent for indoxyl sulfate alkaline phosphatase.

[0107] (1) A detection kit for indoxyl sulfate. In this experiment, the principle of a competitive chemiluminescent immunoassay reagent was adopted. The analyte in the sample and the antigen labeled with a luminescent label 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. After the reaction system reaches equilibrium, a substrate solution is added to detect the amount of the antigen labeled with the luminescent label bound to the antibody. A standard curve is made with known standards, and the concentration of indoxyl sulfate in the unknown sample can be obtained by calibrating the luminescence value of the unknown sample on the standard curve. It is characterized by including indoxyl sulfate antigen labeled with a chemiluminescent label and indoxyl sulfate monoclonal antibody coated on a fixed carrier; (2) In the indoxyl sulfate labeled with the chemiluminescent label, the chemiluminescent label is alkaline phosphatase. (3) The antibody coated on the fixed carrier is an anti-indoxyl sulfate antibody coated on magnetic beads.

[0108] Obtaining the standard curve: Set the reaction parameters of the alkaline phosphatase chemiluminescent analyzer (see Table 1). The on-machine detection process is as follows: Add the sample (or standard), Reagent 1 and Reagent 2 simultaneously, incubate at 37 °C for 10 minutes; add the luminescent substrate solution, measure the luminescence value, and the machine automatically calculates and fits the calibration curve and calculates the sample concentration. The calibration curve is as attached Figure 2 as shown

[0109] Table 1: Reaction Parameters of the Alkaline Phosphatase Chemiluminescent Analyzer

[0110]

[0111] Samples were compared using the indoxyl sulfate alkaline phosphatase chemiluminescent detection reagent of the present invention and the indoxyl sulfate ELISA detection reagent of well-known foreign manufacturers. The detection data and data analysis are shown in the attachment Figure 3 .

[0112] Example 11: Cross-Reaction Test of Analogs

[0113] Four common indoxyl sulfate structural analogs were selected for cross-reaction tests and measured using the alkaline phosphatase chemiluminescent detection reagent. Four common indoxyl sulfate structural analogs and the cross-reaction rates are shown in Table 2.

[0114] Table 2: Cross-reaction results of 4 common indoxyl sulfate structural analogs

[0115] Indoxyl sulfate Cross-reactivity (%) Indoxyl sulfate 100 Indican 0.10 Indole-3-propionic acid 0.00 Indole 0.30

[0116] Determination result: From the cross-reaction results of the above 4 common indoxyl sulfate structural analogs, it can be seen that the antibody prepared by the present invention has good specificity.

[0117] From the above results, it can be seen that the indoxyl sulfate complete immunogen prepared from the indoxyl sulfate derivative provided by the present invention has strong immunogenicity, the produced antibody has high specificity, good affinity with indoxyl sulfate, and the alkaline phosphatase chemiluminescence reagent prepared using the above antibody has good stability and high sensitivity, and can achieve high-throughput and rapid detection of indoxyl sulfate on a fully automatic chemiluminescence analyzer, and has the advantages of simple operation, high sensitivity, strong specificity, accurate results, etc., and can also effectively reduce the detection cost of indoxyl sulfate, which is beneficial to clinical promotion and use.

[0118] 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 structural or equivalent process transformation made using the specification and drawings of the present invention, or directly or indirectly applied in other related technical fields, shall be similarly included in the patent protection scope of the present invention.

Claims

1. An indoxyl sulfate 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 an integer between 1 and 10.

2. An indoxyl sulfate immunogen, characterized in that: It has the structural formula shown in formula (II): Where R is —(CH2) n —COOH, n is an 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 poly-lysine.

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

4. A method for preparing an indoxyl sulfate immunogen, characterized in that: The preparation method comprises: preparing the indoxyl sulfate derivative according to claim 1; and connecting the indoxyl sulfate derivative with a carrier to obtain the indoxyl sulfate immunogen; wherein the carrier is a protein or polypeptide with immunogenicity; the steps of preparing the indoxyl sulfate derivative comprise: (1) subjecting indoxyl sulfate to a substitution reaction with halogenated -(CH2)n-COO-tert-butyl ester in an N,N-dimethylformamide (DMF) environment to obtain 2-(CH2)n-COO-tert-butyl indoxyl sulfate; (2) dissolving the 2-(CH2)n-COO-tert-butyl indoxyl sulfate in a strong acid solution to obtain a reaction solution, and stirring the reaction solution at 40-60°C for 2.5-6.5 hours to obtain the indoxyl sulfate derivative.

5. The preparation method according to claim 7, characterized in that: The step (1) comprises the following steps: 1-1, dissolving indoxyl sulfate and halogenated-(CH2)n-COO-tert-butyl ester in di-N,N-dimethylformamide (DMF) to obtain a mixed solution; wherein the molar ratio of the indoxyl sulfate to the halogenated-(CH2)n-COO-tert-butyl ester is 0.8-1.6:1; preferably, after obtaining the mixed solution, the step of heating the mixed solution to a constant temperature is further included, and more preferably, the temperature of heating to the constant temperature is 70-100° C., and the constant temperature time is greater than or equal to 32 hours; 1-2, adding ethyl acrylate to the mixed solution to obtain a reaction mixture, and stirring and mixing the reaction mixture at a low temperature of 0 to 8° C. to obtain a solid-liquid mixture; 1-3, filtering, washing, purifying and vacuum drying the reaction mixture to obtain the 2-(CH2)n-COO-tert-butyl indoxyl sulfate.

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

7. The preparation method according to claim 4, characterized in that: The coupling step (2) of the carrier and the indoxyl sulfate derivative comprises: 2-1, preparing a carrier solution and an indoxyl sulfate derivative solution, wherein the carrier is preferably serum protein, keyhole limpet hemocyanin, thyroglobulin or poly-lysine; 2-2, adding the indoxyl sulfate derivative solution to the carrier solution to obtain a mixed solution of the activated indoxyl sulfate derivative and the carrier; 2-3, stirring the mixed solution at 2-10° C. overnight (or reacting at room temperature for 2 hours) to obtain the crude indoxyl sulfate immunogen; 2-4, purifying the crude indoxyl sulfate immunogen to obtain the pure indoxyl sulfate immunogen, preferably by dialysis bag dialysis.

8. The preparation method according to claim 7, characterized in that: In step 2-1, the step of preparing the carrier solution includes dissolving the carrier in a phosphate buffer solution of 0.05 to 0.2 M and a pH of 8.0 to 9.5 to obtain the carrier solution; and the step of preparing the indoxyl sulfate derivative solution includes placing the indoxyl sulfate derivative, 1-ethyl-3-(-3-dimethylaminopropyl)carbodiimide (EDAc) and N-hydroxysulfosuccinimide (Sulfo-NHS) in N,N-dimethylformamide, methanol and 5 to 20 mM, pH 4.0 to 6.0 phosphate buffer solution and stirring at room temperature to obtain a solution of the indoxyl sulfate derivative in an activated state; wherein the mass ratio of the carrier to the indoxyl sulfate derivative is 1 to 8:

1.

9. An indoxyl sulfate detection kit, characterized in that: The kit contains the indoxyl sulfate derivative according to claim 1 and the antibody according to any one of claims 3.

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