Preparation method of p-cresol sulfate derivative, immunogen, antibody and p-cresol sulfate detection kit

Abstract

The invention relates to a preparation method of a p-cresol sulfate (PCS) detection kit by using a chemiluminescence immunoassay technology, and particularly relates to a preparation method of a p-cresol sulfate (PCS) detection kit. P-cresol sulfate is a protein-bound uremic toxin in a human body, has small molecular weight and poor immunogenicity, and when a corresponding antibody is prepared, a hapten and a specific macromolecular carrier need to be coupled 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 sulfuric acid p-cresol hapten, preparation of a sulfuric acid p-cresol complete antigen and an anti-sulfuric acid p-cresol antibody, a method for measuring the concentration of sulfuric acid p-cresol, and composition and components of a reagent. The design and synthesis of the hapten mainly comprise design and chemical synthesis of a sulfuric acid p-cresol derivative. The p-cresol 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 4.

Description

Technical Field

[0001] The present invention relates to the field of biomedical immunoassay, and particularly to a p-cresyl sulfate derivative, a p-cresyl sulfate immunogen, its specific antibody, and a p-cresyl sulfate detection kit. Background Art

[0002] p-Cresyl sulfate (PCS), whose structural formula is shown in Formula (Ⅲ):

[0003]

[0004] Uremic toxins refer to substances that cannot be cleared through urine in end-stage renal disease, accumulate in the body, and have toxic effects. Uremic toxins can be classified according to their physicochemical properties into: (1) water-soluble small molecules that cannot bind to proteins, such as urea, creatinine, etc.; (2) protein-bound substances: most of them have a small molecular weight and are difficult to be cleared by hemodialysis, such as p-cresyl sulfate and indoxyl sulfate; (3) middle molecules, such as parathyroid hormone and β2-microglobulin. Uremic toxins can be classified according to their sources into: (1) endogenous metabolites, such as asymmetric dimethylarginine; (2) microbial metabolites, mainly metabolites of intestinal flora, such as indoles and phenols; (3) exogenous intake substances, such as oxalates. Among them, p-cresyl sulfate and indoxyl sulfate are the most studied enterogenic uremic toxins at present. p-Cresyl sulfate mainly comes from phenylalanine and tyrosine in food, and is generated into 4-hydroxyphenylacetic acid by the action of anaerobic bacteria in the intestine. 4-Hydroxyphenylacetic acid is decarboxylated to p-cresol, absorbed through the intestinal mucosa, and converted into p-cresyl sulfate by the action of sulfotransferase in intestinal epithelial cells. It enters the urine through the renal tubules and is excreted. Due to the decline of renal function in uremic patients, it cannot be effectively excreted from the body. Coupled with the mutual competition of various urinary toxins for excretion pathways, it is easy to cause the accumulation of p-cresyl sulfate in the body, which is one of the most critical toxins leading to renal fibrosis. Clinical observations show that p-cresyl 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 p-cresyl 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 at the same time. 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 p-cresyl 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 terms of reagent performance. Summary of the Invention

[0006] The object of the present invention is to provide a p-cresol sulfate derivative, a p-cresol sulfate immunogen, its specific antibody and a p-cresol sulfate detection kit, so as to improve the defects in the prior art that the p-cresol sulfate determination method has poor specificity, low sensitivity and cannot be automatically analyzed.

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

[0008]

[0009] Wherein, R is a linking group -(CH2)n-COOH and n is 4. The p-cresol sulfate derivative of the present invention has the basic structure for preparing a p-cresol sulfate immunogen with immunogenicity, providing a structural basis for preparing a new p-cresol sulfate detection reagent.

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

[0011]

[0012] Wherein, R is a linking group -(CH2)n-COOH and n is 4, and the carrier is a protein or polypeptide with immunogenicity. The p-cresol sulfate immunogen of the present invention has high immunogenicity, can stimulate the animal body to produce an immune response, produce anti-p-cresol sulfate specific antibodies with high titers, and has strong antibody affinity, and is suitable for preparing a p-cresol sulfate competitive immunoassay reagent with high sensitivity and high specificity.

[0013] When n = 4, R in the above p-cresol sulfate immunogen is -(CH2)4-COOH. The carrier suitable for the above p-cresol 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 still another aspect of the present invention, there is also provided an anti-p-cresol 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 p-cresol 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 of the present invention can be a polyclonal antibody, a monoclonal antibody or a recombinant antibody, and is preferably a monoclonal antibody.

[0015] The antibodies of the present invention can be prepared by the prior art. A typical method for obtaining polyclonal antibodies is to use a single immunogen, with or without adjuvant, to immunize an animal at one or more sites. The host animals include: rabbits, goats, mice, sheep, guinea pigs, horses, alpacas or camels. Immunization is continued 5 - 7 times until the antibody titer reaches the highest level. 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 a p - cresol sulfate immunogen is also provided. The preparation method includes the steps of preparing the above - mentioned p - cresol sulfate derivative and connecting the above - mentioned p - cresol sulfate derivative with a carrier to obtain the p - cresol sulfate immunogen. By connecting the p - cresol sulfate derivative prepared by the above method with an immunogenic protein or polypeptide, the p - cresol 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 p - cresol sulfate immunogen of the present invention, a method for preparing the above - mentioned p - cresol sulfate derivative is also provided. The preparation method includes the following steps: Step 1, synthesis of compound 3: Dissolve compound 1 and compound 2 in dimethylformamide (DMF), add potassium carbonate, stir for 12 hours, add purified water, extract 3 times with dichloromethane, dry the obtained organic phase with sodium sulfate, concentrate, and purify through a silica gel drying column to obtain compound 3;

[0018]

[0019] Step 2, synthesis of the p - cresol sulfate derivative: Dissolve compound 3 and benzyltriethylammonium chloride in CHCl3, dropwise add sodium hydroxide solution, stir at 56 °C for 2.5 hours, adjust to pH = 4 with 6 mol / L hydrochloric acid, extract 3 times with ethyl acetate, dry the obtained organic phase, concentrate, and purify through a preparative - scale high - performance liquid chromatograph to obtain a white solid compound, which is the p - cresol sulfate derivative; this preparation method is simple to operate, and the reaction conditions are mild, with high stability and good repeatability.

[0020]

[0021] Specifically, in Step 1 of the preparation of the above-mentioned p-cresol sulfate derivative, the following steps are further included: Weigh 20 g of Compound 1 and 33.2 g of Compound 2, dissolve them together in 300 mL of dimethylformamide (DMF), then add 36.4 g of potassium carbonate to make a reaction mixture solution. Stir the reaction mixture solution at room temperature for 12 hours. After the reaction is completed, add 200 mL of purified water to the reaction solution, and then extract this solution with 300 mL of dichloromethane. The extraction step is repeated 3 times. Dry the obtained organic phase with anhydrous sodium sulfate, and then concentrate it. Purify the residue obtained after concentration through a silica gel drying column (PE:EA = 5:1, that is, the volume ratio of petroleum ether to ethyl acetate = 5:1) to obtain Compound 3;

[0022] In Step 2 of the preparation of the p-cresol sulfate derivative, the following steps are further included: Weigh 3 g of Compound 3 and 0.15 g of benzyltriethylammonium chloride, dissolve them together in 30 mL of CHCl3 to make a reaction solution. Weigh 15 g of sodium hydroxide and dissolve it in 15 mL of deionized water to make an alkaline solution. Dropwise add this alkaline solution to the above-mentioned reaction solution at 56 °C to make a reaction mixture, and then stir the reaction mixture at 56 °C for 2.5 hours. Adjust the residue obtained after the reaction to pH 4.0 with 6 mol / L hydrochloric acid for acidification treatment, and then extract it with 30 mL of EA. The extraction step is repeated 3 times. Dry and concentrate the obtained organic phase. Purify the residue obtained after the above treatment through a preparative high-performance liquid chromatograph to finally obtain a white solid compound, which is the p-cresol sulfate derivative.

[0023] In the above-mentioned preparation method of the p-cresol sulfate immunogen of the present invention, the connection step of the carrier and the p-cresol sulfate derivative can be reasonably improved according to the different carriers in actual operation. In the present invention, the above-mentioned connection steps include: Step 1, prepare a carrier solution and a p-cresol sulfate derivative solution; wherein, the mass ratio of the carrier to the p-cresol sulfate derivative is 1-8:1; preferably, the carrier is serum protein, keyhole limpet hemocyanin, thyroglobulin or polylysine; Step 2, dropwise add the activated p-cresol sulfate derivative solution to the carrier solution to obtain a crude p-cresol sulfate immunogen; Step 3, stir the dropwise addition mixture overnight at 2-10 °C or react at room temperature for 2 hours to obtain a crude p-cresol sulfate immunogen product; Step 4, purify the crude p-cresol sulfate immunogen product to obtain the p-cresol sulfate immunogen. The preparation steps of the present invention can obtain the target product through simple activation, dropwise addition, and purification steps, and the preparation method is simple, the process stability is high, and the reproducibility is good.

[0024] In the above method for preparing the p-cresol sulfate immunogen of the present invention, in the actual operation of the steps of preparing the carrier solution and the p-cresol sulfate derivative solution, appropriate solvent concentrations and pH values are reasonably selected according to different carrier types. In step 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 - 0.20 M and a pH of 8.0 - 9.5 to obtain a carrier solution; the step of preparing the p-cresol sulfate derivative solution is to place the p-cresol sulfate derivative, 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide, and N-hydroxysulfosuccinimide in N,N-dimethylformamide, methanol, and a potassium phosphate buffer solution with a concentration of 5 - 20 mM and a pH of 4.0 - 6.0, and stir at room temperature to obtain an activated p-cresol sulfate derivative solution. Using a phosphate buffer solution with a concentration of 0.10 - 0.25 M 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 p-cresol sulfate derivative to fully react and combine in a slightly alkaline environment. Using a phosphate buffer solution with a concentration in the range of 5 - 20 mM and a pH in the range of 4.0 - 6.0 can fully activate the carboxyl group of the p-cresol sulfate derivative in a slightly acidic solution.

[0025] In the above method for preparing the p-cresol sulfate immunogen of the present invention, in the step of dropping to obtain the crude p-cresol sulfate immunogen, in order to further increase the content of p-cresol sulfate immunogen in the crude p-cresol sulfate immunogen. In steps 2 and 3 of the present invention, through the dropping step, the p-cresol sulfate derivative can react more fully with the carrier; stirring overnight at 2 - 10 °C can further promote the formation of p-cresol sulfate immunogen, thereby increasing the content of p-cresol sulfate immunogen in the crude product.

[0026] In the above method for preparing the p-cresol sulfate immunogen of the present invention, any operation capable of purifying the p-cresol sulfate immunogen from the crude p-cresol sulfate immunogen is applicable to the present invention. Preferably, in step 4 above, the crude p-cresol sulfate immunogen is purified by dialysis to obtain the p-cresol sulfate immunogen. The dialysis method is simple and has a good purification effect.

[0027] According to another aspect of the present invention, there is also provided a p-cresol sulfate detection reagent, which includes an anti-p-cresol sulfate specific antibody, a p-cresol sulfate enzyme-labeled conjugate, and an enzyme substrate, wherein the anti-p-cresol sulfate specific antibody is any one of the above anti-p-cresol sulfate specific antibodies; the p-cresol sulfate enzyme-labeled conjugate contains the above p-cresol sulfate derivative. The p-cresol sulfate enzyme-labeled conjugate is formed by conjugating an enzyme and a hapten, and the hapten therein is the above p-cresol sulfate derivative.

[0028] The above-mentioned p-cresol sulfate detection reagent of the present invention has a high specificity of the anti-p-cresol sulfate specific antibody, a strong binding force with p-cresol sulfate, and a detection sensitivity much higher than that of the corresponding products in the prior art. Preferably, the above-mentioned enzyme-labeled conjugate is an alkaline phosphatase-hapten enzyme-labeled conjugate; the substrate of the above-mentioned enzyme is AMPPD or APS-5. The detection reagent using an 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 p-cresol sulfate in the sample and is suitable for high-throughput automated detection.

[0029] According to another aspect of the present invention, there is also provided a p-cresol sulfate detection kit, which contains the above-mentioned anti-p-cresol sulfate specific antibody and / or an indicator reagent for detecting the anti-p-cresol sulfate specific antibody and p-cresol sulfate complex. The indicator reagent is selected from enzyme reagents, radioisotope reagents, fluorescent reagents, and luminescent reagents. Preferably, the indicator reagent is composed of a p-cresol sulfate enzyme-labeled conjugate and the substrate of the enzyme, and the p-cresol sulfate enzyme-labeled conjugate can be conjugated with the p-cresol sulfate derivative of the present invention, which can conveniently and accurately determine the content of p-cresol sulfate in the sample and is suitable for high-throughput automated detection.

[0030] By applying the technical solution of the present invention, the p-cresol sulfate derivative obtained by substituting the hydrogen (H) at a specific site with a specific R group and the p-cresol sulfate immunogen formed by connecting with a specific carrier have high immunogenicity. The antibody produced by immunizing animals has high specificity and a strong specific binding ability with p-cresol sulfate. The high-throughput and rapid detection of p-cresol sulfate can be realized on a fully automated 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 p-cresol sulfate and is beneficial to clinical promotion and use. BRIEF DESCRIPTION OF THE DRAWINGS

[0031] Figure 1 : The standard curve of p-cresol sulfate ELISA detection.

[0032] Figure 2 : The calibration curve of p-cresol sulfate alkaline phosphatase chemiluminescence.

[0033] Figure 3 : The comparison result of samples between the p-cresol sulfate alkaline phosphatase chemiluminescence detection reagent and the p-cresol sulfate ELISA detection reagent of a well-known foreign manufacturer. DETAILED DESCRIPTION OF THE EMBODIMENTS

[0034] It should be noted that, without conflict, the embodiments in the present 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.

[0035] Example 1: Synthesis and Structure Confirmation of p-Cresol Sulfate Derivative

[0036] The chemical structure of the p-cresol sulfate derivative used in the following examples is shown in Formula (IV):

[0037]

[0038] The specific synthesis steps of the p-cresol sulfate derivative shown in Formula (IV) are as follows:

[0039]

[0040] The preparation process of the p-cresol sulfate derivative is as follows:

[0041] 1. Synthesis of Compound 3

[0042] Weigh 20 g of Compound 1, namely hydroquinone monosulfate (purchased from Shanghai Aladdin Biochemical Technology Co., Ltd.) and 33.2 g of Compound 2 (purchased from Merck), dissolve them together in 300 mL of DMF, then add 36.4 g of potassium carbonate to make a reaction mixture solution, and stir the above reaction mixture solution at room temperature for 12 hours. After the reaction is completed, add 200 mL of purified water to the above reaction solution, and then extract this solution with 300 mL of DCM. The extraction step is repeated 3 times. The extracted organic phase is dried with sodium sulfate, concentrated, and the residue obtained after concentration is purified through a silica gel drying column (PE:EA = 5:1) to obtain 29.5 g of Compound 3, with a yield of 70.2%.

[0043] 2. Synthesis of p-Cresol Sulfate Derivative

[0044] Weigh 3 g of Compound 3 and 0.15 g of benzyltriethylammonium chloride, dissolve them together in 30 mL of CHCl3 to make a reaction solution. Weigh 15 g of sodium hydroxide and dissolve it in 15 mL of water to make a sodium hydroxide solution. Add this sodium hydroxide solution dropwise to the above reaction solution at 56 °C to make a reaction mixture, and then stir this reaction mixture at 56 °C for 2.5 hours. The residue obtained after the reaction is adjusted to pH = 4 with 6 mol / L hydrochloric acid for acidification treatment, and then extracted with 30 mL of EA. The extraction step is repeated 3 times, and then the extracted organic phase is dried and concentrated. The residue obtained after the above treatment is purified by pre-HPLC, and finally 0.75 g of a white solid compound, namely the p-cresol sulfate derivative, is obtained, with a yield of 23.9%.

[0045] 3. Structure Identification of the Above-Purified Product

[0046] The above compound was scanned by nuclear magnetic resonance spectroscopy using Varian III plus 300MHz, with TMS as the internal standard, and the result was characterized as the target product.

[0047] Example 2: Synthesis of BSA-p-cresol sulfate derivative immunogen

[0048] The BSA-p-cresol sulfate immunogen is formed by linking the -(CH2)n-COO- group of bovine serum albumin (BSA) with the p-cresol sulfate derivative shown in formula (Ⅰ). In this example, n = 4. The synthesis method of this immunogen is described in detail as follows:

[0049] 1. Dissolve bovine serum albumin (20 mg) in 5 ml of 0.2 M phosphate buffer with pH 8.5;

[0050] 2. Add the following chemicals into a small beaker and stir to dissolve: 20 mg of the synthesized p-cresol 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;

[0051] 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 the p-cresol sulfate immunogen.

[0052] Example 3: Synthesis of KLH-p-cresol sulfate derivative immunogen

[0053] The KLH-acetyl p-cresol sulfate immunogen is formed by linking the -(CH2)n-COO- group of keyhole limpet hemocyanin (KLH) with the acetyl p-cresol sulfate derivative shown in formula (Ⅰ). In this example, n = 4. The synthesis method of this immunogen is described in detail as follows:

[0054] 1. Dissolve keyhole limpet hemocyanin (20 mg) in 5 ml of 0.18 M phosphate buffer with pH 8.5;

[0055] 2. Add the following chemicals into a small beaker and stir to dissolve: 20 mg of the synthesized p-cresol 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 for reaction.

[0056] 3. Drop the activated solution into the KLH solution and stir overnight at 2 - 10 °C to obtain the crude p-cresol sulfate immunogen; Purify the synthesized antigen by dialysis to obtain the p-cresol sulfate immunogen.

[0057] Example 4: Synthesis of Thyroglobulin-p-Cresol Sulfate Derivative Immunogen

[0058] The thyroglobulin-p-cresol sulfate immunogen is formed by connecting thyroglobulin with the -(CH2)n-COO- group of the p-cresol sulfate derivative shown in formula (I). In this example, n = 4. The synthesis method of this immunogen is described in detail, and the specific steps are as follows:

[0059] 1. Dissolve thyroglobulin (20 mg) in 5 mL of 0.20 M phosphate buffer with pH 9.0.

[0060] 2. Add the following chemicals into a small beaker and stir to dissolve: 10 mg of the synthesized p-cresol 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 for reaction.

[0061] 3. Drop the activated solution drop by drop into the thyroglobulin solution and stir overnight at 2 - 10 °C to obtain the complete antigen; Purify the synthesized antigen by dialysis to obtain the p-cresol sulfate immunogen.

[0062] Example 5: Synthesis of Polylysine-p-Cresol Sulfate Derivative Immunogen

[0063] The polylysine-p-cresol sulfate immunogen is formed by connecting polylysine with the -(CH2)n-COO- group of the p-cresol sulfate derivative shown in formula (I). In this example, n = 4. The synthesis method of this immunogen is described in detail, and the specific steps are as follows:

[0064] 1. Dissolve polylysine (20 mg) in 5 ml of 0.20 M phosphate buffer at pH 9.0;

[0065] 2. Add the following chemicals into a small beaker and stir to dissolve: 10 mg of synthesized p-cresol 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 30 min;

[0066] 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 p-cresol sulfate immunogen.

[0067] Example 6: Preparation of anti-p-cresol sulfate specific monoclonal antibody

[0068] This example presents a method for preparing a p-cresol sulfate monoclonal antibody, which includes the following steps:

[0069] 1. Animal immunization Immunize two 8-week-old Balb / C female mice with the artificial antigen prepared in Example 3 by the multi-point subcutaneous injection method on the back of the neck. For the primary immunization, use the immunogen emulsified with Freund's complete adjuvant. Mix the same volume of immunogen and Freund's complete adjuvant for emulsification, and 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 tail of the mouse 7 days after each immunization, centrifuged to obtain the antiserum, and stored at -20 °C for serum titer determination and specificity determination.

[0070] 2. Analysis of antiserum effect by ELISA indirect enzyme-linked immunosorbent assay Using the conventional antibody titer determination method, with the blank serum without antibody as the control, dilute the antiserum by a certain multiple and then perform ELISA detection. Finally, the titer of the anti-p-cresol sulfate specific antibody of this invention is detected to be 1:30000 - 1:50000, indicating that the antibody prepared by this invention has strong specificity and high sensitivity.

[0071] 3. Cell fusion and screening of positive hybridomas

[0072] (1) Recovery 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 to disperse 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. During this period, the medium needs to be changed several times. When the cells cover the bottom of each culture dish, they can be used for cell fusion.

[0073] (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 screen for grinding the spleen. Blow off 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 the cell screen, 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 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 of spleen cells, add them to myeloma cells through a cell sieve, blow evenly, add basal culture medium to 20mL, and centrifuge at 1200r / min for 5 minutes. Take out the mixed cells after centrifugation and discard the supernatant, suck off the excess culture medium with a gun, shake the precipitated cells, and then incubate them in a 37℃ cell culture incubator for 5 minutes.

[0074] (3) Cell fusion: After the incubation, place the centrifuge tube in 37°C warm water and keep it rotating. Use the pipette tip to absorb 1 mL 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 1 mL within the third minute, 3 mL within the fourth minute, and 16 mL within the fifth to sixth minutes. Add along the wall while stirring gently to separate the PEG. Seal the centrifuge tube and centrifuge at 900 r / min for 8 minutes. Pour off the supernatant, 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.

[0075] (4) Screening of positive hybridomas: Half of the medium was replaced with HT medium within 4 days after fusion, and the medium was fully replaced with HT medium in each well after 8 days. 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, and 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, and 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, and 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 of each plate was positive and the titer and inhibition were similar after detection. At this point, 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 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.

[0076] 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°C for standby use to obtain the p-cresol sulfate monoclonal antibody.

[0077] Example 7: p-Cresol Sulfate ELISA Test The titer and specificity of the antiserum were determined by ELISA indirect enzyme-linked immunosorbent assay, and the steps are as follows: The antibody prepared in Example 6 was used to perform an ELISA test for p-cresol sulfate. This test utilizes competitive immunoassay to determine the p-cresol sulfate content in a liquid sample. The principle is that the p-cresol sulfate in the sample competes with the coupled p-cresol sulfate derivative (HRP-p-cresol sulfate derivative enzyme conjugate) for binding to the limited sites on the antibody coated in the ELISA plate. If there is almost no or no p-cresol sulfate in the liquid sample, the p-cresol sulfate derivative coupled with the HRP enzyme 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 p-cresol sulfate, the enzyme-p-cresol sulfate derivative conjugate will reduce the binding with the antibody, thereby weakening the color development signal. Therefore, the absorbance generated by the test is inversely proportional to the p-cresol sulfate content in the liquid sample. The specific steps are as follows:

[0078] 1. Preparation of Standards

[0079] P-cresol sulfate powder (purchased from MCE) was dissolved in methanol solution to prepare a 1 mg / ml stock solution. The stock solution was diluted with ELISA buffer to 400.00 ng / mL, 200.00 ng / mL, 100.00 ng / mL, 50.00 ng / mL, 25.00 ng / mL, 12.50 ng / mL, 6.25 ng / mL and 0.00 ng / mL standard solutions. The ELISA buffer contained 50.0 mM Tris, 100 mM NaCl and 0.2% BSA.

[0080] 2. Preparation of standard curve using ELISA test method of p-cresol sulfate

[0081] Use PBS to dilute the anti-p-cresol sulfate antibody prepared in Example 6 to a final concentration solution of 1:10000, and coat 100 μL / well on a 96-well ELISA plate and place at 4°C for 12-24 hours; wash the 96-well ELISA plate coated with the anti-p-cresol sulfate antibody three times with PBS, add 200 μL / well of 0.5% BSA solution, seal and place at 4°C for 8-16 hours. Then wash three times with PBS and add 20 μL / well of standard. Then add 100 μL / well of HRP-p-cresol sulfate conjugate at a working concentration; incubate at room temperature for 30 minutes and wash the plate 5 times with PBS; then add 100 μL of TMB substrate to each well and incubate 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, and make a standard curve. The results are as follows. Figure 1 shown.

[0082] Example 8: Preparation of p-cresol sulfate alkaline phosphatase chemiluminescent detection reagent

[0083] The antibody obtained in Example 6 was used to prepare a chemiluminescent detection reagent for p-cresol sulfate alkaline phosphatase as follows:

[0084] 1. A detection kit for p-cresol sulfate. This experiment uses the principle of competitive chemiluminescent immunoassay reagents. 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 antigen labeled with a luminescent label bound to the antibody. A standard curve is made with known standards, and the luminescence value of the unknown sample is calibrated on the standard curve to obtain the concentration of p-cresol sulfate in the unknown sample. It is characterized by comprising p-cresol sulfate labeled with a chemiluminescent label and a monoclonal antibody against p-cresol sulfate coated on a fixed carrier; 2. In the p-cresol sulfate labeled with a chemiluminescent label, the chemiluminescent label is alkaline phosphatase. 3. The antibody coated on the fixed carrier is an anti-p-cresol sulfate antibody coated on magnetic beads.

[0085] 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 Figure 2 shown.

[0086] Table 1: Reaction parameters of the alkaline phosphatase chemiluminescent analyzer

[0087]

[0088] The samples were compared between the p-cresol sulfate alkaline phosphatase chemiluminescent detection reagent of the present invention and the p-cresol sulfate ELISA detection reagent of well-known foreign manufacturers. The detection data and data analysis are shown in Figure 3 .

[0089] Example 9: Cross-reaction test of analogs

[0090] Four common p-cresol sulfate structural analogs were selected for cross-reaction tests and measured using the alkaline phosphatase chemiluminescent detection reagent. Four common p-cresol sulfate structural analogs and the cross-reaction rates are shown in Table 2.

[0091] Table 2: Cross-reaction results of four common p-cresol sulfate structural analogs

[0092] p-Cresol Sulfate Cross-Reaction (%) p-Cresol Sulfate 100 Cresol Sulfonic Acid 0.01 o-Cresol 0.05 4-Methylcalcium Methanol-Sulfuric Acid 0.02

[0093] Determination result: From the cross-reaction results of the above 4 common structural analogs of p-cresol sulfate, it can be seen that the antibody prepared by the present invention has good specificity.

[0094] From the above results, it can be seen that the p-cresol sulfate complete immunogen prepared from the p-cresol sulfate derivative provided by the present invention has strong immunogenicity, the produced antibody has high specificity, good affinity with p-cresol 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 p-cresol 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 p-cresol sulfate, which is beneficial to clinical promotion and use.

[0095] 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 accordingly. Any equivalent structural or equivalent process transformation made by using the content of the specification and drawings of the present invention, or directly or indirectly applied in other related technical fields, shall be equally included in the patent protection scope of the present invention.

Claims

1. A p-cresol 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 4.

2. A p-cresol sulfate immunogen, characterized in that: It has the structural formula shown in formula (II): Where R is —(CH2) n —COOH, n is 4, and the carrier is a protein or polypeptide with immunogenicity, wherein the carrier is serum protein, keyhole limpet hemocyanin, thyroglobulin or poly-lysine.

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

4. A method for preparing a p-cresol sulfate immunogen, characterized in that: The preparation method comprises: preparing the p-cresol sulfate derivative according to claim 1; and connecting the p-cresol sulfate derivative with a carrier to obtain the p-cresol sulfate immunogen; wherein the carrier is a protein or polypeptide with immunogenicity; the steps of preparing the p-cresol sulfate derivative comprise: (1) Step 1, synthesis of compound 3: Compound 1 and compound 2 were dissolved in dimethylformamide (DMF), potassium carbonate was added, stirred for 12 hours, purified water was added, and extracted with dichloromethane three times. The organic phase obtained by extraction was dried over sodium sulfate, concentrated, and purified by a silica gel drying column to obtain compound 3; (2) Step 2, synthesis of p-cresol sulfate derivatives: Compound 3 and benzyltriethylammonium chloride were dissolved in CHCl3, sodium hydroxide solution was added dropwise, and the mixture was stirred at 56°C for 2.5 hours. The pH value of HCl was adjusted to 4, and the mixture was extracted with ethyl acetate three times. The organic phase obtained by the extraction was dried and concentrated, and purified by preparative high performance liquid chromatography to obtain a white solid compound, which is a p-cresol sulfate derivative.

5. The preparation method according to claim 4, characterized in that: The coupling step of the carrier and the p-cresol sulfate derivative comprises: (1) Step 5-1, preparing a carrier solution and a solution of a p-cresol sulfate derivative; preferably, the carrier is serum protein, keyhole limpet hemocyanin, thyroglobulin or poly-lysine; (2) Step 5-2, adding the p-cresol sulfate derivative solution to the carrier solution to obtain a mixed solution of the activated p-cresol sulfate derivative and the carrier; (3) Step 5-3, stirring the mixed solution at 2-10° C. overnight (or reacting at room temperature for 2 hours) to obtain the crude product of p-cresol sulfate immunogen; (4) Step 5-4, purifying the crude p-cresol sulfate immunogen to obtain the pure p-cresol sulfate immunogen; preferably, purification is performed by dialysis bag dialysis.

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

1.

7. A p-cresol sulfate detection kit, characterized in that, The kit contains the p-cresol sulfate derivative according to claim 1 or the antibody according to any one of claim 3.

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