Kynurenine derivative, immunogen and specific antibody thereof, and preparation method of kynurenine detection kit

Abstract

The invention relates to a preparation method of a kynurenine detection kit by using a chemiluminescence immunoassay technology. The kynurenine detection kit is used for detecting kynurenine by using a chemiluminescence immunoassay technology. Kynurenine is one of important metabolic intermediates of tryptophan in a human body, and the abnormal metabolic pathway of kynurenine is closely related to mental disorder, type II diabetes, obesity, insulin resistance and the like. As kynurenine is small in molecular weight and poor in immunogenicity, when a corresponding antibody is prepared, a hapten needs to be coupled with a specific macromolecular carrier to prepare a complete antigen. The invention mainly relates to design and synthesis of a kynurenine hapten, preparation of a kynurenine complete antigen and an anti-kynurenine antibody, a method for determining the concentration of kynurenine, and composition and components of a reagent. The design and synthesis of the hapten mainly comprise design and chemical synthesis of a kynurenine derivative. Kynurenine derivative provided by the invention has a structure as shown in formula (I): # imgabs0 #

Description

Technical Field

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

[0002] Kynurenine, whose structural formula is shown in formula (III):

[0003]

[0004] Tryptophan (TRP) is an essential amino acid for the human body. Ingested TRP is primarily metabolized through the kynurenine pathway, with kynurenine being one of the most important intermediate metabolites in this pathway. Most metabolites produced by the kynurenine pathway are neuroactive and play a crucial role in regulating N-methyl-D-aspartate (NMDA) receptor function and free radical production. The NMDA receptor has been implicated in various neurodegenerative diseases, such as Parkinson's disease, Alzheimer's disease, and depression. Furthermore, the production of free radicals and excitatory amino acids can damage brain cells and exacerbate brain damage in stroke. Cerebrospinal fluid, a colorless, transparent fluid found in the ventricles and subarachnoid space, contains numerous brain tissue metabolites, reflecting brain metabolism. The kynurenine pathway is involved in immune activation and inflammation regulation and is associated with obesity and insulin resistance. Furthermore, tryptophan is metabolized by the gut microbiota to produce various indole derivatives. In human studies, plasma levels of tryptophan and kynurenine metabolites have been associated with an increased risk of type 2 diabetes (T2D), while indolepropionic acid, a microbial metabolite of tryptophan, has been associated with a reduced risk of T2D. Therefore, studying the changes in kynurenine levels in the body is of great significance.

[0005] At present, the main methods for kynurenine detection are high performance liquid chromatography mass spectrometry (HPLC MS / MS) and enzyme-linked immunosorbent assay (ELISA). Due to the small molecular structure of kynurenine, the fragments produced after proton impact using high performance liquid chromatography mass spectrometry are also small, which affects the selectivity and sensitivity of the detection, and because the equipment is expensive and the detection cost is high, it is not suitable for widespread application. The ELISA method is cumbersome to operate, time-consuming and labor-intensive, requires manual operation, has poor repeatability, and is not conducive to the promotion of clinical testing. The present invention starts with antigen design and antibody preparation, and adopts an advanced fully automatic chemiluminescence detection platform to completely solve the pain points of kynurenine clinical testing and has broad application prospects. This detection reagent can be used for high-throughput, automated, and multi-type sample determination, and has the outstanding advantages of high sensitivity, specificity, and good stability in reagent performance. Summary of the Invention

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

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

[0008]

[0009] The kynurenine derivative of the invention has a basic structure for preparing a kynurenine immunogen with immunogenicity, and provides a structural basis for preparing a new kynurenine detection reagent.

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

[0011]

[0012] The carrier is an immunogenic protein or polypeptide. The kynurenine immunogen of the present invention has high immunogenicity, can stimulate the animal body to produce an immune response, and produce high-titer anti-kynurenine-specific antibodies with strong antibody affinity, and is suitable for preparing a highly sensitive and specific kynurenine competitive detection reagent.

[0013] Kynurenine derivatives suitable for use as carriers of the aforementioned kynurenine immunogens are preferably protein carriers, but other immunogenic substances with sufficiently large molecular weight and sufficient reactive groups can also serve as carriers. The most commonly used immunogenic carriers include serum proteins, keyhole limpet hemocyanin (KLH), thyroglobulin, and polylysine. The carrier in the present invention is preferably keyhole limpet hemocyanin.

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

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

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

[0017] In the method for preparing the above-mentioned kynurenine immunogen of the present invention, a method for preparing the above-mentioned kynurenine derivative is also provided, which comprises the following steps: dissolving 100 mg of compound 1 in 10 mL of dimethylformamide, then adding 0.1 g of compound 2, 0.6 g of triethylamine and 0.12 g of 2-(7-azobenzotriazole)-N,N,N',N'-tetramethyluronium hexafluorophosphate at 0°C to prepare a reaction mixture solution, stirring the reaction mixture solution at room temperature overnight, filtering out the solid matter after the reaction, concentrating the filtrate, and finally purifying the residue obtained by concentration by flash column chromatography to obtain the kynurenine derivative.

[0018] In the preparation method of the kynurenine derivatives of the present invention, the preparation steps of the kynurenine derivatives are as follows:

[0019]

[0020] In the above-mentioned method for preparing the kynurenine immunogen of the present invention, the step of connecting the carrier and the kynurenine derivative can be reasonably improved according to the different carriers in actual operation. In the present invention, the above-mentioned connection step includes: step a, preparing a carrier solution and a kynurenine derivative solution; wherein the mass ratio of the carrier to the kynurenine derivative is 1 to 8:1; preferably, the carrier is serum protein, keyhole limpet hemocyanin, thyroglobulin or polylysine; step b, adding the activated kynurenine derivative solution dropwise to the carrier solution to obtain a crude kynurenine immunogen; step c, stirring the added mixture at 2 to 10°C overnight or reacting at room temperature for 2 hours to obtain a crude kynurenine immunogen; step d, purifying the crude kynurenine immunogen to obtain the kynurenine immunogen. The preparation steps of the present invention can obtain the target product through simple dissolution, dropwise addition, and purification steps. The preparation method is simple, the process is highly stable, and the reproducibility is good.

[0021] In the method for preparing the kynurenine immunogen described above, during the steps of preparing the carrier solution and the kynurenine derivative solution, appropriate solvent concentrations and pH values are selected based on the type of carrier. In step a, the carrier solution is prepared by dissolving the carrier in a 0.05-0.20 M phosphate buffer solution at a pH of 8.0-9.5 to obtain a carrier solution. The kynurenine derivative solution is prepared by placing the kynurenine derivative in N,N-dimethylformamide, methanol, and a 0.5-0.20 M potassium phosphate buffer solution at a pH of 8.0-9.5, stirring at room temperature to obtain a kynurenine derivative solution. Dissolving the carrier in a phosphate buffer solution at a concentration of 0.10-0.25 M and a pH of 8.0-9.5 allows the amino groups on the carrier and the ester groups of the activated kynurenine derivative to fully react and bond in a slightly alkaline environment.

[0022] In the method for preparing the kynurenine immunogen of the present invention, in the step of dropwise addition to obtain a crude kynurenine immunogen, in order to further increase the kynurenine immunogen content in the crude kynurenine immunogen, the dropwise addition in steps b and c allows the kynurenine derivative to react more fully with the carrier; and stirring overnight at 2-10° C. further promotes the formation of the kynurenine immunogen, thereby increasing the kynurenine immunogen content in the crude product.

[0023] In the above-mentioned method for preparing the kynurenine immunogen of the present invention, any operation capable of purifying the crude kynurenine immunogen to obtain the kynurenine immunogen is applicable to the present invention. Preferably, in step d above, the crude kynurenine immunogen is purified by dialysis to obtain the kynurenine immunogen. Dialysis is a simple method and has a good purification effect.

[0024] According to another aspect of the present invention, a kynurenine detection reagent is provided, comprising an anti-kynurenine-specific antibody, a kynurenine-enzyme conjugate, and an enzyme substrate, wherein the anti-kynurenine-specific antibody is any of the anti-kynurenine-specific antibodies described above; and the kynurenine-enzyme conjugate contains the kynurenine derivative described above. The kynurenine-enzyme conjugate is formed by coupling an enzyme and a hapten, wherein the hapten is the kynurenine derivative described above.

[0025] The kynurenine detection reagent of the present invention has a detection sensitivity far superior to that of existing products due to the high specificity of the anti-kynurenine-specific antibody and its strong binding affinity to kynurenine. Preferably, the enzyme-labeled conjugate is an alkaline phosphatase-hapten enzyme-labeled conjugate; and the enzyme substrate is AMPPD or APS-5. Detection reagents using the alkaline phosphatase-hapten enzyme-labeled conjugate and AMPPD or APS-5 as enzyme substrates can conveniently and accurately determine the kynurenine content in a sample and are suitable for high-throughput automated detection.

[0026] According to another aspect of the present invention, a kynurenine detection kit is also provided, comprising the above-mentioned anti-kynurenine-specific antibody and an indicator reagent for detecting the anti-kynurenine-specific antibody and the kynurenine complex. The indicator reagent is selected from an enzyme reagent, a radioisotope reagent, a fluorescent reagent, and a luminescent reagent. Preferably, the indicator reagent comprises a kynurenine enzyme-labeled conjugate and an enzyme substrate, wherein the kynurenine enzyme-labeled conjugate can be coupled to the kynurenine derivative of the present invention, enabling convenient and accurate determination of the kynurenine content in a sample, and being suitable for high-throughput automated detection.

[0027] The technical solution of the present invention, which connects a kynurenine derivative to a specific carrier via an ester bond, creates a kynurenine immunogen with high immunogenicity. The antibodies produced by immunized animals are highly specific and have strong specific binding ability to kynurenine. High-throughput and rapid kynurenine detection can be achieved on a fully automated chemiluminescence immunoassay using alkaline phosphatase chemiluminescence immunoassay technology. This method offers advantages such as ease of operation, high sensitivity, strong specificity, and accurate results. It also effectively reduces the cost of kynurenine detection, facilitating its widespread clinical use. BRIEF DESCRIPTION OF THE DRAWINGS

[0028] Figure 1 : Standard curve of kynurenine ELISA detection.

[0029] Figure 2 : Calibration curve of chemiluminescence of kynurenine alkaline phosphatase.

[0030] Figure 3 : The results of sample comparison between the kynurenine chemiluminescence detection reagent of the present invention and the kynurenine ELISA detection reagent of a well-known foreign manufacturer were obtained. DETAILED DESCRIPTION

[0031] It should be noted that, in the absence of conflict, the embodiments and features of the embodiments of the present invention can be combined with each other. The present invention will be described in detail below with reference to the accompanying drawings and in combination with the embodiments.

[0032] Example 1: Synthesis and structural confirmation of kynurenine derivatives

[0033] The chemical structure of the kynurenine derivatives used in the following examples is shown in formula (IV):

[0034]

[0035] The specific synthetic route of the kynurenine derivative represented by formula (IV) is as follows:

[0036]

[0037] The specific synthesis steps are as follows:

[0038] 100 mg of compound 1 was dissolved in 10 mL of dimethylformamide, and then 0.1 g of compound 2, 0.6 g of triethylamine, and 0.12 g of 2-(7-azobenzotriazole)-N,N,N',N'-tetramethyluronium hexafluorophosphate were added at 0°C to prepare a reaction mixture. The reaction mixture was stirred at room temperature overnight. After the reaction, the solid material was filtered out, and the filtrate was concentrated. Finally, the concentrated residue was purified by flash column chromatography to obtain 52 mg of a kynurenine derivative with a yield of 37.8%.

[0039] The compound was subjected to nuclear magnetic resonance spectroscopy using a Varian III plus 300 MHz spectrometer with TMS as the internal standard. The results showed that the target product was a kynurenine derivative.

[0040] Example 2: Synthesis of BSA-kynurenine derivative immunogens

[0041] The BSA-kynurenine immunogen is formed by ester-linking bovine serum albumin (BSA) and a kynurenine derivative represented by formula (I). In this example, the synthesis method of the immunogen is described in detail. The specific steps are as follows:

[0042] 1. Dissolve bovine serum albumin (20 mg) in 5 ml of 0.2 M phosphate buffer, pH 8.5;

[0043] 2. Add the following chemicals to a small beaker and stir to dissolve: 20 mg of the synthesized kynurenine derivative, 0.3 ml of D,D-dimethylformamide (DMF), 0.3 ml of methanol, and 1.0 ml of 0.2 M, pH 8.5 phosphate buffer. Stir and dissolve these chemicals at room temperature for 10 minutes.

[0044] 3. Add the dissolved solution dropwise to the BSA solution and stir overnight at 2-10°C to obtain a crude antigen product; purify the synthesized antigen by dialysis to obtain a kynurenine immunogen.

[0045] Example 3: Synthesis of KLH-kynurenine derivative immunogens

[0046] The KLH-kynurenine immunogen is formed by ester-linking hemocyanin (KLH) and a kynurenine derivative represented by formula (I). In this example, the synthesis method of the immunogen is described in detail. The specific steps are as follows:

[0047] 1. Dissolve hemocyanin (20 mg) in 5 ml of 0.18 M phosphate buffer, pH 8.5;

[0048] 2. Add the following chemicals to a small beaker and stir to dissolve: 20 mg of the synthesized kynurenine derivative, 0.3 ml of D, D-dimethylformamide (DMF), 0.3 ml of methanol, and 1.0 ml of 0.18 M, pH 8.5 phosphate buffer. Stir and dissolve these chemicals at room temperature for 10 minutes.

[0049] 3. Add the dissolved solution dropwise to the KLH solution and stir overnight at 2-10°C to obtain a crude kynurenine immunogen; purify the synthesized antigen by dialysis to obtain a kynurenine immunogen.

[0050] Example 4: Synthesis of Thyroglobulin-Kynurenic Acid Derivative Immunogens

[0051] The thyroglobulin-kynurenine immunogen is formed by ester-linking thyroglobulin and a kynurenine derivative represented by formula (I). In this example, the synthesis method of the immunogen is described in detail. The specific steps are as follows:

[0052] 1. Dissolve thyroglobulin (20 mg) in 5 ml of 0.20 M phosphate buffer, pH 9.0;

[0053] 2. Add the following chemicals to a small beaker and stir to dissolve: 10 mg of the synthesized kynurenine derivative, 0.3 ml of N,N-dimethylformamide (DMF), 0.3 ml of methanol, and 1.0 ml of 0.20 M, pH 9.0 potassium phosphate buffer. Stir and dissolve these chemicals at room temperature for 30 minutes.

[0054] 3. Add the activated solution dropwise to the thyroglobulin solution and stir overnight at 2-10°C to obtain the complete antigen; purify the synthesized antigen by dialysis to obtain the kynurenine immunogen.

[0055] Example 5: Synthesis of polylysine-kynurenine derivative immunogens

[0056] The polylysine-kynurenine immunogen is formed by linking polylysine with the ester bond group of a kynurenine derivative represented by formula (I). In this example, the synthesis method of the immunogen is described in detail. The specific steps are as follows:

[0057] 1. Dissolve poly-lysine (20 mg) in 5 ml of 0.20 M, pH 9.0 phosphate buffer;

[0058] 2. Add the following chemicals to a small beaker and stir to dissolve: 10 mg of the synthesized kynurenine derivative, 0.3 ml of N,N-dimethylformamide (DMF), 0.3 ml of methanol, and 1.0 ml of 0.20 M, pH 9.0 potassium phosphate buffer. Stir and dissolve these chemicals at room temperature for 30 minutes.

[0059] 3. Add the activated solution dropwise to the poly-lysine solution and stir overnight at 2-10°C to obtain the complete antigen; centrifuge the synthesized antigen to obtain the supernatant, and purify the supernatant by dialyzing to obtain the kynurenine immunogen.

[0060] Similarly, 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.

[0061] Example 6: Preparation of anti-kynurenine-specific monoclonal antibodies

[0062] This example provides a method for preparing a kynurenine monoclonal antibody, which comprises the following steps:

[0063] 1. Animal immunization

[0064] Two eight-week-old female Balb / C mice were immunized with the artificial antigen prepared in Example 5, using multiple subcutaneous injections at the nape of the neck. The primary immunization used the immunogen emulsified in Freund's complete adjuvant (CFA). Equal volumes of the immunogen and CFA were mixed and emulsified, with a single mouse dose of 0.1 mg. After the primary immunization, booster immunizations were administered every 14 days at a dose of 0.1 mg using the same emulsification method. Five immunizations were performed, starting with the third booster immunization. Seven days after each immunization, 30 μL of blood was collected from the tail of the mice, centrifuged, and stored at -20°C for titer and specificity determination.

[0065] 2. ELISA indirect enzyme-linked immunosorbent assay to analyze the effect of antiserum

[0066] Using a conventional antibody titer determination method, blank serum without antibody was used as a control, and the antiserum was diluted a certain multiple and then subjected to ELISA detection. The final detection showed that the titer of the anti-kynurenine-specific antibody of the present invention was 1:30,000-1:50,000, indicating that the antibody prepared by the present invention has strong specificity and high sensitivity.

[0067] 3. Cell fusion and screening of positive hybridomas

[0068] (1) Resuscitation of myeloma cells: Take the myeloma cells out of liquid nitrogen and quickly thaw them in a 37°C water bath. Centrifuge at 1000 rpm for 5 minutes after thawing. Pour off the supernatant in a clean bench and add about 1 mL of complete culture medium to the cell pellet. Blow away the cells. Use a pipette to take out the cells and mix them with complete culture medium. Place them in a 10 cm diameter culture dish and expand to 4 to 6 dishes. Change the medium several times during this period. When the cells cover the bottom of each culture dish, they can be used for cell fusion.

[0069] (2) Cell preparation: Take out two small culture dishes, pour part of the culture medium into one to cool the dissection tools, and absorb a small amount of culture medium into the other and put it into a cell sieve for grinding the spleen. Blow down the revived myeloma cells and transfer them to a 50mL centrifuge tube, seal it, and centrifuge it at 1200r / min for 5 minutes. Kill the mice after five immunizations, soak them in 75% alcohol for about 1 minute, put them in a clean bench, take blood from the heart, warm them at 37℃ for 30 minutes, centrifuge them for 15 minutes, and take the serum and store it at -20℃. After taking the mouse spleen cells, grind them thoroughly in a cell sieve, wash them with the pre-packaged basic solution, and transfer them to a 50mL centrifuge tube, seal it, discard the supernatant of the centrifuged myeloma cells, add the basic solution to wash them again, and centrifuge them together with the spleen cells at 1200r / min for 5 minutes. After the second centrifugation, discard the supernatant of the myeloma cells, add 2mL of basic solution, and blow them evenly. Remove the supernatant from the spleen cells, pass them through a cell sieve, and add them to the myeloma cells. After pipetting evenly, add basal medium to 20 mL and centrifuge at 1200 rpm for 5 minutes. Remove the centrifuged mixed cells and discard the supernatant. Aspirate the excess medium with a pipette and shake the precipitated cells to disperse. Then, incubate the mixture in a 37°C cell culture incubator for 5 minutes.

[0070] (3) Cell fusion: After the incubation, place the centrifuge tube in 37°C warm water and keep rotating. Use the pipette tip to absorb 1mL of PEG preheated to 37°C. Slowly add PEG to the precipitated cells within the first minute. Let it stand for 1 minute. Preheat the basal culture medium. Add 1mL within the third minute, 3mL within the fourth minute, and 16mL within the fifth to sixth minutes. Stir gently and add along the wall to separate the PEG. Seal the centrifuge tube and centrifuge at 900r / min for 8 minutes. Pour off the supernatant and add the fused cells to HAT complete culture medium. Stir gently and evenly add to four 24-well culture plates. Keep the volume of HAT culture medium containing fused cells the same in each well.

[0071] (4) Screening of positive hybridomas: within 4 days after fusion, half of the medium was replaced with HT medium. After 8 days, the medium was replaced with HT medium in each well. On the 10th day, the supernatant in the multi-well culture plate was extracted and the specific antibodies in the culture medium were detected by indirect ELISA. The positive hybridoma cells with high titer and strong affinity were selected. The positive wells with the best fusion effect were screened and marked. Under sterile conditions, the cells were transferred to a new 96-well culture plate. Each original well was cloned into two 96-well plates. After the cells adhered to the wall and covered 1 / 4 of the bottom of the well, the supernatant was taken and tested by ELISA. The titer and inhibition rate were also used as measurement indicators. The strong positive cells were subcloned by limiting dilution method. This was repeated 3 to 4 times (note that the positive well cells selected in each round need to be expanded and cultured and then frozen for future use). Until each well on each plate is positive and the titer and inhibition are similar after detection, the hybridoma cell line was successfully established and a hybridoma cell line that can stably secrete uniform antibodies was obtained. Single cell clones were picked and those that tested positive were transferred to 24-well cell culture plates, 6-well cell culture plates, and 10 cm cell culture dishes for expansion and freezing.

[0072] 4. Large-scale preparation of monoclonal antibodies

[0073] After obtaining hybridoma cell clones that secrete specific monoclonal antibodies, large-scale monoclonal antibodies are typically produced using in vitro culture and in vivo monoclonal antibody induction methods. Preliminary, ten or more Balb / c mice aged eight weeks or older are injected intraperitoneally with liquid lysate at a dose of 0.5 mL per mouse. One to two weeks later, hybridoma cells are injected intraperitoneally into the mice. Following cell inoculation, the mice are observed daily. Starting on day 7, the abdominal cavity will swell. Before the mice die, ascites is collected using a sterile disposable syringe. The collected ascites is centrifuged at 12,000 rpm for 10 minutes to remove the upper fat layer and the lower fibrin layer. The middle layer is collected and the titer and inhibition rate are determined using ELISA. After purification, the antibody is stored at -20°C until further use. This yields the kynurenine monoclonal antibody.

[0074] Example 7: Kynurenine ELISA test

[0075] The titer and specificity of the antiserum were determined by ELISA indirect enzyme-linked immunosorbent assay, and the steps are as follows:

[0076] 1. ELISA test for kynurenine using the antibody prepared in Example 6

[0077] This test uses a competitive immunoassay to measure the kynurenine content in a liquid sample. The principle is that kynurenine in the sample competes with a conjugated kynurenine derivative (HRP-kynurenine derivative enzyme conjugate) for binding to the limited sites on the antibody coated on the ELISA plate. If the liquid sample contains little or no kynurenine, the HRP enzyme-conjugated kynurenine derivative will bind to the antibody on the ELISA plate. Conversely, if the liquid sample contains a large or constant amount of kynurenine, the enzyme-kynurenine derivative conjugate will bind less to the antibody, resulting in a weaker color signal. Therefore, the absorbance generated by the test is inversely proportional to the kynurenine content in the liquid sample.

[0078] 2. The specific steps for establishing the standard curve for kynurenine ELISA are as follows:

[0079] (1) Preparation of standard products

[0080] Kynurenine powder (purchased from Merck) was dissolved in methanol to prepare a 1 mg / mL stock solution. The stock solution was diluted sequentially with ELISA buffer containing 50.0 mM Tris, 100 mM NaCl, and 0.2% BSA to prepare standard solutions of 12.50 μmol / L, 6.25 μmol / L, 3.13 μmol / L, 1.56 μmol / L, 0.78 μmol / L, 0.39 μmol / L, 0.10 μmol / L, and 0.00 μmol / L.

[0081] (2) Preparation of standard curve using kynurenine ELISA test method

[0082] Use PBS to dilute the anti-kynurenine antibody prepared in Example 7 to a final concentration solution of 1:10000, and coat 100 μL / well on a 96-well enzyme-labeled plate and place it at 4°C for 12-24 hours; wash the 96-well enzyme-labeled plate coated with the anti-kynurenine antibody 3 times with PBS, add 200 μL / well of 0.5% BSA solution, block it at 4°C and place it for 8-16 hours. Then wash it 3 times with PBS and add 20 μL / well of standard. Then add 100 μL / well of HRP-kynurenine conjugate at a working concentration; incubate at room temperature for 30 minutes and then wash the plate 5 times with PBS; then add 100 μL of TMB substrate to each well and incubate at room temperature for 30 minutes. Then add 100 μL of stop solution (2M sulfuric acid) to each well. Measure the absorbance at 450 nm. Calibrate according to the absorbance at 450 nm corresponding to each standard to make a standard curve. The results are as shown below. Figure 1 shown.

[0083] Example 8: Preparation of Kynurenine Alkaline Phosphatase Chemiluminescent Detection Reagent

[0084] The antibody prepared in Example 8 was used to prepare a chemiluminescent detection reagent for kynurenine alkaline phosphatase.

[0085] 1. A kynurenine detection kit. This experiment adopts the principle of competitive chemiluminescent immunoassay reagent. The analyte in the sample and the antigen labeled with a luminescent marker competitively bind to a certain number of antibody binding sites. The amount of antigen bound to the antibody is inversely proportional to the concentration of the analyte in the sample. When the reaction system reaches equilibrium, a substrate solution is added to detect the amount of antigen labeled with a luminescent marker bound to the antibody. A standard curve is made using a known standard substance, and the luminescence value of the unknown sample is calibrated on the standard curve to obtain the concentration of kynurenine in the unknown sample. It is characterized in that it includes kynurenine labeled with a chemiluminescent marker and a kynurenine monoclonal antibody coated on a fixed carrier.

[0086] 2. The chemiluminescent marker in the kynurenine labeled with the chemiluminescent marker is alkaline phosphatase.

[0087] 3. The antibody coated on the fixed carrier is an anti-kynurenine antibody coated on magnetic beads.

[0088] (1) Obtaining the standard curve: Set the reaction parameters of the alkaline phosphatase chemiluminescence analyzer (see Table 1). The on-machine detection process is as follows: add the sample (or standard), reagent 1 and reagent 2 at the same time, incubate at 37°C for 10 minutes; add the luminescent substrate solution, measure the luminescence value, and the machine automatically calculates the calibration curve and calculates the sample concentration. The calibration curve is as follows: Figure 2 shown.

[0089] Table 1: Alkaline phosphatase chemiluminescence analyzer reaction parameters

[0090]

[0091] (2) Sample comparison was performed using the kynurenine alkaline phosphatase chemiluminescence detection reagent of the present invention and the kynurenine ELISA detection reagent of a well-known foreign manufacturer. The detection data and data analysis are shown in Figure 3 .

[0092] Example 9: Analog cross-reaction test

[0093] Five common kynurenine analogs were selected for cross-reactivity testing and assayed using alkaline phosphatase chemiluminescence detection reagent. The five common kynurenine analogs and their cross-reactivity rates are shown in Table 2.

[0094] Table 2: Cross-reactivity results of common kynurenine structural analogs

[0095] Kynurenine Cross-reaction (%) Kynurenine 100％ 5-HT 0.01％ Tryptophan 0.02％ 5-Hydroxyindoleacetic acid 0.00％ Melatonin 0.00％

[0096] Determination results: The cross-reaction results of the above five common kynurenine structural analogs show that the antibody prepared by the present invention has good specificity.

[0097] From the above results, it can be seen that the complete kynurenine immunogen prepared from the kynurenine derivative provided by the present invention has strong immunogenicity, the antibodies produced are highly specific, and have good affinity for kynurenine. The alkaline phosphatase chemiluminescent reagent prepared using the above antibody has good stability and high sensitivity, and can achieve high-throughput and rapid detection of kynurenine on a fully automatic chemiluminescence analyzer. It has the advantages of simple operation, high sensitivity, strong specificity, and accurate results. It can also effectively reduce the cost of kynurenine detection, which is conducive to clinical promotion and use.

[0098] It should be noted that the above are only embodiments of the present invention and do not limit the patent scope of the present invention. Any equivalent structure or equivalent process transformation made using the contents of the present invention description and drawings, or directly or indirectly applied in other related technical fields, are also included in the patent protection scope of the present invention.

Claims

1. A kynurenine derivative, characterized in that: It has the structure shown in formula (I):

2. A kynurenine immunogen, characterized in that It has the structural formula shown in formula (II): The carrier is a protein or polypeptide with immunogenicity; the carrier is serum protein, keyhole limpet hemocyanin, thyroglobulin or polylysine.

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

4. A method for preparing a kynurenine immunogen, characterized in that: The preparation method comprises: preparing the kynurenine derivative according to claim 1; and connecting the kynurenine derivative to a carrier to obtain the kynurenine immunogen; wherein the carrier is an immunogenic protein or polypeptide; the synthetic route for preparing the kynurenine derivative is as follows: 100 mg of compound 1 was dissolved in 10 mL of dimethylformamide, and then 0.1 g of compound 2, 0.6 g of triethylamine, and 0.12 g of 2-(7-azobenzotriazole)-N,N,N',N'-tetramethyluronium hexafluorophosphate were added at 0°C to prepare a reaction mixture. The reaction mixture was stirred at room temperature overnight. After the reaction was completed, the solid material was filtered out, and the filtrate was concentrated. Finally, the concentrated residue was purified by flash column chromatography to obtain a kynurenine derivative.

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

6. The preparation method according to claim 5, characterized in that In step a, the step of preparing the carrier solution includes dissolving the carrier in a 0.05-0.2M phosphate buffer solution with a pH of 8.0-9.5 to obtain the carrier solution; and the step of preparing the kynurenine derivative solution includes placing the kynurenine derivative in N,N-dimethylformamide, methanol and a 0.05-0.2M phosphate buffer solution with a pH of 8.0-9.5 and stirring at room temperature to obtain the kynurenine derivative solution; wherein the mass ratio of the carrier to the kynurenine derivative is 1-8:

1.

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

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