Bilirubin derivative, immunogen, specific antibody thereof and bilirubin detection kit preparation method

Abstract

The invention relates to a preparation method of a bilirubin detection kit by using a chemiluminescence immunoassay technology. Bilirubin is an important basis for clinically judging jaundice and is also an important index of liver functions. As bilirubin is small in molecular weight and poor in immunogenicity, when a corresponding antibody is prepared, a proper derivative site needs to be selected firstly, and a derived hapten is coupled with a specific macromolecular carrier to prepare a complete antigen. According to the invention, the antibody with good specificity and strong affinity is successfully prepared by designing and transforming the hapten. The invention mainly relates to design and synthesis of a bilirubin hapten, preparation of a bilirubin complete antigen and an anti-bilirubin antibody, a method for determining the concentration of bilirubin, and composition and components of a reagent. The design and synthesis of the hapten mainly comprise design and chemical synthesis of bilirubin derivatives. The bilirubin derivative disclosed by the invention has a structure as shown in a formula (I) which is described in the specification.

Description

Technical Field

[0001] This invention relates to the field of biomedical immunoassay, specifically to the preparation of a bilirubin derivative, a bilirubin immunogen, its specific antibody, and a bilirubin detection kit. Background Technology

[0002] Bilirubin, its structural formula is shown in formula (Ⅲ): Formula (Ⅲ).

[0003] Bilirubin is a type of bile pigment, the main pigment in human bile, and is orange-yellow in color. It is a major metabolite of iron porphyrin compounds in the body, is toxic, and can cause irreversible damage to the brain and nervous system. However, it also has antioxidant functions, inhibiting the oxidation of linoleic acid and phospholipids. Bilirubin in the body is divided into direct bilirubin and indirect bilirubin. The main difference between indirect and direct bilirubin lies in their chemical structure, metabolic pathway, and clinical significance. Indirect bilirubin is lipid-soluble, does not bind with glucuronic acid, and cannot be directly excreted through the kidneys. Direct bilirubin, on the other hand, is water-soluble, forms after binding with glucuronic acid, and can be excreted through bile and urine. Elevated indirect bilirubin is usually associated with increased red blood cell destruction, such as in hemolytic anemia. Elevated direct bilirubin may indicate impaired excretory function of the liver or biliary system, such as in obstructive jaundice. Clinically, direct bilirubin and total bilirubin are mainly measured, with total bilirubin being the sum of direct and indirect bilirubin. Bilirubin is an important basis for clinically diagnosing jaundice and is also an important indicator of liver function.

[0004] Currently, the main methods for bilirubin detection are: high-performance liquid chromatography (HPLC), chemical oxidation (diazo reaction method), enzymatic methods, dry chemistry methods, and transdermal assays. Each method has its advantages and disadvantages, but all have limitations in large-scale clinical application: HPLC is time-consuming, costly, and difficult to implement in batches. Chemical oxidation methods have unstable reagents, poor specificity, and are easily affected by hemolysis and lipemia. Enzymatic methods are easily affected by antioxidants and have high reagent costs. Dry chemistry methods are easily affected by environmental factors, have poor accuracy, and are costly. Transdermal assays are affected by various factors. This invention starts with antigen design and antibody preparation, and adopts an advanced fully automated chemiluminescence detection platform, completely solving the pain points of clinical bilirubin testing and has broad application prospects. This test reagent can perform high-throughput, automated, and multi-type sample determination, and has outstanding advantages in terms of high sensitivity, specificity, and stability. Summary of the Invention

[0005] The purpose of this invention is to provide a bilirubin derivative, bilirubin immunogen and its specific antibody and bilirubin detection kit, so as to improve the shortcomings of poor specificity and low sensitivity of existing bilirubin measurement methods. This invention is of great significance for scientific research, precision medicine for newborns and specific clinical situations where the accuracy of detection is extremely important.

[0006] According to one aspect of the present invention, a bilirubin derivative is provided having the structural formula shown in formula (I): Equation (Ⅰ).

[0007] The bilirubin derivative of the present invention possesses the basic structure for preparing an immunogenic bilirubin immunogen, providing a structural basis for the preparation of new bilirubin detection reagents.

[0008] According to another aspect of the present invention, a bilirubin immunogen is also provided, having the structural formula shown in formula (II): Formula (II).

[0009] The carrier is an immunogenic protein or polypeptide. The bilirubin immunogen of this invention has high immunogenicity, can stimulate the animal body to produce an immune response, generate high-titer anti-bilirubin specific antibodies, and has strong antibody affinity, making it suitable for preparing highly sensitive and highly specific bilirubin competitive detection reagents.

[0010] For bilirubin derivatives, protein carriers are preferred as carriers for the aforementioned bilirubin immunogens; however, other types of immunogenic substances with sufficiently large molecular weights and a sufficient number of active groups can also be used as carriers. The most commonly used immunogenic carriers include serum proteins, keyhole hemocyanin (KLH), thyroglobulin, and polylysine. The carrier used in this invention is preferably keyhole hemocyanin.

[0011] According to another aspect of the present invention, an anti-bilirubin specific antibody is also provided, produced by immunizing animals with an immunogen. The antibody is produced by immunizing animals with any of the aforementioned bilirubin immunogens. The term "antibody" as used in this invention refers not only to complete protein molecules but also to polypeptide fragment antibodies or derivatives of polypeptide fragment antibodies that retain the specific binding ability of complete antibodies. The antibody of the present invention can be a polyclonal antibody, a monoclonal antibody, or a recombinant antibody, preferably a monoclonal antibody.

[0012] The antibodies of this invention can be prepared using existing technologies. 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. Host animals include rabbits, goats, mice, sheep, guinea pigs, horses, alpacas, or camels. Continuous immunization is performed 5-7 times until the antibody titer reaches its maximum. Blood is collected from the animal at regular intervals to obtain appropriate amounts of specific antiserum. Monoclonal antibodies can be prepared using hybridoma cell technology. Recombinant antibodies can be prepared using gene engineering expression technology.

[0013] According to another aspect of the present invention, a method for preparing a bilirubin immunogen is also provided. This method includes the steps of preparing the aforementioned bilirubin derivative and linking the bilirubin derivative to a carrier to obtain a bilirubin immunogen. By linking the bilirubin derivative prepared by the above method to an immunogenic protein or polypeptide, a highly immunogenic bilirubin immunogen of the present invention can be obtained, and the preparation method is simple to operate.

[0014] In the above-mentioned method for preparing bilirubin immunogen of the present invention, a method for preparing the above-mentioned bilirubin derivative is also provided.

[0015] In the preparation method of the above-mentioned bilirubin derivative of the present invention, the preparation steps of the above-mentioned bilirubin derivative are as follows: .

[0016] In the above-mentioned method for preparing bilirubin immunogen of the present invention, the linking step of the carrier and the bilirubin derivative can be reasonably modified according to different carriers in actual operation. In the present invention, the linking step includes: a) preparing a carrier solution and a bilirubin derivative solution; wherein, the mass ratio of the carrier to the bilirubin derivative is 1-8:1; preferably, the carrier is serum protein, keyhole hemocyanin, thyroglobulin, or polylysine; b) adding the activated bilirubin derivative solution dropwise to the carrier solution to obtain crude bilirubin immunogen; c) stirring the dropwise mixture overnight at 2-10°C or reacting at room temperature for 2 hours to obtain crude bilirubin immunogen; d) purifying the crude bilirubin immunogen to obtain bilirubin 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, has high process stability, and good reproducibility.

[0017] In the above-described method for preparing bilirubin immunogen of the present invention, the actual operation of the steps for preparing the carrier solution and the bilirubin derivative solution involves rationally selecting suitable solvent concentrations and pH values ​​according to the different types of carriers. In step a of the present invention, the step of preparing the carrier solution involves dissolving the carrier in a 0.05–0.20 M phosphate buffer solution with a pH of 7.0–8.5 to obtain the carrier solution; the step of preparing the bilirubin derivative solution involves placing the bilirubin derivative in an N,N-dimethylformamide and methanol solution and stirring at room temperature to obtain the bilirubin derivative solution. Using a 0.10–0.25 M phosphate buffer solution with a pH of 7.0–8.5 to dissolve the carrier allows the amino groups on the carrier and the maleimide groups of the activated bilirubin derivative to fully react and bind in a slightly alkaline environment.

[0018] In the above-described method for preparing bilirubin immunogen of the present invention, in the step of adding the above-described bilirubin immunogen to obtain crude bilirubin immunogen, in order to further increase the bilirubin immunogen content in the crude bilirubin immunogen, in steps b and c of the present invention, the dropwise addition step allows the bilirubin derivative to react more fully with the carrier; stirring overnight at 2-10°C further promotes the generation of bilirubin immunogen, thereby increasing the bilirubin immunogen content in the crude product.

[0019] In the above-described method for preparing bilirubin immunogen of the present invention, any operation capable of purifying bilirubin immunogen from crude bilirubin immunogen is applicable to the present invention. In step d above, the crude bilirubin immunogen is purified by dialysis to obtain bilirubin immunogen. Dialysis is a simple method and provides good purification results.

[0020] According to another aspect of the present invention, a bilirubin detection reagent is also provided, comprising an anti-bilirubin specific antibody, a bilirubin enzyme-labeled conjugate, and an enzyme substrate, wherein the anti-bilirubin specific antibody is any one of the aforementioned anti-bilirubin specific antibodies; the bilirubin enzyme-labeled conjugate contains the aforementioned bilirubin derivative. The bilirubin enzyme-labeled conjugate is formed by coupling an enzyme and a hapten, wherein the hapten is the aforementioned bilirubin derivative.

[0021] The bilirubin detection reagent of the present invention has a detection sensitivity far exceeding that of corresponding products in the prior art due to the high specificity and strong binding affinity of the anti-bilirubin specific antibody. Preferably, the enzyme-labeled conjugate is an alkaline phosphatase-hapten enzyme-labeled conjugate; the substrate of the enzyme is AMPPD or APS-5. The detection reagent using an alkaline phosphatase-hapten enzyme-labeled conjugate and AMPPD or APS-5 as the enzyme substrate can conveniently and accurately determine the bilirubin content in a sample, and is suitable for high-throughput automated detection.

[0022] According to another aspect of the present invention, a bilirubin detection kit is also provided, comprising the aforementioned anti-bilirubin-specific antibody and an indicator reagent for detecting the anti-bilirubin-specific antibody and bilirubin complexes. The indicator reagent is selected from enzyme reagents, radioisotope reagents, fluorescent reagents, and luminescent reagents. Preferably, the indicator reagent consists of a bilirubin enzyme-labeled conjugate and an enzyme substrate, wherein the bilirubin enzyme-labeled conjugate can be coupled with the bilirubin derivative of the present invention, enabling convenient and accurate determination of the bilirubin content in the sample, and is suitable for high-throughput automated detection.

[0023] The bilirubin immunogen, formed by linking a bilirubin derivative to a specific carrier via ester bonds, exhibits high immunogenicity. The antibodies induced in immunized animals show high specificity and strong binding affinity to bilirubin. High-throughput, rapid detection of bilirubin can be achieved using alkaline phosphatase chemiluminescence immunoassay technology on a fully automated chemiluminescence immunoassay analyzer. This method offers advantages such as ease of operation, high sensitivity, strong specificity, and accurate results, while also effectively reducing the cost of bilirubin detection, thus facilitating its widespread clinical application. Attached Figure Description

[0024] Figure 1 : Standard curve for bilirubin ELISA detection.

[0025] Figure 2 Chemiluminescence calibration curve of bilirubin alkaline phosphatase.

[0026] Figure 3 The results of sample comparison between the bilirubin chemiluminescence detection reagent of the present invention and the bilirubin enzymatic detection reagent of a well-known foreign manufacturer are as follows. Detailed Implementation

[0027] It should be noted that, unless otherwise specified, the embodiments and features described in the present invention can be combined with each other. The present invention will now be described in detail with reference to the accompanying drawings and embodiments.

[0028] Example 1: Synthesis and structural confirmation of bilirubin derivatives

[0029] The chemical structures of the bilirubin derivatives used in the following examples are shown in formula (Ⅳ): Formula (Ⅳ).

[0030] The specific synthetic route for the bilirubin derivative shown in formula (Ⅳ) is as follows:

[0031] The specific synthesis steps are as follows: .

[0032] Synthesis of bilirubin derivatives:

[0033] 1.5 g of compound 1 (bilirubin), 900 mg of compound 2, 500 mg of TEA, and 1600 mg of HATU were dissolved in 80 mL of DCM to prepare a reaction solution. This reaction solution was stirred at room temperature for 3 hours, then quenched with 150 mL of sodium carbonate aqueous solution. Extraction was performed with EA, and the resulting organic layer was washed with 70 mL of concentrated brine three times. The mixture was dried with anhydrous sodium sulfate, filtered, and the filtrate was removed by vacuum evaporation. The crude product was purified by preparative high-performance liquid chromatography (HPLC). Finally, 520 mg of a red solid bilirubin derivative was obtained, with a yield of 34.6%.

[0034] Example 2: Synthesis of BSA-bilirubin derivative immunogen

[0035] The BSA-bilirubin immunogen is composed of bovine serum albumin (BSA) linked by an ester bond to a bilirubin derivative shown in formula (I). In this embodiment, the synthesis method of this immunogen is described in detail, and the specific steps are as follows:

[0036] 1. Dissolve bovine serum albumin (20 mg) in 5 mL of 0.2 M phosphate buffer, pH 8.5;

[0037] 2. Add the following chemicals to a small beaker and stir to dissolve: 20 mg of the synthesized bilirubin derivative, 0.3 mL of D,D-dimethylformamide (DMF), and 0.3 mL of methanol. Stir these chemicals thoroughly at room temperature for 10 minutes. An ultrasonic device can be used to accelerate the dissolution.

[0038] 3. Add the dissolved solution dropwise to the BSA solution and stir overnight at 2-10℃ to obtain the crude antigen; purify the synthesized antigen by dialysis to obtain bilirubin immunogen.

[0039] Example 3: Synthesis of KLH-bilirubin derivative immunogen

[0040] The KLH-acetylbilirubin immunogen is composed of hemocyanin (KLH) linked by an ester bond to a bilirubin derivative represented by formula (I). In this embodiment, the synthesis method of this immunogen is described in detail, and the specific steps are as follows:

[0041] 1. Dissolve hemocyanin (20 mg) in 5 mL of 0.18 M phosphate buffer, pH 8.5;

[0042] 2. Add the following chemicals to a small beaker and stir to dissolve: 20 mg of the synthesized bilirubin derivative, 0.3 mL of D,D-dimethylformamide (DMF), and 0.3 mL of methanol. Stir these chemicals to dissolve at room temperature for 10 min.

[0043] 3. Add the dissolved solution dropwise to the KLH solution and stir overnight at 2-10℃ to obtain the crude bilirubin immunogen; purify the synthesized antigen by dialysis to obtain the bilirubin immunogen.

[0044] Example 4: Synthesis of thyroglobulin-bilirubin derivative immunogen

[0045] The thyroglobulin-bilirubin immunogen is formed by ester bonds between thyroglobulin and the bilirubin derivative shown in formula (I). In this embodiment, the synthesis method of this immunogen is described in detail, and the specific steps are as follows:

[0046] 1. Dissolve thyroglobulin (20 mg) in 5 mL of 0.20 M phosphate buffer, pH 9.0;

[0047] 2. Add the following chemicals to a small beaker and stir to dissolve: 10 mg of the synthesized bilirubin derivative, 0.3 mL of N,N-dimethylformamide (DMF), and 0.3 mL of methanol. Stir these chemicals thoroughly at room temperature for 10 min to dissolve.

[0048] 3. Add the dissolved solution dropwise to the thyroglobulin solution and stir overnight at 2-10℃ to obtain the complete antigen; purify the synthesized antigen by dialysis to obtain the bilirubin immunogen.

[0049] Example 5: Synthesis of poly-L-lysine-bilirubin derivative immunogen

[0050] The poly-L-lysine-bilirubin immunogen is formed by linking the amino group of poly-L-lysine to the maleimide group of the bilirubin derivative shown in formula (I). In this embodiment, the synthesis method of this immunogen is described in detail, and the specific steps are as follows:

[0051] 1. Dissolve 20 mg of poly-L-lysine in 5 mL of 0.20 M phosphate buffer solution at pH 9.0;

[0052] 2. Add the following chemicals to a small beaker and stir to dissolve: 10 mg of the synthesized bilirubin derivative, 0.3 mL of N,N-dimethylformamide (DMF), and 0.3 mL of methanol. Stir these chemicals to dissolve at room temperature for 10 min.

[0053] 3. Add the completely dissolved solution dropwise to the poly-L-lysine solution and stir overnight at 2-10℃ to obtain the complete antigen; centrifuge the synthesized antigen to collect the supernatant, and purify the supernatant by dialysis to obtain the bilirubin immunogen.

[0054] Similarly, the carrier is still an immunogenic protein, which can be a serum protein, keyhole hemocyanin (KLH), thyroglobulin, and polylysine. Preferably, the carrier is keyhole hemocyanin.

[0055] Example 6: Preparation of anti-bilirubin specific monoclonal antibody

[0056] This embodiment presents a method for preparing bilirubin monoclonal antibodies, which includes the following steps:

[0057] 1. Animal immunization

[0058] The artificial antigen prepared in Example 5 was used to immunize two approximately 8-week-old female Balb / C mice via subcutaneous injection at multiple points on the neck and back. For the initial immunization, an immunogen emulsified with Freund's complete adjuvant was used. Equal volumes of immunogen and Freund's complete adjuvant were mixed and emulsified, with a single mouse immunogen dose of 0.1 mg. After the initial immunization, booster immunizations were administered every 14 days at a dose of 0.1 mg, using the same emulsification method. A total of 5 immunizations were performed. Starting from the third booster immunization, 30 μL of blood was collected from the mouse tail 7 days after each immunization. The antiserum was collected by centrifugation and stored at -20°C for serum titer and specificity assays.

[0059] 2. ELISA indirect enzyme-linked immunosorbent assay (ELISA) was used to analyze the antiserum efficacy.

[0060] Using conventional antibody titer determination methods, with blank serum without antibodies as a control, the antiserum was diluted a certain number of times and then subjected to ELISA detection. The final detection yielded a titer of 1:30000-1:50000 for the anti-bilirubin specific antibody of this invention, indicating that the antibody prepared by this invention has high specificity and high sensitivity.

[0061] 3. Screening for cell fusion and positive hybridomas

[0062] (1) Resuscitating myeloma cells: Take myeloma cells out of liquid nitrogen and quickly place them in a 37°C water bath to thaw. After thawing, centrifuge at 1000 r / min for 5 minutes. Discard the supernatant in a clean bench and add about 1 mL of complete culture medium to the cell pellet. Disperse the cells, take them out with a pipette and mix them with the complete culture medium. Place them in a 10 cm diameter culture dish and expand to 4-6 dishes. Change the medium several times during this period. When the cells in each culture dish cover the bottom, they can be used for cell fusion.

[0063] (2) Cell preparation: Take out two small culture dishes. Pour some culture medium into one dish to cool the dissecting tools, and aspirate a small amount of culture medium into the other dish and place it in a cell sieve for grinding the spleen. Blow down the revived myeloma cells and transfer them to a 50mL centrifuge tube, seal the tube, and centrifuge at 1200 r / min for 5 minutes. Sacrifice the mice after five immunizations, soak them in 75% alcohol for about 1 minute, place them in a clean bench, collect blood from the heart, incubate at 37℃ for 30 minutes, centrifuge for 15 minutes, and collect the serum for storage at -20℃. After taking the mouse spleen cells, grind them thoroughly in a cell sieve, wash them with pre-allocated basal solution, transfer them to a 50mL centrifuge tube, seal the tube, discard the supernatant from the centrifuged myeloma cells, add basal solution to wash them again, and centrifuge them together with the spleen cells at 1200 r / min for 5 minutes. After the second centrifugation, discard the supernatant from the myeloma cells, add 2mL of basal solution, and mix well. Spleen cells were cleaned of supernatant, passed through a cell sieve, and added to myeloma cells. After mixing thoroughly by pipetting, basal culture medium was added to a final volume of 20 mL, and the mixture was centrifuged at 1200 r / min for 5 minutes. The centrifuged mixture was then removed, the supernatant was discarded, excess culture medium was aspirated, and the precipitated cells were dispersed by shaking. The mixture was then incubated at 37°C for 5 minutes.

[0064] (3) Cell fusion: After incubation, place the centrifuge tube in 37°C warm water and keep it rotating throughout the process. Use a pipette tip to draw 1 mL of PEG preheated to 37°C and slowly add the PEG to the precipitated cells within the first minute. Let it stand for 1 minute, preheat the basal medium, add 1 mL within the third minute, 3 mL within the fourth minute, and 16 mL within the fifth and sixth minutes, while gently stirring and adding along the wall to separate the PEG. Seal the centrifuge tube and centrifuge at 900 r / min for 8 minutes. Discard the supernatant and add the fused cells to HAT complete medium. Gently stir and evenly distribute the mixture into four 24-well culture plates. Ensure that the volume of HAT medium containing fused cells is the same in each well.

[0065] (4) Screening of positive hybridomas: Within 4 days after fusion, the medium was partially replaced with HT medium, and after 8 days, the medium was completely 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. Positive hybridoma cells with high titers 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 and covered 1 / 4 of the bottom of the well, the supernatant was taken and detected by ELISA. The titer and inhibition rate were used as indicators. The cells with strong positive results were selected and subcloned using the limiting dilution method. This process was repeated 3-4 times (note that the positive cells selected in each round need to be expanded and then frozen for later use) until each well of each plate was positive and the titer and inhibition rate were similar. At this point, the hybridoma cell line was successfully established and a hybridoma cell line that could stably secrete uniform antibodies was obtained. Single-cell clones were selected, and those that tested positive for all cells were transferred to 24-well cell culture plates, 6-well cell culture plates, and 10cm cell culture dishes for further culture and then frozen in time.

[0066] 4. Large-scale preparation of monoclonal antibodies

[0067] After obtaining hybridoma cell clones that secrete specific monoclonal antibodies, monoclonal antibodies are typically prepared in large quantities using in vitro culture and in vivo animal induction methods. Liquid calcite (0.5 mL / mouse) was injected intraperitoneally into more than ten Balb / c mice over 8 weeks old beforehand. Hybridoma cells were injected intraperitoneally into the mice 1-2 weeks later. The mice were observed daily after cell inoculation, especially from day 7 onwards, when the peritoneum would swell. Ascites fluid was aseptically collected using a disposable syringe before the mice died. The collected ascites fluid was centrifuged at 12000 rpm for 10 minutes to remove the upper fat and lower fibrin layers, collecting the middle layer. The titer and inhibition rate were determined using ELISA. After purification, the bilirubin monoclonal antibody was stored at -20°C for later use.

[0068] Example 7: Bilirubin ELISA Test

[0069] The titer and specificity of the antiserum were determined using an indirect enzyme-linked immunosorbent assay (ELISA). The steps are as follows:

[0070] 1. ELISA test for bilirubin using the antibody prepared in Example 6.

[0071] This test utilizes a competitive immunoassay to determine the bilirubin content in liquid samples. The principle is that bilirubin in the sample competes with a conjugated bilirubin derivative (HRP-bilirubin derivative enzyme conjugate) for binding to a limited number of sites on the antibody coated on the ELISA plate. If the liquid sample contains almost no or no bilirubin, the HRP-conjugated bilirubin derivative will bind to the antibody on the ELISA plate. Conversely, if the liquid sample contains a large amount or a certain quantity of bilirubin, the binding of the enzyme-bilirubin derivative conjugate to the antibody will decrease, thus weakening the colorimetric signal. Therefore, the absorbance produced by the test is inversely proportional to the bilirubin content in the liquid sample.

[0072] 2. The establishment of the standard curve for bilirubin ELISA detection is as follows:

[0073] (1) Preparation of standard products

[0074] Bilirubin powder (purchased from Merck) was dissolved in dimethyl sulfoxide (DMSO) solution to prepare a 1 mg / mL stock solution, which was stored protected from light. The stock solution was then sequentially diluted with ELISA buffer to prepare standard solutions of 300.00 µg / mL, 100.00 µg / mL, 33.30 µg / mL, 11.10 µg / mL, 3.70 µg / mL, 1.23 µg / mL, 0.41 µg / mL, and 0.00 µg / mL. The ELISA buffer contained 50.0 mM Tris, 100 mM NaCl, and 0.2% BSA.

[0075] (2) Prepare a standard curve using the ELISA test method for bilirubin.

[0076] The anti-bilirubin antibody prepared in Example 6 was diluted to a final concentration of 1:5000 with PBS, and 100 μL / well was coated onto a 96-well ELISA plate and incubated at 4°C for 12-24 h. The 96-well ELISA plate coated with the anti-bilirubin antibody was washed three times with PBS, and then 200 μL / well of 0.5% BSA solution was added. The plate was then blocked at 4°C for 8-16 h. After washing three times with PBS, 20 μL / well of standard was added. Then, 100 μL / well of working concentration HRP-bilirubin conjugate was added. After incubation at room temperature for 30 min, the plate was washed five times with PBS. Then, 100 μL of TMB substrate was added to each well and incubated at room temperature for 30 min. Finally, 100 μL of stop solution (2M sulfuric acid) was added to each well. The absorbance at 450 nm was measured. A standard curve was constructed based on the absorbance at 450 nm corresponding to each standard, and the results are shown in the attached figure. Figure 1 As shown.

[0077] Example 8: Preparation of chemiluminescent detection reagent for bilirubin alkaline phosphatase

[0078] The antibody obtained in Example 6 was used to prepare a chemiluminescent detection reagent for bilirubin alkaline phosphatase.

[0079] 1. A bilirubin detection kit, wherein sample preparation (using an accelerator to accelerate the dissolution of poorly soluble indirect bilirubin in the sample) is part of sample dilution. When determining total bilirubin, each sample is incubated with the accelerator separately to obtain fully dissolved indirect bilirubin. No accelerator is needed when detecting direct bilirubin. This experiment employs a competitive chemiluminescent immunoassay principle, where 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. After the reaction system reaches equilibrium, a substrate solution is added to detect the amount of antigen labeled with the luminescent marker bound to the antibody. A standard curve is constructed using known standards, and the luminescence value of the unknown sample is calibrated on the standard curve to obtain the concentration of bilirubin in the unknown sample. The kit is characterized by comprising an accelerator, chemiluminescently labeled bilirubin, and a bilirubin monoclonal antibody coated on a fixed carrier.

[0080] 2. The accelerator includes caffeine / sodium benzoate and surfactant.

[0081] 3. The accelerator contains caffeine reagent at a concentration of 0.1~5 mg / mL and sodium benzoate at a concentration of 10 mg / mL; the surfactant is Brij-35.

[0082] 4. The chemiluminescent marker in the bilirubin labeled with the chemiluminescent marker is alkaline phosphatase.

[0083] 5. The antibody coated on the immobilized carrier is an anti-bilirubin antibody coated on magnetic beads.

[0084] (1) Obtaining the standard curve: Set the reaction parameters for the alkaline phosphatase chemiluminescence analyzer (see Table 1). The instrument detection procedure is as follows: simultaneously add the sample (or standard), reagent 1 and reagent 2, incubate at 37℃ for 10 minutes; add the luminescent substrate solution, measure the luminescence value, the machine automatically calculates the fitted calibration curve, calculates the sample concentration, and the calibration curve is attached. Figure 2 As shown.

[0085] Table 1: Reaction parameters of the alkaline phosphatase chemiluminescence analyzer are as follows:

[0086] (2) Sample comparison was performed using the bilirubin alkaline phosphatase chemiluminescent detection reagent and enzymatic method of the present invention. The detection data and data analysis are shown in the appendix. Figure 3 .

[0087] Example 9: Cross-reactivity test of analogues

[0088] Nine common bilirubin structural analogs were selected for cross-reactivity testing, and the results were determined using an alkaline phosphatase chemiluminescence assay. The nine common bilirubin structural analogs and their cross-reactivity rates are shown in Table 2.

[0089] Table 2: Cross-reactivity results of 9 common bilirubin structural analogs are as follows:

[0090] Results: Based on the cross-reactivity results of the above nine common bilirubin structural analogs, the antibody prepared in this invention has good specificity.

[0091] The results above show that the bilirubin complete immunogen prepared from the bilirubin derivatives provided by this invention has strong immunogenicity, produces antibodies with high specificity and good affinity for bilirubin. The alkaline phosphatase chemiluminescence reagent prepared using the above antibodies has good stability and high sensitivity, enabling high-throughput and rapid detection of bilirubin on a fully automated chemiluminescence analyzer. It also has the advantages of simple operation, high sensitivity, strong specificity, and accurate results, and can effectively reduce the cost of bilirubin detection, which is conducive to its clinical application.

[0092] It should be noted that the above are merely embodiments of the present invention and do not limit the patent scope of the present invention. Any equivalent structural or procedural transformations made using the content of the present invention's specification and drawings, or direct or indirect applications in other related technical fields, are similarly included within the patent protection scope of the present invention.

Claims

1. A bilirubin derivative, characterized in that, It has the structure shown in equation (Ⅰ): Equation (Ⅰ).

2. A bilirubin immunogen, characterized in that, It has the structural formula shown in equation (Ⅱ): Equation (II); The carrier is an immunogenic protein or polypeptide; the carrier is a serum protein, keyhole hemocyanin, thyroglobulin, or polylysine.

3. An anti-bilirubin specific antibody, produced by immunizing animals with an immunogen, characterized in that, The anti-bilirubin specific antibody is obtained by immunizing animals with the bilirubin immunogen described in claim 2.

4. A method for preparing a bilirubin immunogen, characterized in that, The preparation method includes: preparing the bilirubin derivative according to claim 1; and linking the bilirubin derivative to a carrier to obtain the bilirubin immunogen; wherein the carrier is an immunogenic protein or polypeptide; the synthetic route for preparing the bilirubin derivative is as follows: 。 5. The preparation method according to claim 4, characterized in that, The coupling step between the carrier and the bilirubin derivative includes: a. Prepare a carrier solution and a bilirubin derivative solution; the carrier is serum protein, keyhole hemocyanin, thyroglobulin, or polylysine; b. Add the bilirubin derivative solution to the carrier solution to obtain a mixed solution of bilirubin derivative and carrier; c. Stir the mixed solution overnight at 2-10°C or react at room temperature for 2 hours to obtain the crude bilirubin immunogen product; d. The crude bilirubin immunogen is purified to obtain the pure bilirubin immunogen.

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 0.05–0.2 M phosphate buffer solution with a pH of 7.0–8.5 to obtain the carrier solution; The step of preparing the bilirubin derivative solution includes placing the bilirubin derivative in an N,N-dimethylformamide and methanol solution and stirring at room temperature to obtain a solution of the bilirubin derivative; wherein the mass ratio of the carrier to the bilirubin derivative is 1 to 8:

1.

7. A bilirubin detection kit, characterized in that, The kit contains the bilirubin derivative of claim 1 and / or the antibody of any one of claims 3.

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