Galactose derivatives, immunogens and antibodies specific thereto and methods of making galactose detection kits

Abstract

The application relates to a preparation of a galactose detection kit by using a chemiluminescence immunoassay technique. Since the galactose has small molecular weight, poor immunogenicity, strong immunological tolerance, and has a D / L chiral structure, exists in a six-membered ring form in a solution, and can be converted into alpha and beta anomers. Therefore, when preparing a corresponding antibody, firstly, a suitable anomer and a derivative site need to be selected, and a derived hapten and a specific macromolecular carrier are coupled to prepare a complete antigen. The application successfully prepares an antibody with good specificity and strong affinity by designing and reforming the hapten. The application mainly relates to the design and synthesis of a galactose hapten, the preparation of a galactose complete antigen and an anti-galactose antibody, and a method for determining the concentration of galactose, the composition and ingredients of a reagent. The galactose derivative has the structure shown in formula (I).

Description

Technical Field

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

[0002] Galactose, with the structural formula shown in formula (Ⅲ): Formula (Ⅲ).

[0003] Galactose is a monosaccharide and a component of important biomolecules such as lactose, cerebrosides, and gangliosides. In the human body, galactose is mainly produced by the hydrolysis of lactose and converted into glucose in the liver for energy. Accurate measurement of galactose is crucial for the diagnosis and treatment of galactosemia (hypergalactosemia). Galactosemia (-mia) is of paramount importance. Galactosemia is an autosomal recessive genetic disorder in which patients lack the enzyme to metabolize galactose, leading to its accumulation in the body and subsequent serious complications such as liver damage, cataracts, and intellectual disability. Furthermore, monitoring serum or urinary galactose levels is clinically valuable for assessing liver function and certain metabolic syndromes. Urinary galactose testing, in particular, can detect the body's lactose metabolism level, thereby determining lactose tolerance.

[0004] Currently, methods for determining galactose in biological samples mainly involve in vitro detection of lactose intolerance, including clinical diagnostic methods, the "breath hydrogen test" instrumental method, and urinary galactose detection methods. Among these, the clinical diagnostic method is simple, but results relying solely on clinical manifestations are unreliable. The "breath hydrogen test" instrumental method requires specialized equipment and places high demands on the test subjects, especially those with a smoking history, leading to inaccurate results. Enzymatic methods have relatively poor specificity; the specificity of lactose oxidase is not ideal, and the presence of significant amounts of reducing substances (such as vitamin C) or enzyme inhibitors in human urine can interfere, causing color changes in the test strip even when galactose-free urine samples are added, resulting in false negatives. Therefore, in actual testing, sample pretreatment is required to remove interfering substances, which not only adds inconvenience but also increases testing costs, limiting its application to qualitative or semi-quantitative detection. This invention, starting with antigen design and antibody preparation, employs an advanced fully automated chemiluminescence detection platform, completely solving the pain points of clinical galactose testing and possessing broad application prospects. This test reagent can perform high-throughput, automated, and multi-type sample testing, 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 galactose derivative, a galactose immunogen and its specific antibody and a galactose detection kit, so as to improve the problems of poor specificity and narrow application scope of existing galactose determination methods.

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

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

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

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

[0010] Galactose derivatives are preferred carriers for the aforementioned galactose immunogens, with protein carriers being the preferred option. 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-galactose-specific antibody is also provided, produced by immunizing animals with an immunogen, wherein the antibody is produced by immunizing animals with any of the aforementioned galactose immunogens. The term "antibody" as used in this invention refers not only to complete protein molecules but also to polypeptide fragment antibodies or polypeptide fragment antibody derivatives 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 galactose immunogen is also provided. This method includes the steps of preparing the aforementioned galactose derivative and linking the aforementioned galactose derivative to a carrier to obtain a galactose immunogen. The galactose derivative prepared by the above method, when linked to an immunogenic protein or polypeptide, yields the highly immunogenic galactose immunogen of the present invention, and the preparation method is simple to operate.

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

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

[0016] In the above-described method for preparing galactose immunogen of the present invention, the linking step between the carrier and the galactose 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 galactose derivative solution; wherein the mass ratio of the carrier to the galactose derivative is 1-8:1; preferably, the carrier is serum protein, keyhole hemocyanin, thyroglobulin, or polylysine; b) adding the activated galactose derivative solution dropwise to the carrier solution to obtain crude galactose immunogen; c) stirring the dropwise mixture overnight at 2-10°C or reacting at room temperature for 2 hours to obtain crude galactose immunogen; d) purifying the crude galactose immunogen to obtain galactose 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 the galactose immunogen of the present invention, the actual operation of the steps for preparing the carrier solution and the galactose 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 8.0–9.5 to obtain the carrier solution; the step of preparing the galactose derivative solution involves placing the galactose derivative in N,N-dimethylformamide, methanol, and a 0.05–0.20 M potassium phosphate buffer solution with a pH of 8.0–9.5 and stirring at room temperature to obtain the galactose derivative solution. Using a 0.10–0.25 M phosphate buffer solution with a pH of 8.0–9.5 to dissolve the carrier allows the amino groups on the carrier and the ester groups of the activated galactose derivative to fully react and bind in a slightly alkaline environment.

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

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

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

[0021] The galactose detection reagent of the present invention exhibits significantly higher detection sensitivity than corresponding products in the prior art due to the high specificity and strong binding affinity of the anti-galactose 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 galactose content in a sample, making it suitable for high-throughput automated detection.

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

[0023] The galactose immunogen, formed by linking a galactose 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 galactose. High-throughput, rapid detection of galactose 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 galactose detection, thus facilitating its widespread clinical application. Attached Figure Description

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

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

[0026] Figure 3 The results of a sample comparison between the galactose chemiluminescence detection reagent of the present invention and the galactase detection reagent of a well-known foreign manufacturer. 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 galactose derivatives

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

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

[0031] The specific synthesis steps are as follows:

[0032] Synthesis of compound 2: .

[0033] 1. Weigh 3.2 g (19.5 mmol) of compound 1 (β-D-galactopyranose), dissolve it in 100 mL of N,N-dimethylformamide (DMF), add 15 g (375 mmol) of sodium hydride (NaH, purity 55% w / t, soluble in mineral oil) under nitrogen protection, cool the mixture to 0 °C, add 25 mL (212.5 mmol) of benzyl bromide (BnBr) dropwise, stir at room temperature for 20 hours, slowly add 120 mL of methanol (MeOH), then add 140 mL of purified water, and adjust the pH of the solution to neutral with hydrochloric acid (6 M);

[0034] 2. The above solution was extracted with dichloromethane (CH2Cl2), the organic phase was washed with deionized water, anhydrous magnesium chloride (MgSO4) was added for drying, filtered and concentrated, and the product was purified by silica gel column chromatography (mobile phase: PE / EtOAc=10:1) to finally obtain 8.7 g of green oily compound 2, with a yield of 84%.

[0035] Synthesis of compound 3: .

[0036] 1. Weigh 8.7 g (16 mmol) of compound 2 and dissolve it in 200 mL of dry toluene. Under nitrogen protection, add 160 mL of diisobutylaluminum hydride (DIBAL, 1 M dissolved in toluene) dropwise. Increase the temperature of this mixture to 55 °C and stir for 2 hours. Place the solution at 0 °C, add 200 mL of HCl (1 N), and stir the mixture rapidly and evenly for 35 minutes.

[0037] 2. The above mixture was diluted with ethyl acetate (EtOAc), and the aqueous phase was extracted with ethyl acetate (EtOAc). The bound organic phase was washed with brine, and anhydrous magnesium chloride (MgSO4) was added for drying. After two filtrations and concentrations, the product was mixed with methyl tert-butyl ether / hexane (MTBE / hexane) and stirred to finally obtain 4.7 g of white amorphous solid compound 3, with a yield of 80%.

[0038] Synthesis of compound 4: .

[0039] 1. Weigh 4.7 g (10.6 mmol) of compound 3 and dissolve it in 22 mL of N,N-dimethylformamide (DMF). Under nitrogen protection, add 2.0 g (49.2 mmol) of sodium hydride (NaH, purity 55% w / t, dissolved in mineral oil). Stir the mixture at room temperature for 1.5 hours. Add 7.8 g (37.3 mmol) of ethyl 1-bromopentanoate dropwise at 0 °C. Then stir the mixture at 25 °C for 2 hours.

[0040] 2. The above mixture was diluted with deionized water and then extracted with ethyl acetate (EtOAc). The organic phase was washed with brine, dried with anhydrous magnesium sulfate (MgSO4), filtered, concentrated, and the product was purified by silica gel column chromatography (mobile phase: PE / EtOAc=10:1). Finally, 4.2 g of colorless oily compound 4 was obtained, with a yield of 88%.

[0041] Synthesis of compound 5: .

[0042] 1. Weigh 4.2g (7.5mmol) of compound 4 and add it to 60mL of 2N alkaline solution (i.e., sodium hydroxide) and then add 6mL of ethanol. Mix well and reflux the mixture for 2 hours.

[0043] 2. After cooling the above reaction mixture to room temperature, acidify it with 1N dilute hydrochloric acid, extract it with ethyl acetate (EtOAc), rinse the organic layer with brine, dry it with anhydrous magnesium sulfate (MgSO4), filter and concentrate it to finally obtain 2.2g of compound 5, with a yield of 89%.

[0044] Synthesis of compound 6: .

[0045] 1. Weigh 2.2 g (4.2 mmol) of compound 5 and 1.1 g of palladium / carbon (Pd / C, 10%) and dissolve them in 60 mL of methanol to prepare a mixture. Stir the mixture at 55 °C overnight.

[0046] 2. The mixture was filtered and the filtrate was concentrated to obtain 0.9 g of compound 6, with a yield of 79.2% and a purity of >99%.

[0047] Synthesis of galactose derivatives:

[0048] The specific synthesis steps are as follows: .

[0049] 900 mg of compound 1 was dissolved in 90 mL of dimethylformamide. Then, 0.9 g of compound 2, 5.4 g of triethylamine, and 1.1 g of 2-(7-azobenzotriazole)-N,N,N',N'-tetramethylurea hexafluorophosphate were added at 0 °C to prepare a reaction mixture. This reaction mixture was stirred overnight at room temperature. After the reaction was completed, the solid matter was removed by filtration, and the filtrate was concentrated. Finally, the residue obtained by concentration was purified by rapid column chromatography to obtain 660 mg of galactose derivative, with a yield of 53.1%.

[0050] Example 2: Synthesis of BSA-galactose derivative immunogen

[0051] The BSA-galactose immunogen is formed by linking bovine serum albumin (BSA) with a galactose derivative shown in formula (I) via an ester bond. In this embodiment, the synthesis method of this immunogen is described in detail, and the specific steps are as follows:

[0052] 1. Dissolve bovine serum albumin (20 mg) in 5 mL of 0.2 M phosphate buffer, pH 8.5;

[0053] 2. Add the following chemicals to a small beaker and stir to dissolve: 20 mg of the synthesized galactose 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 react these chemicals at room temperature for 10 min.

[0054] 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 the galactose immunogen.

[0055] Example 3: Synthesis of KLH-galactose derivative immunogen

[0056] The KLH-galactose immunogen is formed by an ester bond between hemocyanin (KLH) and a galactose derivative as shown in formula (I). In this embodiment, the synthesis method of this immunogen is described in detail, and the specific steps are as follows:

[0057] 1. Dissolve hemocyanin (20 mg) in 5 mL of 0.18 M phosphate buffer, pH 8.5;

[0058] 2. Add the following chemicals to a small beaker and stir to dissolve: 20 mg of the synthesized galactose 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 min.

[0059] 3. Add the dissolved solution dropwise to the KLH solution and stir overnight at 2-10℃ to obtain the crude galactose immunogen; purify the synthesized antigen by dialysis to obtain the galactose immunogen.

[0060] Example 4: Synthesis of thyroglobulin-galactose derivative immunogen

[0061] The thyroglobulin-galactose immunogen is formed by ester bonds between thyroglobulin and the galactose 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:

[0062] 1. Dissolve thyroglobulin (20 mg) in 5 mL of 0.20 M, pH 9.0 phosphate buffer;

[0063] 2. Add the following chemicals to a small beaker and stir to dissolve: 10 mg of the synthesized galactose 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 min.

[0064] 3. Add the activated 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 galactose immunogen.

[0065] Example 5: Synthesis of poly-L-lysine-galactose derivative immunogen

[0066] The poly-L-lysine-galactose immunogen is formed by linking poly-L-lysine with an ester bond group of the galactose derivative shown in formula (I). In this embodiment, the synthesis method of the immunogen is described in detail, and the specific steps are as follows:

[0067] 1. Dissolve 20 mg of poly-L-lysine in 5 mL of 0.20 M, pH 9.0 phosphate buffer;

[0068] 2. Add the following chemicals to a small beaker and stir to dissolve: 10 mg of the synthesized galactose 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 min.

[0069] 3. Add the activated 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 galactose immunogen.

[0070] 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.

[0071] Example 6: Preparation of anti-galactose-specific monoclonal antibody

[0072] This embodiment proposes a method for preparing galactose monoclonal antibodies, which includes the following steps:

[0073] 1. Animal immunization

[0074] 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.

[0075] 2. ELISA indirect enzyme-linked immunosorbent assay (ELISA) was used to analyze the antiserum efficacy.

[0076] 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-galactose specific antibody of this invention, indicating that the antibody prepared by this invention has high specificity and high sensitivity.

[0077] 3. Screening for cell fusion and positive hybridomas

[0078] (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.

[0079] (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.

[0080] (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.

[0081] (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.

[0082] 4. Large-scale preparation of monoclonal antibodies

[0083] 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. Its titer and inhibition rate were determined using ELISA. After purification, it was stored at -20℃ for later use, yielding the galactose monoclonal antibody.

[0084] Example 7: Galactose ELISA Test

[0085] The titer and specificity of the antiserum were determined using an indirect enzyme-linked immunosorbent assay (ELISA). The steps are as follows:

[0086] 1. The antibody prepared in Example 6 was used for ELISA testing of galactose.

[0087] This test utilizes a competitive immunoassay to determine the galactose content in a liquid sample. The principle is that galactose in the sample competes with a conjugated galactose derivative (HRP-galactose 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 galactose, the HRP-conjugated galactose derivative will bind to the antibody on the ELISA plate. Conversely, if the liquid sample contains a large amount or a certain quantity of galactose, the enzyme-galactose derivative conjugate will bind less to the antibody, thus weakening the colorimetric signal. Therefore, the absorbance produced by the test is inversely proportional to the galactose content in the liquid sample.

[0088] 2. The establishment of the standard curve for galactose ELISA detection is as follows:

[0089] (1) Preparation of standard products

[0090] β-D-galactopyranose powder (purchased from Aladdin) was dissolved in deionized water to prepare a stock solution of 50 mg / mL. The stock solution was then sequentially diluted with ELISA buffer to prepare standard solutions of 8.00 mg / mL, 4.00 mg / mL, 2.00 mg / mL, 1.00 mg / mL, 0.50 mg / mL, 0.25 mg / mL, 0.125 mg / mL, and 0.00 ng / mL. The ELISA buffer contained 50.0 mM Tris, 100 mM NaCl, and 0.2% BSA.

[0091] (2) Prepare a standard curve using the ELISA test for galactose.

[0092] The anti-galactose antibody prepared in Example 6 was diluted to a final concentration of 1:10000 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-galactose 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 of HRP-galactose 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.

[0093] Example 8: Preparation of chemiluminescent detection reagent for galactose alkaline phosphatase

[0094] The antibody obtained in Example 6 was used to prepare a chemiluminescent detection reagent for galactose alkaline phosphatase.

[0095] 1. A galactose detection kit, wherein sample preparation (converting α-D-galactopyranose in the sample to β-D-galactopyranose) is part of sample dilution, and β-D-galactopyranose is obtained by incubating each sample with galactotropic enzyme reagent. This experiment uses the principle of competitive chemiluminescent immunoassay, 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 plotted using known standards, and the concentration of galactose in the unknown sample is obtained by calibrating the luminescence value of the unknown sample on the standard curve. The kit is characterized by comprising a sample processing solution, chemiluminescently labeled galactose, and a galactose monoclonal antibody coated on a fixed carrier. The sample processing solution includes mutagenin and a buffer solution; the sample processing solution contains galactose mutagenin at a concentration of 0.1~10 U / mL, preferably 8 U / mL; the buffer solution is 50 mM Tris buffer, pH 7.2. The chemiluminescent label in the galactose is alkaline phosphatase. The antibody coated on the immobilization carrier is an anti-galactose antibody coated on magnetic beads.

[0096] 2. Obtaining the standard curve: Set the reaction parameters for the alkaline phosphatase chemiluminescence analyzer (see Table 1). The instrumentation procedure is as follows: simultaneously add the sample (or standard), reagent 1, and reagent 2, and incubate at 37°C for 10 minutes; add the luminescent substrate solution, measure the luminescence value, and the machine automatically calculates the fitted calibration curve and calculates the sample concentration. The calibration curve is attached. Figure 2 As shown.

[0097] Table 1: Reaction parameters of alkaline phosphatase chemiluminescence analyzer

[0098] 3. Samples were compared using the chemiluminescent detection reagent for galactose alkaline phosphatase and the enzymatic galactose detection reagent of the present invention. The detection data and data analysis are shown in the appendix. Figure 3 .

[0099] Example 9: Cross-reactivity test of analogues

[0100] Nine common galactose structural analogs were selected for cross-reactivity testing, and the results were determined using an alkaline phosphatase chemiluminescence assay. The nine common galactose structural analogs and their cross-reactivity rates are shown in Table 2.

[0101] Table 2: Cross-reactivity results of 9 common galactose structural analogues

[0102] Results: Based on the cross-reactivity results of the above nine common galactose structural analogs, the antibody prepared in this invention has good specificity.

[0103] The results above show that the complete galactose immunogen prepared from the galactose derivative provided by this invention has strong immunogenicity, produces antibodies with high specificity and good affinity for galactose. The alkaline phosphatase chemiluminescence reagent prepared using the above antibody has good stability and high sensitivity, enabling high-throughput and rapid detection of galactose 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 galactose detection, which is conducive to its clinical application.

[0104] 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 galactose derivative, characterized in that, It has the structure shown in equation (Ⅰ): Equation (I); The main structure of the galactose derivative is β-D-galactopyranose configuration.

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

4. A method for preparing a galactose immunogen, characterized in that, The preparation method includes: preparing the galactose derivative according to claim 1; and linking the galactose derivative to a carrier to obtain the galactose immunogen; wherein the carrier is an immunogenic protein or polypeptide; the synthetic route for preparing the galactose derivative is as follows: 。 5. The preparation method according to claim 4, characterized in that, The coupling step between the carrier and the galactose derivative includes: a. Prepare a carrier solution and a galactose derivative solution; the carrier is serum protein, keyhole hemocyanin, thyroglobulin, or polylysine; b. Add the galactose derivative solution to the carrier solution to obtain a mixed solution of galactose 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 galactose immunogen product; d. The crude galactose immunogen is purified to obtain the pure galactose 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 8.0–9.5 to obtain the carrier solution; and the step of preparing the galactose derivative solution includes placing the galactose derivative in N,N-dimethylformamide, methanol, and a 0.05–0.2 M phosphate buffer solution with a pH of 8.0–9.5 and stirring at room temperature to obtain the galactose derivative solution; wherein the mass ratio of the carrier to the galactose derivative is 1–8:

1.

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

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