Levodopa derivatives, immunogens and antibodies specific thereto and methods of preparing levodopa test kits

Abstract

This invention relates to a method for preparing a levodopa detection kit using chemiluminescent immunoassay. Due to the small molecular weight and poor immunogenicity of levodopa, and its presence of catechol in its structure, it is easily oxidized in neutral or alkaline environments. Therefore, when preparing the corresponding antibody, it is necessary to first select a suitable derivatization site and conjugate the derived hapten to a specific macromolecular carrier to prepare the complete antigen. This invention successfully prepared antibodies with good specificity and strong affinity through the design and modification of the hapten. This invention mainly relates to the design and synthesis of levodopa haptens, the preparation of complete levodopa antigens and anti-levodopa antibodies, and the methods, reagent composition, and components for determining levodopa concentration. The hapten design and synthesis of this invention mainly includes the design and chemical synthesis of levodopa derivatives. The levodopa derivative of this invention has the structure shown in formula (I): Formula (I).

Description

Technical Field

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

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

[0003] Levodopa is the core and most effective drug for treating Parkinson's disease, considered the "gold standard" for controlling motor symptoms. As a prodrug, levodopa itself is inactive. Its function is to enter the brain and be converted into the deficient neurotransmitter dopamine, directly supplementing the insufficient dopamine in the brains of Parkinson's patients and improving symptoms such as tremor, rigidity, and bradykinesia. Monitoring levodopa levels in the body is crucial: for example, monitoring adverse drug reactions, as long-term use of levodopa may lead to elevated homocysteine ​​levels, increasing the risk of peripheral neuropathy (such as polyneuropathy). By monitoring blood drug concentrations and combining them with other indicators, dosage can be optimized to prevent such complications. It also helps adjust medication regimens, especially when using levodopa / carbidopa enteric gel for continuous infusion therapy. Monitoring blood drug concentrations is essential to ensure stable and effective drug levels and can guide combination therapy (such as COMT inhibitors). Because levodopa has a narrow therapeutic window and its plasma concentration is easily affected by factors such as metabolic level, diet and drug interactions, accurate monitoring of drug concentration in biological fluids such as blood or plasma is of great clinical significance for optimizing dosing regimens and avoiding adverse reactions such as motor impairment and "on-off phenomenon".

[0004] Currently, the main methods for levodopa detection are: Liquid Chromatography-Tandem Mass Spectrometry (LC-MS / MS): the gold standard, with extremely high sensitivity and specificity; High Performance Liquid Chromatography-Electrochemical / Fluorescence (HPLC-Electrochemical / Fluorescence): HPLC coupled with a high-sensitivity detector, a classic and mainstream low-cost method; and HPLC-UV: the most widely used routine analytical method, mainly used for routine content determination. Other methods include Enzyme-Linked Immunosorbent Assay (ELISA) and Capillary Electrophoresis (CE). Each of these methods has its advantages and disadvantages, but all have limitations in large-scale clinical application. ELISA is cumbersome, time-consuming, labor-intensive, requires manual operation, and has poor repeatability, hindering its widespread adoption in clinical testing. HPLC is time-consuming, costly, and difficult to implement in batches. This invention addresses the pain points of levodopa clinical testing by starting with antigen design and antibody preparation, and employing an advanced fully automated chemiluminescence detection platform, thus offering broad application prospects. This reagent can perform high-throughput, automated, and multi-type sample determination, and boasts outstanding advantages in terms of high sensitivity, specificity, and stability. Summary of the Invention

[0005] The purpose of this invention is to provide a levodopa derivative, a levodopa immunogen and its specific antibody and a levodopa detection kit, so as to improve the shortcomings of the existing levodopa determination methods, which are cumbersome, inefficient, costly and unable to be automated.

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

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

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

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

[0010] Levodopa derivatives are preferred carriers for the aforementioned levodopa immunogen, 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, a specific antibody against levodopa is also provided, produced by immunizing animals with an immunogen, wherein the antibody is produced by immunizing animals with any of the aforementioned levodopa 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 levodopa immunogen is also provided. This method includes the steps of preparing the aforementioned levodopa derivative and linking the levodopa derivative to a carrier to obtain a levodopa immunogen. The levodopa derivative prepared by the above method, when linked to an immunogenic protein or polypeptide, yields the highly immunogenic levodopa immunogen of the present invention, and the preparation method is simple to operate.

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

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

[0016] In the above-described method for preparing levodopa immunogen of the present invention, the linking step between the carrier and the levodopa 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 levodopa derivative solution; wherein the mass ratio of the carrier to the levodopa derivative is 1-8:1; preferably, the carrier is serum protein, keyhole hemocyanin, thyroglobulin, or polylysine; b) adding the activated levodopa derivative solution dropwise to the carrier solution to obtain crude levodopa immunogen; c) stirring the dropwise mixture overnight at 2-10°C or reacting at room temperature for 2 hours to obtain crude levodopa immunogen; d) purifying the crude levodopa immunogen to obtain levodopa 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 levodopa immunogen of the present invention, the actual operation of the steps for preparing the carrier solution and the levodopa 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 levodopa derivative solution involves placing the levodopa derivative in N,N-dimethylformamide, methanol, and a 0.5–0.20 M potassium phosphate buffer solution with a pH of 8.0–9.5 and stirring at room temperature to obtain the levodopa 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 levodopa derivative to fully react and bind in a slightly alkaline environment.

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

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

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

[0021] The levodopa 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-levodopa 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 levodopa content in a sample, making it suitable for high-throughput automated detection.

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

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

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

[0025] Figure 2 Chemiluminescence calibration curve of L-DOPA alkaline phosphatase.

[0026] Figure 3 The results of comparing the levodopa chemiluminescence detection reagent of the present invention with those of high performance liquid chromatography (HPLC) are shown. 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 levodopa derivatives

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

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

[0031] The specific synthesis steps are as follows:

[0032] Synthesis of compound 2: .

[0033] 1. Weigh 29.55 g (150.0 mmol) of compound 1, 25.63 g (147.9 mmol) of benzyl bromide, and 30.62 g (221.9 mmol) of K₂CO₃, and dissolve them together in 250 mL of acetone. Stir overnight at room temperature. Thin-layer chromatography (TLC) showed that the reactants had reacted completely. Filter the resulting solution after reaction, concentrate the filtrate under vacuum, and dilute the residue with 350 mL of EA. Wash the organic layer with 250 mL of purified water and 250 mL of brine, dry with Na₂SO₄, and then concentrate under vacuum to obtain the crude product. Finally, purify the crude product by silica gel column chromatography to obtain 17.2 g of white solid compound 2, with a yield of 31.2%.

[0034] 2. The above white solid compound was subjected to nuclear magnetic resonance spectroscopy using a Bruker Avance III plus 400MHz VARIAN MERCURY plus 300M spectrometer, with TMS as an internal standard. The results identified the compound as the target product, compound 2.

[0035] Synthesis of compound 3: .

[0036] 1. Weigh 17.52 g (56.2 mmol) of compound 2, 43.39 g (223.68 mmol) of ethyl 4-bromobutyrate, and 20.58 g (149.12 mmol) of K₂CO₃, and dissolve them together in 200 mL of acetonitrile. Stir overnight at 80 °C. TLC analysis showed that the reaction was complete. Filter the resulting solution, concentrate the filtrate under vacuum to obtain the crude product. Finally, purify the crude product by silica gel column chromatography to obtain 15.7 g of yellow oily compound 3, with a yield of 44.8%.

[0037] 2. The above-mentioned brown oily compound was subjected to nuclear magnetic resonance spectroscopy using a Bruker Avance III plus 400MHz VARIAN MERCURY plus 300M spectrometer, with TMS as an internal standard. The result was characterized as compound 3 shown above.

[0038] Synthesis of compound 4: .

[0039] 1. Weigh 15.7 g (40.4 mmol) of compound 3 and 2 g of Pd / C catalyst, dissolve them together in 150 mL of ethanol, and then stir at room temperature for 2 hours. Filter the resulting synthesis solution, concentrate the filtrate under vacuum to obtain the crude product, and finally purify the crude product by silica gel column chromatography to obtain 5.9 g of white solid compound 4, with a yield of 33.3%.

[0040] 2. The above-mentioned white solid compound was subjected to nuclear magnetic resonance spectroscopy using a Bruker Avance III plus 400MHz VARIAN MERCURY plus 300M spectrometer, with TMS as an internal standard. The result was characterized as compound 4 shown above.

[0041] Synthesis of L-DOPA derivatives: .

[0042] 1. Weigh 5.9 g (168.9 mmol) of compound 8 and 3.5 g (83 mmol) of LiOH, dissolve them together in 80 mL of methanol, and then stir at 50 °C for 4 hours. Dilute this reaction mixture with 150 mL of purified water at 0 °C, and then adjust the pH to 3.0 with 2 mol hydrochloric acid. Extract the mixture after reaction with 3 × 100 mL of DCM. Concentrate the bound organic layer under vacuum to obtain the crude product. Finally, purify the crude product by silica gel column chromatography to obtain 5.3 g of brown solid L-DOPA derivative, with a yield of 56.4%.

[0043] 2. The above-mentioned white solid compound was subjected to nuclear magnetic resonance spectroscopy using a Bruker Avance III plus 400MHz VARIAN MERCURY plus 300M spectrometer, with TMS as an internal standard. The results characterized it as the levodopa derivative shown above.

[0044] Example 2: Synthesis of BSA-L-DOPA derivative immunogen

[0045] The BSA-L-DOPA immunogen is composed of bovine serum albumin (BSA) linked by an ester bond to a L-DOPA 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 bovine serum albumin (20 mg) in 5 mL of 0.2 M phosphate buffer, pH 8.5;

[0047] 2. Add the following chemicals to a small beaker and stir to dissolve: 20 mg of the synthesized levodopa derivative, 0.3 mL of LD, D-dimethylformamide (DMF), 0.3 mL of methanol, 1.0 mL of 10 mM, pH 5.0 phosphate buffer, 20 mg of 1-ethyl-3-(-3-dimethylaminopropyl)carbodiimide (EDAc), and 2.5 mg of N-hydroxythiosuccinimide (Sulfo-NHS). Stir and dissolve these chemicals at room temperature for 10 min.

[0048] 3. Add the activated 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 levodopa immunogen.

[0049] Example 3: Synthesis of KLH-L-DOPA derivative immunogen

[0050] The KLH-acetyl-L-DOPA immunogen is formed by an ester bond between hemocyanin (KLH) and the L-DOPA 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 hemocyanin (20 mg) in 5 mL of 0.18 M phosphate buffer, pH 8.5;

[0052] 2. Add the following chemicals to a small beaker and stir to dissolve: 20 mg of the synthesized levodopa derivative, 0.3 mL of D-dimethylformamide (DMF), 0.3 mL of methanol, 1.0 mL of 10 mM phosphate buffer (pH 5.0), 20 mg of 1-ethyl-3-(-3-dimethylaminopropyl)carbodiimide (EDAc), and 2 mg of N-hydroxythiosuccinimide (Sulfo-NHS). Stir and dissolve these chemicals at room temperature for 10 min.

[0053] 3. Add the dissolved solution dropwise to the KLH solution and stir overnight at 2-10℃ to obtain the crude levodopa immunogen; purify the synthesized antigen by dialysis to obtain the levodopa immunogen.

[0054] Example 4: Synthesis of thyroglobulin-levodopa derivative immunogen

[0055] The thyroglobulin-levodopa immunogen is formed by ester bonds between thyroglobulin and the levodopa 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:

[0056] 1. Dissolve thyroglobulin (20 mg) in 5 mL of 0.20 M phosphate buffer, pH 9.0;

[0057] 2. Add the following chemicals to a small beaker and stir to dissolve: 10 mg of the synthesized levodopa derivative, 0.3 mL of N,N-dimethylformamide (DMF), 0.3 mL of methanol, 1.0 mL of 20 mM, pH 5.0 potassium phosphate buffer, 20 mg of 1-ethyl-3-(-3-dimethylaminopropyl)carbodiimide (EDAc), and 2.0 mg of N-hydroxythiosuccinimide (Sulfo-NHS). Stir and react these chemicals at room temperature for 30 min.

[0058] 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 levodopa immunogen.

[0059] Example 5: Synthesis of poly-L-L-DOPA derivative immunogen

[0060] The poly-L-L-DOPA immunogen is formed by linking poly-L ...

[0061] 1. Dissolve 20 mg of poly-L-lysine in 5 mL of 0.20 M phosphate buffer solution at pH 9.0;

[0062] 2. Add the following chemicals to a small beaker and stir to dissolve: 10 mg of the synthesized levodopa derivative, 0.3 mL of N,N-dimethylformamide (DMF), 0.3 mL of methanol, 1.0 mL of 20 mM, pH 5.0 potassium phosphate buffer, 20 mg of 1-ethyl-3-(-3-dimethylaminopropyl)carbodiimide (EDAc), and 2.0 mg of N-hydroxythiosuccinimide (Sulfo-NHS). Stir and react these chemicals at room temperature for 30 min.

[0063] 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 levodopa immunogen.

[0064] 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.

[0065] Example 6: Preparation of anti-levodopa specific monoclonal antibody

[0066] This embodiment proposes a method for preparing levodopa monoclonal antibody, which includes the following steps:

[0067] 1. Animal immunization

[0068] 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.

[0069] 2. ELISA indirect enzyme-linked immunosorbent assay (ELISA) was used to analyze the antiserum efficacy.

[0070] 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 specific anti-levodopa antibody of this invention, indicating that the antibody prepared by this invention has high specificity and high sensitivity.

[0071] 3. Screening for cell fusion and positive hybridomas

[0072] (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.

[0073] (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.

[0074] (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.

[0075] (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.

[0076] 4. Large-scale preparation of monoclonal antibodies

[0077] 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 levodopa monoclonal antibody.

[0078] Example 7: Levodopa ELISA Test

[0079] The titer and specificity of the antiserum were determined using an indirect enzyme-linked immunosorbent assay (ELISA). The steps are as follows:

[0080] 1. The antibody prepared in Example 6 was used for ELISA testing of levodopa.

[0081] This test utilizes a competitive immunoassay to determine the levodopa content in a liquid sample. The principle is that levodopa in the sample competes with its conjugated levodopa derivative (HRP-levodopa 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 levodopa, the HRP-conjugated levodopa derivative will bind to the antibody on the ELISA plate. Conversely, if the liquid sample contains a large amount or a certain quantity of levodopa, the enzyme-levodopa 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 levodopa content in the liquid sample.

[0082] 2. The specific steps for establishing the standard curve for levodopa ELISA detection are as follows:

[0083] (1) Preparation of standard products

[0084] Levodopa powder (purchased from Aladdin) was dissolved in methanol to prepare a stock solution of 1 mg / mL. The stock solution was then sequentially diluted with ELISA buffer to prepare standard solutions of 10.00 µg / mL, 5.00 µg / mL, 2.50 µg / mL, 1.25 µg / mL, 0.625 µg / mL, 0.313 µg / mL, 0.156 µg / mL, and 0.00 µg / mL. The ELISA buffer contained 50.0 mM Tris, 100 mM NaCl, and 0.2% BSA.

[0085] (2) Prepare a standard curve using the ELISA test for levodopa.

[0086] The anti-levodopa 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 microplate and incubated at 4°C for 12-24 h. The 96-well microplate coated with the anti-levodopa antibody was washed three times with PBS, and then 200 μL / well of 0.5% BSA solution was added. The plate was blocked at 4°C for 8-16 h. The plate was then washed three times with PBS, and 20 μL / well of standard was added. Then, 100 μL / well of working concentration of HRP-levodopa 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 the plate was 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.

[0087] Example 8: Preparation of a chemiluminescent detection reagent for L-DOPA alkaline phosphatase

[0088] The antibody obtained in Example 6 was used to prepare a chemiluminescent detection reagent for levodopa alkaline phosphatase.

[0089] 1. A levodopa detection kit, which employs a competitive chemiluminescent immunoassay principle. The analyte in the sample and an 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 concentration of levodopa 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 levodopa labeled with a chemiluminescent marker and a levodopa monoclonal antibody coated on a fixed carrier. The chemiluminescent marker in the levodopa labeled with the chemiluminescent marker is alkaline phosphatase; the antibody coated on the fixed carrier is an anti-levodopa antibody coated on magnetic beads.

[0090] 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.

[0091] Table 1: Reaction parameters of alkaline phosphatase chemiluminescence analyzer

[0092] 3. Samples were compared using the chemiluminescent detection reagent for L-DOPA alkaline phosphatase of this invention and high-performance liquid chromatography (HPLC). The detection data and data analysis are attached. Figure 3 .

[0093] Example 9: Cross-reactivity test of analogues

[0094] Six common levodopa structural analogs were selected for cross-reactivity testing, and the results were determined using an alkaline phosphatase chemiluminescence assay. The six common levodopa structural analogs and their cross-reactivity rates are shown in Table 2.

[0095] Table 2: Cross-reactivity results of 6 common levodopa structural analogues

[0096] Results: Based on the cross-reactivity results of the above six common levodopa structural analogs, the antibody prepared in this invention has good specificity.

[0097] The results above show that the levodopa complete immunogen prepared from the levodopa derivative provided by this invention has strong immunogenicity, produces antibodies with high specificity and good affinity for levodopa. The alkaline phosphatase chemiluminescence reagent prepared using the above antibody has good stability and high sensitivity, enabling high-throughput and rapid detection of levodopa 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 levodopa detection, which is conducive to its clinical application.

[0098] 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 levodopa derivative, characterized in that, It has the structure shown in equation (Ⅰ): Equation (Ⅰ).

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

4. A method for preparing a levodopa immunogen, characterized in that, The preparation method includes: preparing the levodopa derivative according to claim 1; and linking the levodopa derivative to a carrier to obtain the levodopa immunogen; wherein the carrier is an immunogenic protein or polypeptide; the synthetic route for preparing the levodopa derivative is as follows: 。 5. The preparation method according to claim 4, characterized in that, The coupling steps between the carrier and the levodopa derivative include: a. Prepare a carrier solution and a levodopa derivative solution; the carrier is serum protein, keyhole hemocyanin, thyroglobulin, or polylysine; b. Add the L-DOPA derivative solution to the carrier solution to obtain a mixed solution of activated L-DOPA 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 levodopa immunogen; purify the crude levodopa immunogen to obtain the pure levodopa immunogen; purify 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 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 levodopa derivative solution includes placing the levodopa derivative with 1-ethyl-3-(-3-dimethylaminopropyl)carbodiimide (EDAc) and N-hydroxythiosuccinimide (Sulfo-NHS) in N,N-dimethylformamide, methanol, and 5–20 mM phosphate buffer solution with a pH of 4.0–6.0 and stirring at room temperature to obtain a solution of the levodopa derivative in an activated state; wherein the mass ratio of the carrier to the levodopa derivative is 1–8:

1.

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

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