Deramanide hapten, deramanide antigen, deramanide antibody, and preparation method and application of deramanide hapten, deramanide antigen

Abstract

The invention discloses a Deramanide hapten, an antigen, an antibody and a preparation method and application thereof, and belongs to the technical field of small molecule immunodetection.A connecting arm with a specific length is introduced to a tertiary amine nitrogen atom of a Deramanide molecule to form a quaternary ammonium salt structure; according to the present invention, with the application of the nitroimidazole oxazole ring system, the molecule is endowed with the chemical operability on the premise of not damaging the core pharmacophore (the nitroimidazole oxazole ring system) so as to efficiently couple the carrier protein and induce the generation of the high-affinity and high-specificity antibody, such that the immunodetection system suitable for the therapeutic drug monitoring (TDM) and the bedside instant detection (POCT) scene is finally constructed;

Description

Technical Field

[0001] This invention belongs to the field of small molecule immunoassay technology, specifically relating to a Delamanid hapten, antigen, antibody, their preparation methods and applications. Background Technology

[0002] Delamanide (DLMD, structural formula shown in Formula 1), a novel nitrodihydroimidazole oxazole anti-tuberculosis drug, has played a crucial role in the treatment of multidrug-resistant tuberculosis (MDR-TB) and extensively drug-resistant tuberculosis (XDR-TB) in recent years. It exerts a potent bactericidal effect by specifically inhibiting the biosynthesis of mycolic acid, an important component of the mycobacterial cell wall, and has been included as one of the core recommended drugs by the World Health Organization. Clinical studies have shown a high correlation between the blood concentration of delamanide and its therapeutic efficacy and safety: too low a concentration easily leads to treatment failure and induces increased drug resistance; while too high a concentration significantly increases the risk of QT interval prolongation and related cardiotoxicity. Therefore, implementing therapeutic drug monitoring (TDM) to achieve personalized and precise drug administration has become an important trend in the current clinical management of tuberculosis.

[0003] Formula 1.

[0004] Currently, the clinical detection of delamanid blood concentrations primarily relies on liquid chromatography-tandem mass spectrometry (LC-MS / MS). While this method boasts high sensitivity and specificity, its application is limited by expensive equipment, complex pretreatment procedures, and a long detection cycle (typically 2-4 hours), making it difficult to meet the needs of primary healthcare institutions for rapid, low-cost testing. More importantly, this technology is not suitable for emerging application scenarios such as home follow-up or point-of-care testing (POCT), severely hindering the promotion and implementation of TDM strategies in resource-constrained areas. Against this backdrop, antibody recognition-based immunoassay methods, due to their ease of operation, low cost, and rapid response, are considered the most promising alternative approach.

[0005] However, successfully applying immunoassay technology to delamanid faces fundamental obstacles. This is because the delamanid molecule itself is highly hydrophobic, structurally complex, and lacks naturally occurring active functional groups suitable for chemical conjugation. Specifically, its core pharmacophore (nitrodihydroimidazole oxazole ring system) not only has significant steric hindrance but also requires extremely high chemical stability; any inappropriate modification can disrupt its conformational characteristics, thereby affecting the subsequent antibody's recognition ability of the original drug molecule. Furthermore, since small molecule haptens must be artificially introduced with a linker arm and conjugated with a carrier protein to induce an immune response, and the number of modifiable sites in the delamanid molecule is extremely limited, traditional conjugation strategies often lead to problems such as low conjugation efficiency, antigenic epitope masking, or insufficient immunogenicity. Consequently, even if the immunogen is successfully prepared, the induced antibodies often exhibit defects such as strong cross-reactivity and low affinity due to the recognition site deviating from the true pharmacophore structure. To date, no reports have been found in domestic or international publicly available literature or patent databases regarding the design of Delamanid specific haptens, the development of high-affinity antibodies, or mature immunoassay systems, indicating a significant technological gap in this field.

[0006] A deeper analysis reveals that the essence of the current technological predicament does not simply stem from the choice of detection platform, but rather from the inherent contradiction between the physicochemical properties of the delamanide molecule and its role as an immunoassay target: on the one hand, to maintain the antibody's high specificity in recognizing the parent drug, the integrity of its pharmacophore must be preserved to the maximum extent; on the other hand, to achieve effective protein conjugation to construct an immunogen, exogenous linker structures must be introduced into its molecule. This tension between "structure preservation" and "conjugability" constitutes the core technological bottleneck that has long remained unresolved in this field. Focusing solely on downstream optimization of the detection method while neglecting the rational design at the upstream hapten molecule level makes it difficult to fundamentally solve the imbalance between antibody specificity and detection sensitivity. Therefore, how to accurately introduce a linker arm that combines chemical operability and immune exposure advantages without interfering with the key pharmacophore structure of delamanide, thereby constructing a hapten-antibody system with both high immunogenicity and high recognition fidelity, has become a crucial prerequisite for achieving rapid, reliable, and universal immunoassay of delamanide. Therefore, how to overcome the molecular structural limitations of de la Manid and construct a novel hapten that can effectively couple with a carrier protein and induce the production of highly specific antibodies, and how to establish a rapid immunoassay method suitable for primary care and POCT scenarios based on this, has become a key challenge and a technical problem that needs to be solved by those skilled in the art. Summary of the Invention

[0007] The purpose of this invention is to provide a delamanide hapten, which can be coupled with a carrier protein to prepare an antigen. This antigen can then be used to prepare antibodies suitable for colloidal gold competitive immunochromatography (LFA) and quantitative immunofluorescence assay (FIA), thus solving the problem of difficult point-of-care detection of delamanide in existing technologies. To achieve the above-mentioned objective, this invention introduces a linker arm of a specific length onto the tertiary amine nitrogen atom of the delamanide molecule, forming a quaternary ammonium salt structure. This endows the molecule with chemical operability without destroying its core pharmacophore (nitrodihydroimidazole oxazole ring system), enabling it to efficiently couple with a carrier protein and induce the production of high-affinity, high-specificity antibodies. Ultimately, this constructs an immunoassay system suitable for therapeutic drug monitoring (TDM) and point-of-care testing (POCT) scenarios.

[0008] To achieve the above objectives, the technical solution adopted by the present invention is as follows: Firstly, the core objective of this invention is to provide a Delamanid hapten, the structural formula of which is shown in Formula I: Formula I In Formula 1, n is 2 to 8, and the active group X is -COOH or -NH2.

[0009] Secondly, the present invention also provides a method for preparing the hapten described in the first aspect, comprising the following steps: (1) Prepare the quaternary ammonium salt intermediate as shown in Formula II; Formula II In Equation II, R is -(CH2). n -COOEt or -(CH2) n -NH-Boc); (2) Deprotection treatment of ester group or Boc protecting group, wherein the ester group is deprotected by hydrolyzing the ester group with sodium hydroxide aqueous solution, and the target hapten is obtained after acidification and precipitation, and the Boc protecting group is deprotected by removing the Boc protecting group with trifluoroacetic acid, and the target hapten is obtained by neutralization and extraction purification.

[0010] Thirdly, the present invention also provides the use of the hapten described in the first aspect in the preparation of the Delamanid antigen.

[0011] Fourthly, the present invention also utilizes the hapten described in the first aspect to couple with a carrier protein to obtain a Delamanid antigen, wherein the carrier protein is one of bovine serum albumin, ovalbumin, or keyhole cyanobacterium hemocyanin. In the present invention, the coupling method between the hapten and the carrier protein depends on the type of the terminal functional group A of the hapten. When A is a carboxyl group, an activation system consisting of 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (EDC) and N-hydroxysuccinimide (NHS) is used. In phosphate-buffered saline (PBS) at pH 7.0–7.4, the carboxyl group of the hapten is activated to an NHS ester at 4°C. Subsequently, bovine serum albumin (BSA, molecular weight approximately 66 kDa) or keyhole cyanobacterium hemocyanin (KLH, molecular weight approximately 450 kDa) is added, causing the ε-amino group of its lysine residue to undergo a nucleophilic substitution reaction with the activated ester, forming a stable amide bond. After the reaction lasted 12–24 hours, the reaction solution was dialyzed using a dialysis bag with a molecular weight cutoff of 10 kDa (6 changes of medium, 4 hours each time), or Sephadex G-25 gel filtration chromatography was used to thoroughly remove unreacted small molecule impurities and obtain purified complete antigen. When A is amino, two coupling strategies can be used: one is the glutaraldehyde cross-linking method, in which the hapten and carrier protein are dissolved separately in PBS, a 0.2% glutaraldehyde solution is added, and the reaction is carried out overnight at 4°C to form a Schiff base structure, which is then stabilized by reduction with sodium cyanoborohydride; the other is to first derivatize the surface carboxyl groups of the carrier protein with succinic anhydride to introduce additional carboxyl groups, and then activate it with EDC / NHS before coupling it with the amino group of the hapten. Both methods were carried out at pH 7.2 and 4°C for 12–24 hours, and the post-processing was the same as above.

[0012] Fifthly, the present invention also provides the use of the antigen described in the fourth aspect in the preparation of Delamanid antibodies.

[0013] Sixthly, the present invention also provides an antibody against Delamanid, which is prepared by immunizing animals with the Delamanid antigen described in the fourth aspect. The antibody is a polyclonal antibody or a monoclonal antibody, prepared by the following steps: emulsifying the above-mentioned complete antigen with Freund's adjuvant and injecting it subcutaneously at multiple sites into BALB / c mice or New Zealand white rabbits; using complete Freund's adjuvant for the initial immunization and incomplete Freund's adjuvant for the booster immunization, with a total of 4 immunizations at 2-week intervals; collecting blood 7-10 days after the final immunization, separating serum, and purifying polyclonal antibodies by Protein A affinity chromatography; or obtaining monoclonal antibodies by fusing spleen cells with SP2 / 0 myeloma cells, screening for positive clones, expanding culture, and preparing ascites fluid.

[0014] In a seventh aspect, the present invention also provides the application of the delamanid antigen as described in the fourth aspect and the anti-delamanid antibody as described in the sixth aspect in a method for monitoring delamanid therapeutic drugs, wherein the method for monitoring delamanid therapeutic drugs is a competitive immune detection method.

[0015] Furthermore, the competitive immunoassay method is colloidal gold immunochromatography (LFA) or quantitative immunofluorescence assay (FIA).

[0016] In an eighth aspect, the present invention also provides a kit for detecting Delamanid, the kit comprising the Delamanid antigen as described in the fourth aspect and an anti-Delamanid antibody as described in the sixth aspect.

[0017] Compared with the prior art, the present invention has the following advantages and beneficial effects: 1. This invention introduces a linker arm of a specific length onto the tertiary amine nitrogen atom of the delamanide molecule to form a quaternary ammonium salt structure. This chemical modification does not involve the core pharmacophore of delamanide (nitrodihydroimidazole oxazole ring system), ensuring that the conformational integrity of the pharmacophore is preserved, thereby solving the technical bottleneck that the drug cannot be detected by immunoassay. The prepared antibody has high affinity and excellent specificity. Its application scenarios cover the entire chain of monitoring systems from central laboratories to bedside, home and mobile follow-up, significantly improving the accessibility and compliance of precision treatment for tuberculosis.

[0018] 2. The immune detection system composed of antigens and antibodies described in this invention can be directly applied to therapeutic drug monitoring (TDM) of Delamanid, ensuring that the plasma drug concentration is within the range during treatment, thus achieving dual indicators of efficacy and patient safety. Detailed Implementation

[0019] The present invention will be described in detail below with reference to specific embodiments, thereby making its advantages and various effects more clearly apparent. Those skilled in the art should understand that these specific embodiments and examples are for illustrative purposes only and are not intended to limit the invention.

[0020] Throughout this specification, unless otherwise specified, the terminology used herein should be understood as having the meaning commonly used in the art. Therefore, unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention pertains. In the event of any conflict, this specification shall prevail.

[0021] Unless otherwise specified, all raw materials, reagents, instruments, and equipment used in this invention are commercially available or can be prepared using existing methods. All examples used Delamand (CAS No. 681492-22-8) as a starting material with a purity ≥98%, which was commercially available.

[0022] Example 1

[0023] The present invention provides the Delamanid hapten as shown in Formula III.

[0024] Formula III.

[0025] The preparation process of the DLMD hapten, as shown in Formula III, is as follows: DLMD (1.0 g, 1.87 mmol) was dissolved in 20 mL of anhydrous acetonitrile, and ethyl 3-bromopropionate (0.49 g, 2.25 mmol) was added. The mixture was stirred at 60 °C under nitrogen protection for 12 hours. After cooling to room temperature, the mixture was concentrated under reduced pressure. The residue was precipitated with 50 mL of diethyl ether and filtered to obtain a yellow solid. This solid was dissolved in a mixture of 10 mL THF and 10 mL methanol, and 2 mL of 1 mol / L NaOH aqueous solution was added. The mixture was stirred at room temperature for 4 hours. The reaction solution was adjusted to pH 3.5 with 1 mol / L HCl, extracted with ethyl acetate (3 × 30 mL), and the organic phases were combined, dried over anhydrous Na₂SO₄, and concentrated to obtain the crude product. The crude product was purified by silica gel column chromatography (dichloromethane / methanol = 15:1) and lyophilized to obtain 0.51 g of the white powder hapten DLMD-C₂-COOH, with a yield of 44.9%. ¹H NMR (400 MHz, DMSO-d6) δ:11.92 (s, 1H),7.28 (d,1H),7.12 (m, 4H), 7.02 (m, 4H), 4.85 (d, J = 6.2 Hz, 1H), 4.71 (t, J = 6.0Hz, 2H), C NMR (101 MHz, DMSO-d6) δ: 177.3, 159.3, 149.7, 147.5, 142.3, 138.4, 130.1, 121.8, 120.4, 118.3, 115.2, 114.7, 81.2, 79.5, 77.4, 69.8, 59.3, 58.1, 29.2, 26.7, 23.6.

[0026] Antigen 1 was prepared using DLMD-C2-COOH. The preparation process was as follows: Bovine serum albumin (BSA, Sigma-Aldrich, catalog number A7906) was selected as the carrier protein. The specific procedure was as follows: The hapten (0.1 mmol) was dissolved in N,N-dimethylformamide (DMF, 2 mL), and 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride (EDC·HCl, 23 mg, 0.12 mmol) and N-hydroxysuccinimide (NHS, 14 mg, 0.12 mmol) were added. The mixture was activated at room temperature for 30 minutes. Subsequently, bovine serum albumin (BSA, 50 mg) was dissolved in 0.01 mol / L phosphate buffer (PBS, pH 7.4, 5 mL) and slowly added dropwise to the activation solution. The mixture was stirred at 4°C in the dark for 12 hours. The reaction solution was placed in a dialysis bag with a molecular weight cutoff of 10 kDa and dialyzed against PBS (pH 7.4) at 4°C for 3 days, with the buffer changed 3 times daily. The dialysate was lyophilized to obtain a white powdery complete antigen. The coupling ratio was determined by ultraviolet spectrophotometry: the average number of delamanid hapten molecules coupled to each BSA molecule was calculated to be 7.3. Furthermore, matrix-assisted laser desorption / ionization time-of-flight mass spectrometry (MALDI-TOF MS) analysis showed that the molecular weight of the main peak of the conjugate was approximately 70.8 kDa, which is about 4.4 kDa higher than that of unmodified BSA (66.4 kDa), and is in high agreement with the theoretical increase (7.3 × 0.607 kDa ≈ 4.43 kDa).

[0027] Example 2 The present invention provides the Delamanid hapten as shown in Formula IV.

[0028] Formula IV.

[0029] The preparation process of the Delamanid hapten, as shown in Formula IV, is the same as in Example 1, except that ethyl 3-bromopropionate is replaced with ethyl 5-bromopentanoate (0.55 g, 2.25 mmol). After the same post-treatment and deprotection steps, the pure target hapten, DLMD-C4-COOH (0.61 g, yield 51.4%), is obtained. Its ¹H NMR (400 MHz, DMSO-d6) δ: 11.75 (s, 1H), 7.28 (d, 1H), 7.10 (m, 4H), 6.94 (m, 4H), 4.46 (d, J = 6.2 Hz, 1H), 4.11 (t, J = 6.2 Hz, 2H), 3.91 (d, J = 6.3 Hz, 1H), 3.64 (m, 1H), 3.25 (m, 4H). 3.11 (m,2H),2.24 (m, 4H), 1.96 (m, 2H), 1.73 (m, 2H), 1.52 (m, 2H) ,1.45 (m, 3H);¹³CNMR (101 MHz, DMSO-d6) δ: 178.3, 159.4, 152.3, 149.9, 147.3, 142.3, 138.1,129.5, 121.6, 120.1, 118.2, 115.3, 114.9, 81.7, 79.1, 77.3, 63.4, 59.6, 33.7,23.6, 23.1, 20.4.

[0030] Antigen 2 was prepared using DLMD-C4-COOH, following the preparation process described in Example 1. The conjugation ratio was determined by UV spectrophotometry: the average number of DLA molecules conjugated to each molecule of DLA was calculated to be 10.9 DLA hapten molecules. Furthermore, matrix-assisted laser desorption / ionization time-of-flight mass spectrometry (MALDI-TOF MS) analysis showed that the main peak molecular weight of the conjugate was approximately 73.3 kDa, an increase of approximately 6.9 kDa compared to unmodified BSA (66.4 kDa), which is highly consistent with the theoretical increase (10.9 × 0.635 kDa ≈ 6.92 kDa).

[0031] Example 3 The present invention provides the Delamanid hapten as shown in Formula V.

[0032] Formula V.

[0033] The preparation process of the Delamanid hapten, as shown in Formula V, is the same as in Example 1, except that ethyl 3-bromopropionate is replaced with ethyl 9-bromononanoate (0.68 g, 2.27 mmol). After the same post-treatment and deprotection steps, the target hapten, DLMD-C8-COOH (0.51 g, yield 40.7%), was obtained. Its ¹H NMR (400 MHz, DMSO-d6) δ: 11.71 (s, 1H), 7.19 (d, 1H), 7.14 (m, 4H), 6.91 (m, 4H), 4.63 (d, J = 6.1 Hz, 1H), 4.29 (t, J = 6.5 Hz, 2H), 4.12 (d, J = 5.3 Hz, 1H), 3.50 (m, 1H), 3.29 (m, 4H). C NMR (101 MHz, DMSO-d6) δ: 178.2, 159.4,152.1, 149.5, 147.3, 142.3, 138.1, 129.6, 121.9, 120.1, 118.3, 115.5, 114.8,81.4, 79.3, 77.3, 63.2, 59.6, 34.1, 29.4, 29.0, 26.9, 24.5, 23.2, 20.5.

[0034] Antigen 3 was prepared using DLMD-C8-COOH, following the preparation process described in Example 1. The conjugation ratio was determined by UV spectrophotometry: an average of 8.4 Delamanid hapten molecules were conjugated per BSA molecule. Furthermore, matrix-assisted laser desorption / ionization time-of-flight mass spectrometry (MALDI-TOF MS) analysis showed that the main peak molecular weight of the conjugate was approximately 72.4 kDa, an increase of approximately 6.0 kDa compared to unmodified BSA (66.4 kDa), which is highly consistent with the theoretical increase (8.4 × 0.692 kDa ≈ 6.02 kDa).

[0035] Example 4 The present invention provides the Delamanid hapten as shown in Formula VI.

[0036] Formula VI.

[0037] The preparation of the delamanid hapten, as shown in Formula VI, is as follows: Delamanid (1.0 g, 1.87 mmol) was dissolved in anhydrous acetonitrile (20 mL), and N-Boc-1,5-diiodopentane (0.48 g, 2.25 mmol) was added. The mixture was refluxed at 80 °C for 18 hours. The post-treatment was the same as before, yielding a Boc-protected quaternary ammonium salt intermediate. This intermediate was dissolved in dichloromethane (10 mL), and trifluoroacetic acid (5 mL) was added. The mixture was stirred at room temperature for 4 hours. Most of the solvent was removed under reduced pressure. The residue was neutralized to pH 8 with saturated NaHCO3 solution, extracted with dichloromethane, dried, and concentrated to give DLMD-C4-NH2 (0.42 g, yield 37%). Its ¹H NMR (400 MHz, DMSO-d6) δ: 7.93 (d, 1H), 7.36 (m, 6H), 7.32 (d, J = 7.4 Hz, 2H), 4.66 (d, J = 7.0 Hz, 1H), 4.42 (t, J = 8.6 Hz, 2H), 4.26 (d, J = 7.0 Hz, 1H), 3.75 (m, 1H), 3.45 (m, 4H), 3.35 (m, 2H), 2.85 (t, J = 7.6 Hz, 2H), 2.46 (m, 2H), 2.21 (m,2H), 1.74 (m, 2H), 1.88 (m, 2H), 1.52 (br s, 2H),1.45(m, 3H);¹³C NMR (101MHz, DMSO-d6) δ: 158.7, 152.4, 148.8, 147.4, 142.6, 138.5, 121.9, 118.5,115.4, 114.7, 81.4, 78.6, 77.5, 63.9, 59.4, 41.8, 31.2, 26.7, 23.8, 18.2.

[0038] Antigen 4 was prepared using DLMD-C4-NH2. The preparation process was as follows: 25 mg of DLMD-C4-NH2 and 50 mg of BSA were dissolved in 5 ml of PBS, and 0.2% glutaraldehyde solution was added. The pH of the system was controlled at 7.2, and the reaction was carried out overnight at 4°C to form a Schiff base structure. Then, 10 mg of sodium cyanoborohydride was added for reduction and stabilization, and antigen 4 was purified. The coupling ratio was determined by UV spectrophotometry: it was calculated that each BSA molecule was coupled with an average of 8.9 Delamanid hapten molecules. In addition, matrix-assisted laser desorption / ionization time-of-flight mass spectrometry (MALDI-TOF MS) analysis showed that the molecular weight of the main peak of the conjugate was approximately 71.9 kDa, which was about 5.5 kDa higher than that of unmodified BSA (66.4 kDa), and highly consistent with the theoretical increase (8.9 × 0.607 kDa ≈ 5.4 kDa).

[0039] Experimental Example Antigens 1-4 prepared in Examples 1-4 were used to immunize BALB / c mice. New Zealand white rabbits aged 6-8 weeks (weighing 2.0-2.5 kg) were used as immunization animals. For the first immunization, the immunogen (containing BSA-hapten, 100 μg / rabbit or 20 μg / mouse) was thoroughly emulsified with an equal volume of Freund's complete adjuvant (CFA) to form a water-in-oil emulsion, which was injected subcutaneously at multiple points (8 points in total, including the back and axilla of the rabbit). Two weeks later, a first booster immunization was performed, using Freund's incomplete adjuvant (IFA) instead of CFA, at half the dose, and injected in the same manner as before. Thereafter, booster immunizations were performed every two weeks for a total of three booster immunizations. On day 10 after the last immunization, antiserum was collected via the marginal ear vein (rabbit) or the orbital venous plexus (mouse). After inactivating complement by incubating the serum in a 56°C water bath for 30 minutes, the serum was centrifuged at 4°C, 10,000 × g for 10 minutes to remove clots; the supernatant was the crude polyclonal antibody extract. For the preparation of monoclonal antibodies, on day 3 after the last booster immunization, mouse spleens were harvested, spleen cells were isolated, and fused with SP2 / 0 myeloma cells at a ratio of 5:1. Hybridomas were screened using HAT selective medium. The fused cells were cloned using limiting dilution, and positive wells secreting specific antibodies were screened by indirect ELISA. The coating antigen used for screening was the Delamand-OVA conjugate (prepared using the same method as the BSA conjugate, except the carrier protein was replaced with OVA), the blocking medium was 5% skim milk powder / PBST, the primary antibody was the hybridoma supernatant, and the secondary antibody was HRP-labeled goat anti-mouse IgG. Positive clones were subcloned three times and then expanded through culture to prepare ascites fluid or in vitro culture supernatant, which was then purified to obtain monoclonal antibodies.

[0040] The affinity and specificity of the polyclonal or monoclonal antibodies obtained through the above procedure were evaluated by indirect competitive ELISA. The specific method was as follows: Delamanid-OVA (1 μg / mL in PBS) was coated onto a 96-well ELISA plate and incubated overnight at 4°C; the next day, the plate was washed three times with PBST, and blocked with 5% skim milk powder for 1 hour; subsequently, serially diluted Delamanid standards (0.01~100 ng / mL) and a fixed concentration of antibody (diluted to OD200) were added. 450 ≈ 1.0) mixture, competitive reaction at 37℃ for 1 hour; after washing the plate, add HRP-labeled secondary antibody, develop color, and then measure OD. 450 A competition curve was plotted with drug concentration on the x-axis and inhibition rate on the y-axis, and the half-maximal inhibitory concentration (IC50) was calculated. 50 Experimental results show that the antibody prepared in this invention is effective against the IC50 of Delamanid. 50 The concentration ranged from 5.1 to 11 ng / mL, with the highest antibody affinity induced by the carboxyl hapten with a C4 linker arm (IC50). 50 (≈ 5.1 ng / mL). Cross-reactivity tests showed that when the concentrations of bedaquilin (BDQ), pretomanid, para-aminosalicylic acid (PAS), and moxifloxacin (MFX) were as high as 1000 ng / mL, the inhibition rate of the delamanid assay was less than 5%, indicating that the antibodies prepared from the above antigens all had excellent specificity.

[0041] For higher-precision quantification, this experimental example uses antibodies prepared with DLMD-C4-COOH to develop an immunofluorescence quantitative method (FIA). The fluorescent probe was prepared using carboxyl-modified fluorescent microspheres (200 nm in diameter, excitation wavelength 520–620 nm, emission wavelength 620–680 nm). 10 mg of microspheres were suspended in 1 mL of MES buffer (0.1 mol / L, pH 5.5), and EDC (2 mg) and NHS (2.5 mg) were added. The microspheres were activated at room temperature for 30 minutes. After centrifugation to remove the supernatant, the microspheres were resuspended in PBS (pH 7.4) containing 100 μg of anti-Dramanid antibody and coupled at 4°C for 12 hours. Finally, unreacted carboxyl groups were blocked with PBS containing 1% BSA to obtain the fluorescent probe. The detection chip surface was pre-immobilized with a DLMD-OVA conjugate (10 μg / mL), or coated using a 96-well black microplate. During detection, the sample (50 μL) was mixed with the fluorescent probe (50 μL) and added to the detection area, then incubated at 37°C for 10 minutes. The sample was then washed three times with PBST to remove unbound probe. Finally, the residual fluorescence intensity (Ex / Em = 580 / 650 nm) was measured using a fluorescence reader. The fluorescence signal intensity was negatively correlated with the concentration of delamanid in the sample. A quantitative model was established by measuring a series of concentration standards (0.05–20 μg / mL) and fitting a standard curve using a four-parameter logistic equation. The detection limit of this method was 0.1 μg / mL, the linear range was 0.1–15 μg / mL, and the intra- and inter-batch coefficients of variation were both less than 8%. Parallel measurements of 50 clinical serum samples by LC-MS / MS showed a correlation coefficient R0 between the two methods. 2 = 0.932, indicating that FIA has good clinical applicability.

[0042] Comparative Example 1 This comparative example attempted to introduce a carboxymethyl group onto the aromatic ring of the de la Manid molecule using traditional methods. However, because this region is the core of the pharmacophore, the molecular conformation changed significantly after modification, resulting in an reduced IC50 of the prepared antibody against the parent drug. 50 It has a cross-reactivity rate of >100 ng / mL and a cross-reactivity rate of 35% with putomani, and a cross-reactivity rate of more than 10% with other tuberculosis drugs.

[0043] Comparative Example 2 In this comparative example, direct physical immunization with unmodified delamanid and carrier protein failed to induce detectable specific antibodies (IC50). 50 (Unable to be determined).

[0044] Comparative Example 3 This comparative example uses a short-connecting-arm (n=1) hapten, which, although capable of coupling, suffers from excessive steric hindrance and low antibody affinity (IC50). 50= 27.5 ng / mL), and also has a relatively high cross-reactivity rate with other tuberculosis drugs.

[0045] As shown in Table 1, the key performance parameters of Embodiments 1-4 and Comparative Examples 1-3 of the present invention are summarized.

[0046] Table 1. Summary of key performance parameters of Examples 1-4 and Comparative Examples 1-3 of the present invention

[0047] In summary, this invention successfully prepared high-affinity and high-specificity antibodies by introducing a flexible linker of C2–C8 length onto the tertiary amine nitrogen, with the C4 linker exhibiting the best performance. This technical solution not only solves the problem of delamanid's inability to be immunized due to the lack of a coupling group, but also ensures the accurate recognition of the parent drug by the antibody through rational molecular design. Based on this, high-affinity and high-specificity antibodies were successfully prepared, leading to the development of immunoassay methods suitable for different application scenarios. The entire technology chain, from molecular design, chemical synthesis, immunological validation to the construction of the detection platform, has undergone systematic optimization and rigorous validation, possessing high reproducibility and clinical translational value, providing solid technical support for the precision treatment of anti-tuberculosis drugs.

[0048] The above embodiments are merely one of the preferred embodiments of the present invention and should not be used to limit the scope of protection of the present invention. Any modifications or refinements made to the main design concept and spirit of the present invention that are not of substantial significance, but solve the same technical problem as the present invention, should be included within the scope of protection of the present invention.

Claims

1. A Delamanid hapten, characterized in that, Its structural formula is shown in Formula I: A Delamanid hapten, characterized in that its structural formula is shown in Formula I: Formula I; In Formula 1, n is 2 to 8, and the active group X is -COOH or -NH2.

2. The method for preparing the Delamanid hapten as described in claim 1, characterized in that, Includes the following steps: (1) As shown in Formula II, using Delamand as the initial raw material and anhydrous acetonitrile as the solvent, a quaternary ammonium salt intermediate was prepared under nitrogen protection. Formula II In Equation II, R is -(CH2). n -COOEt or -(CH2) n -NH-Boc); (2) Deprotection treatment of ester group or Boc protecting group, wherein the ester group is deprotected by hydrolyzing the ester group with sodium hydroxide aqueous solution, and the target hapten is obtained after acidification and precipitation, and the Boc protecting group is deprotected by removing the Boc protecting group with trifluoroacetic acid, and the target hapten is obtained by neutralization and extraction purification.

3. The preparation method as described in claim 2, characterized in that, In step (1), the molar ratio of RI to de la Manid is 1.2 to 1.5:

1.

4. The use of the Delamanid hapten as described in claim 1 in the preparation of Delamanid antigen.

5. A Delamanid antigen, characterized in that, The Delamanid antigen is obtained by conjugating the Delamanid hapten with a carrier protein as described in claim 1, wherein the carrier protein is one of bovine serum albumin, ovalbumin, or keyhole hemocyanin.

6. The use of the Delamanid antigen as described in claim 5 in the preparation of Delamanid antibodies.

7. An antibody against Dramanid, characterized in that, The antibody is prepared by immunizing animals with the Delamanid antigen as described in claim 5. The antibody is a polyclonal antibody or a monoclonal antibody, and is prepared by the following steps: emulsifying the above-mentioned complete antigen with Freund's adjuvant and injecting it subcutaneously at multiple sites into BALB / c mice or New Zealand white rabbits; using complete Freund's adjuvant for the initial immunization and incomplete Freund's adjuvant for the booster immunization, with a total of 4 immunizations at 2-week intervals; collecting blood 7-10 days after the final immunization, separating the serum, and purifying it by Protein A affinity chromatography to obtain polyclonal antibodies; or obtaining monoclonal antibodies by fusing spleen cells with SP2 / 0 myeloma cells, screening for positive clones, expanding culture, and preparing ascites fluid.

8. The application of the Delamanid antigen as described in claim 5 and the anti-delamanid antibody as described in claim 7 in a method for monitoring Delamanid therapeutic drugs, characterized in that, The method for monitoring the therapeutic drugs of Delamanid is a competitive immune detection method.

9. The application as described in claim 8, characterized in that, The competitive immunoassay method is colloidal gold immunochromatography or quantitative immunofluorescence assay.

10. A kit for detecting Delamanid, characterized in that, Includes the Delamanid antigen as described in claim 5 and the anti-Delamanid antibody as described in claim 7.

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