PA-824 haptens, antigens, antibodies, and methods of making and using the same

Abstract

This invention discloses a PA-824 hapten, antigen, antibody, and their preparation methods and applications, belonging to the field of small molecule immunoassay technology. By designing the PA-824 hapten structure, this invention successfully introduces a conjugable linker arm while retaining its core pharmacodynamic properties, overcoming the long-standing technical bottleneck of preventing immunoassay of this drug due to its molecular inertness. The prepared antibody possesses high affinity and excellent specificity. The LFA and FIA platforms that can be constructed are simple to operate, have rapid response, and are low in cost, with performance indicators meeting clinical TDM requirements. Its application scenarios cover the entire monitoring chain from central laboratories to bedside, home, and mobile follow-up, significantly improving the accessibility and adherence of precision treatment for MDR / XDR-TB.

Description

Technical Field

[0001] This invention belongs to the field of small molecule immunoassay technology, specifically relating to a PA-824 hapten, antigen, antibody, their preparation methods and applications. Background Technology

[0002] Significant progress has been made in the treatment of tuberculosis in recent years in addressing multidrug-resistant tuberculosis (MDR-TB) and extensively drug-resistant tuberculosis (XDR-TB). PA-824 (also known as Pretomanid), a representative drug of the nitroimidazole-oxazine class (its structural formula is shown in Formula 1), has been included in the World Health Organization's core treatment regimen for BPaL / BPaLM. This drug exhibits excellent bactericidal efficacy in hypoxic microenvironments by inhibiting mycobacterial cell wall synthesis and inducing the production of reactive nitrogen oxides, effectively eliminating persistent bacterial flora. However, the highly complex molecular structure of PA-824, combined with strong hydrophobicity and significant inter-individual metabolic variability, leads to drastic fluctuations in its pharmacokinetic behavior and an extremely narrow therapeutic window. Against this backdrop, therapeutic drug monitoring (TMD) has become a crucial clinical approach to ensure efficacy and mitigate toxic reactions. Currently, the determination of PA-824 blood drug concentration mainly relies on liquid chromatography-tandem mass spectrometry (LC-MS / MS). Although this method has high sensitivity and specificity, its operation procedure is cumbersome, requiring professional technicians to perform sample pretreatment. The equipment investment and operating costs are high, making it difficult to popularize in primary healthcare institutions with limited resources. More importantly, this technology cannot achieve point-of-care testing, nor does it support dynamic medication assessment for patients in mobile follow-up or home settings, which seriously restricts the implementation of precision anti-tuberculosis treatment strategies.

[0003] Further examination of existing technologies reveals that immunoassay, due to its ease of operation, low cost, and rapid response, should have been an ideal alternative for monitoring PA-824 therapeutic drugs. However, to date, there have been no publicly reported methods for PA-824 immunoassay, nor any effective patent portfolios for related haptens or specific antibodies. The fundamental reason for this is that the PA-824 small molecule itself lacks naturally occurring active functional groups suitable for chemical conjugation. Its core pharmacodynamic backbone—the nitroimidazole-oxazine ring system—is highly stable and functionally saturated, while the remaining structural parts are mostly inert alkoxy or aromatic fragments, making it difficult to directly introduce linker arms to construct immunogenic hapten-carrier protein conjugates. Forcibly modifying key pharmacodynamic sites with chemicals could potentially disrupt its binding ability to the target protein, causing the induced antibody to fail to effectively recognize the original drug molecule, thus rendering the detection meaningless. Correspondingly, due to the lack of a structurally sound and immunogenic hapten design strategy, the preparation of anti-PA-824 antibodies with high affinity and low cross-reactivity has long been a technological gap. As a result, mainstream rapid detection platforms such as colloidal gold immunochromatography and fluorescence immunoassay lack the necessary core biorecognition elements, making it impossible to build a point-of-care detection system suitable for PA-824.

[0004] A deeper analysis reveals an inherent technical contradiction: on the one hand, the application of immunoassay technology to small molecule drugs is highly dependent on the successful construction of haptens, and the design of haptens must introduce conjugable groups while retaining the key epitopes of the prototype drug; on the other hand, the molecular characteristics of PA-824 determine that its structure almost lacks "non-critical but modifiable" sites, and the traditional small molecule hapten design paradigm encounters a fundamental bottleneck here. Furthermore, even if a conjugate is obtained through non-specific modification, the induced antibody often exhibits low affinity or high cross-reactivity to the prototype drug due to epitope shift. This is especially problematic in clinical settings where PA-824 is frequently used in combination with novel anti-tuberculosis drugs such as bedaquilin and delamanid, where cross-reactivity severely interferes with the accuracy of the detection results. Therefore, the key to overcoming the technical barriers of PA-824 immunoassay lies in how to accurately select non-critical molecular regions and introduce linkers of appropriate length and properties without disturbing the core pharmacodynamic structure of PA-824, in order to achieve the rational design and efficient synthesis of highly immunogenic haptens and thus obtain specific antibodies with excellent recognition performance. This challenge not only concerns the feasibility of monitoring a single drug, but also directly impacts the overall construction efficiency and clinical applicability of therapeutic drug monitoring systems for future multi-drug regimens. Therefore, establishing a PA-824 hapten-antibody system that balances structural fidelity, immunogenicity, and detection specificity, and developing rapid immunoassay methods suitable for primary care and point-of-care settings based on this system, has become a key challenge and an urgent technical problem for those skilled in the art. Summary of the Invention

[0005] The purpose of this invention is to provide a PA-824 hapten, which can be coupled with a carrier protein to prepare an antigen, and this antigen can be used to prepare antibodies that can be used for colloidal gold competitive immunochromatography (LFA) and quantitative immunofluorescence assay (FIA), thereby solving the problem of difficult real-time detection of PA-824 in the prior art.

[0006] 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 PA-824 hapten, the structural formula of which is shown in Formula I: Formula I In Formula 1, n is 2 to 8, and A is one of -COOH, -NH2, and SH.

[0007] Secondly, the present invention also provides a method for preparing the hapten described in the first aspect. The preparation steps are as follows: PA-824 is dissolved in anhydrous tetrahydrofuran or anhydrous N,N-dimethylformamide, and n-butyllithium or potassium tert-butoxide is added under inert gas protection. The benzyl position of the benzyl side chain of PA-824 is deprotonated at -78℃ to 0℃ to generate a benzyl carbanion intermediate. Subsequently, an appropriate derivative of ω-halocarboxylic acid ester, ω-haloamine hydrochloride or ω-halothiol is added dropwise to the reaction system, wherein the halogen is bromine or iodine, and the carbon chain length corresponds to n=2~8. The reaction temperature is raised to room temperature and stirred continuously for 12~24h. After the reaction is completed, the target hapten is obtained by water quenching, organic phase extraction and column chromatography purification. The active group A is deprotected and further purified to obtain the target hapten pure product.

[0008] Thirdly, the present invention also provides the use of the hapten described in the first aspect in the preparation of the PA-824 antigen.

[0009] Fourthly, the present invention also utilizes the hapten described in the first aspect to couple with a carrier protein to obtain a PA-824 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 composed 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 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. When A is a thiol group, directional coupling is performed using maleimide chemistry: First, the carrier protein (e.g., BSA) is activated in PBS at pH 7.0 with N-γ-maleimide butyryloxysulfonyl-succinimide ester (sulfo-GMBS) at room temperature for 1 hour to form a maleimide-activated protein; then, a thiol-containing hapten is added, and the reaction is carried out at 4°C in the dark for 12 hours. The thiol group undergoes a Michael addition reaction with the maleimide ring to form a stable thioether bond. After the reaction, the protein is purified by dialysis or gel filtration. The coupling efficiency of all complete antigens is determined by UV-Vis spectrophotometry: utilizing the characteristic absorbance of BSA at 280 nm (ε = 43,824 M). -1 cm -1 The absorption difference between the antigen and the hapten at a specific wavelength (e.g., 320 nm) is used to calculate the conjugation ratio (hapten / protein ratio) according to the formula. The conjugation ratio of the complete antigen is usually between 8 and 15, which meets the requirements of immunogenicity. The conjugation density of the hapten is moderate, which can effectively stimulate the immune response and avoid epitope masking caused by steric hindrance.

[0010] Fifthly, the present invention also provides the use of the antigen described in the fourth aspect in the preparation of PA-824 antibody.

[0011] Sixthly, the present invention also provides an antibody against PA-824, which is prepared by immunizing animals with the PA-824 antigen described in the fourth aspect, and the antibody is a polyclonal antibody or a monoclonal antibody; the polyclonal antibody is obtained by immunizing rabbits with PA-824-bovine serum albumin conjugate as an immunogen; the monoclonal antibody is obtained by immunizing mice with PA-824-keyhole hemocyanin conjugate as an immunogen, followed by cell fusion, screening and subcloning to obtain hybridoma cell line 3F7, which is then secreted by the cell line.

[0012] In a seventh aspect, the present invention also provides the application of the PA-824 antigen as described in the fourth aspect and the anti-PA-824 antibody as described in the sixth aspect in a PA-824 therapeutic drug monitoring method, wherein the PA-824 therapeutic drug monitoring method is a competitive immunoassay method.

[0013] Eighthly, the present invention also provides a kit for detecting PA-824, the kit comprising the PA-824 antigen as described in the fourth aspect and an anti-PA-824 antibody as described in the sixth aspect.

[0014] In a ninth aspect, the present invention also provides a colloidal gold immunochromatographic test strip for detecting PA-824. The test strip comprises a base plate, a sample pad, a conjugate pad, a nitrocellulose membrane, and an absorbent pad sequentially adhered to the base plate. The conjugate pad is coated with a colloidal gold-labeled antibody, which is the anti-PA-824 antibody as described in the sixth aspect. The colloidal gold particle size is 30 nm, and the labeling density is 15 μg of gold particles bound per microgram of antibody. The nitrocellulose membrane has a T line and a C line, wherein the T line is coated with the PA-824 antigen as described in the fourth aspect, with a coating concentration of 1.0 mg / mL and a stripping volume of 1.0 μL / cm; the C line is coated with goat anti-rabbit IgG or goat anti-mouse IgG, with a coating concentration of 0.8 mg / mL and a stripping volume of 1.0 μL / cm.

[0015] Compared with the prior art, the present invention has the following advantages and beneficial effects: 1. This invention, through the design of the PA-824 hapten structure, successfully introduces a conjugable linker arm while retaining its core pharmacodynamic properties, thus overcoming the technical bottleneck of the drug's long-standing inability to be detected by immunoassay due to its molecular inertness. The prepared antibody possesses high affinity and excellent specificity. The LFA and FIA platforms that can be constructed are simple to operate, have rapid response, and are low in cost, and their performance indicators meet the needs of clinical TDM. 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 MDR / XDR-TB.

[0016] 2. The immunoassay system composed of the antigen and antibody described in this invention can be directly applied to therapeutic drug monitoring (TDM) of PA-824. In existing BPaL / BPaLM anti-tuberculosis treatment regimens, patients need to take PA-824 long-term. Its effective therapeutic concentration window is 0.5–5 μg / mL. Too low a concentration can easily lead to treatment failure, while too high a concentration may cause cardiotoxicity such as QT interval prolongation. The detection method provided by this invention can achieve rapid concentration assessment at primary health stations, community clinics, or patients' homes, guiding dose adjustment. Furthermore, since the cross-reactivity rate of the antibody of this invention with bedaquilin (BDQ) and delamanide (DLM) is less than 5%, this detection system can be integrated with the established BDQ and DLM immunoassay modules to form a "triple-drug TDMPOCT kit," which monitors the blood concentrations of the three drugs separately or simultaneously on the same detection platform, providing personalized medication basis for multidrug combination therapy. In addition, PA-824 concentration data can be used as input parameters to incorporate into QT prolongation risk prediction models, improving the early warning capability of cardiovascular safety. Attached Figure Description

[0017] Figure 1 This is a schematic diagram of the structure of the colloidal gold immunochromatographic test strip provided by the present invention. The numbers in the diagram are: 1, sample pad; 2, conjugation pad; 3, nitrocellulose membrane; 3-1, T line; 3-2, C line; 4, absorbent pad; 5, base plate. Detailed Implementation

[0018] 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.

[0019] 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.

[0020] 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 use PA-824 (CAS No. 187235-37-6) with a purity ≥98%, which was commercially available.

[0021] Example 1 The present invention provides the PA-824 hapten as shown in Formula II.

[0022] Formula II.

[0023] The preparation process of the PA-824 hapten, as shown in Formula II, is as follows: PA-824 (1.0 g, 2.79 mmol) was dissolved in anhydrous THF (20 mL), cooled to -78°C under nitrogen protection, and n-butyllithium (2.0 mL) was slowly added dropwise while stirring for 30 minutes; subsequently, a THF solution of ethyl 6-bromohexanoate (0.58 mL, excess) (5 mL) was added dropwise, and the mixture was allowed to react at room temperature for 18 hours. The reaction solution was poured into ice water (50 mL), extracted with ethyl acetate (3 × 30 mL), the organic phases were combined, dried over anhydrous sodium sulfate, and concentrated under reduced pressure to obtain the crude product. The crude product was purified by silica gel column chromatography (petroleum ether:ethyl acetate = 3:1) to obtain a white solid intermediate, PA-824-(CH2)5-COOEt (0.85 g). The hapten was dissolved in methanol (10 mL) and 1 N NaOH aqueous solution (5 mL), stirred at room temperature for 6 hours, and after the reaction was complete as monitored by TLC, the pH was adjusted to 3 with 1 N HCl, extracted with ethyl acetate, dried and concentrated, purified by column chromatography, and then further purified by chromatographic separation to obtain pure target hapten 1 (300 mg, yield 23%). 1 H NMR (400 MHz, DMSO-d6): δ12.78 (s, 1H), 8.14 (d, J=8.8 Hz, 2H), 7.51 (d, J=8.8 Hz, 2H), 7.37 (s, 1H), 5.20 (m, 2H), 4.36 (t, J=6.0 Hz, 2H), 3.81 (t, J=4.2 Hz, 2H), 3.64 (t, J=6.0Hz, 2H), 2.26 (t, J=7.2 Hz, 2H), 1.58 (m, 2H), 1.45 (m, 1H), 1.24(m, 2H); 13CNMR (100 MHz, DMSO-d6): δ 173.1, 158.2, 148.5, 138.7, 130.2, 129.8, 125.6,122.4, 112.3, 108.9, 68.5, 55.8, 33.8, 28.9, 28.7, 26.2, 24.5.

[0024] Antigen 1 was prepared using the obtained pure hapten 1. The preparation process was as follows: Bovine serum albumin (BSA, Sigma-Aldrich, catalog number A7906) was selected as the carrier protein. Specifically, 25 mg of PA-824 hapten was weighed and dissolved in 10 ml of phosphate-buffered saline (PBS) at pH 7.0–7.4. Then, 11.3 mg of 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride (EDC·HCl) and 6.8 mg of N-hydroxysuccinimide (NHS) were added. The mixture was stirred at room temperature in the dark for 30 minutes to activate the hapten-NHS active ester. Separately, 50 mg of BSA was added to 5 ml of phosphate-buffered saline (PBS) at pH 7.0–7.4. The activated hapten solution was then slowly added dropwise to the BSA solution, controlling the molar ratio of hapten to BSA to be 15:1 (based on the average molecular weight of BSA of 66 kDa). The mixture was stirred at 4°C in the dark for 12 hours. After the reaction, the mixture was transferred to a regenerated cellulose dialysis bag with a molecular weight cutoff of 10 kDa and dialyzed against PBS buffer (pH 7.4) containing 0.02% NaN3 at 4°C for 48 hours, with the dialysate changed every 6 hours for a total of 8 times. After dialysis, the solution was concentrated to approximately 2 mL using an Amicon Ultra-15 ultrafiltration centrifuge tube (10 kDa MWCO) and filtered through a 0.22 μm sterile filter membrane to obtain a clear and transparent PA-824-BSA conjugate solution with a concentration of 8.5 mg / mL. The conjugation ratio was determined by UV spectrophotometry: the average number of PA-824 hapten molecules conjugated per BSA molecule was calculated to be 12.3. 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 72.3 kDa, which is about 5.9 kDa higher than that of unmodified BSA (66.4 kDa), and is in high agreement with the theoretical increase (12.3 × 0.473 kDa ≈ 5.8 kDa).

[0025] Based on the aforementioned PA-824-BSA antigen 1, a polyclonal antibody 1 was prepared. Healthy adult male New Zealand white rabbits (weighing 2.5–3.0 kg, provided by Beijing Vital River Laboratory Animal Technology Co., Ltd.) were selected. For the initial immunization, 150 μg of PA-824-BSA conjugate was emulsified with an equal volume of Freund's complete adjuvant (Sigma-Aldrich, F5881) to a water-in-oil state and injected subcutaneously at multiple sites on the back (a total of 8 sites). A first booster immunization was performed 21 days later, using the same dose of PA-824-BSA emulsified with Freund's incomplete adjuvant (Sigma-Aldrich, F5506) and injected subcutaneously at multiple sites. Thereafter, booster immunizations were performed every 14 days, for a total of 3 booster immunizations. On day 7 after the final immunization, approximately 10 mL of blood was collected from the marginal ear vein, allowed to stand for 2 hours, and then centrifuged (3000 rpm, 10 minutes) to separate the serum. Serum was purified using a Protein A affinity chromatography column (GE Healthcare, HiTrap Protein A HP): the column was first equilibrated with binding buffer (20 mM sodium phosphate, pH 7.0), and unbound proteins were washed away with the same buffer after sample loading. The target IgG was then eluted with elution buffer (0.1 M glycine-HCl, pH 2.7), and immediately neutralized to pH 7.4 with 1 M Tris-HCl (pH 9.0). After dialysis to remove salts, purified polyclonal antibodies were obtained at a concentration of 2.8 mg / mL. SDS-PAGE showed clear heavy chain (~50 kDa) and light chain (~25 kDa) bands with no obvious contaminating proteins.

[0026] Simultaneously, monoclonal antibody 1 was prepared. Six-–8-week-old female BALB / c mice (purchased from Spiford Biotechnology Co., Ltd.) were selected, using PA-824-KLH conjugate as the immunogen (KLH conjugation method was the same as BSA, conjugation ratio was 15:1). For the first immunization, 50 μg of PA-824-KLH and Freund's complete adjuvant emulsion was injected intraperitoneally. Subsequent three booster immunizations used Freund's incomplete adjuvant, spaced 14 days apart. Three days after the final immunization, mice were sacrificed, and the spleen was aseptically removed. A single-cell suspension was prepared and mixed with SP2 / 0 myeloma cells at a 5:1 ratio. 1 mL of PEG 1500 (Roche, 50% w / v, preheated to 37°C) was added slowly with continuous stirring for 1 minute, followed by dropwise addition of preheated RPMI-1640 medium to terminate the fusion. The fusion cell suspension was seeded into 96-well plates (100 μL HAT selective medium per well) and incubated at 37°C in a 5% CO2 incubator. On day 7, indirect competitive ELISA screening was performed using PA-824-OVA (coating concentration 1 μg / mL, 100 μL / well, overnight at 4°C) as the coating antigen: 50 μL of sample containing 10 ng / mL PA-824 and 50 μL of diluted cell supernatant (1:2) were added to each well, incubated at 37°C for 1 hour, washed, and then HRP-labeled goat anti-mouse IgG (1:5000) was added. After color development, OD was read. 450 Values ​​were determined. Positive wells with inhibition rates >70% were selected for limiting dilution subcloning. This process was repeated three times to obtain a stable hybridoma cell line secreting anti-PA-824 antibody, named 3F7. After expanding the culture of 3F7 cells, some were cryopreserved in liquid nitrogen, and the rest were injected into the peritoneal cavity of syngeneic mice (pre-injected with 0.5 mL of liquid paraffin). Ascites fluid was collected 10 days later. The ascites fluid was precipitated with 45% saturated ammonium sulfate, dialyzed, and purified with Protein G to obtain the monoclonal antibody 3F7 at a concentration of 3.2 mg / mL. The IgG subtype was identified as IgG1.

[0027] The performance of the obtained polyclonal and monoclonal antibodies was evaluated. The affinity for PA-824 was determined using a competitive ELISA method: 96-well plates were coated with PA-824-OVA (1 μg / mL, 100 μL / well, overnight at 4°C). After blocking, serially diluted PA-824 standards (0.01–100 ng / mL) and fixed concentrations of antibodies (polyclonal antibody 1:2000, monoclonal antibody 1:5000) were added, and the mixture was incubated at 37°C for 1 hour. After washing, the corresponding enzyme-labeled secondary antibodies were added, and the assay was performed. Data were fitted using a four-parameter logistic regression (4-PL) algorithm, and the half-maximal inhibitory concentration (IC50) was calculated. 50 The results showed that the IC50 of the polyclonal antibody was... 50 The IC50 value of monoclonal antibody 3F7 was 2.8 ng / mL. 50The concentration was 1.3 ng / mL. Cross-reactivity was tested using the same ELISA system, with betaquine (BDQ), delamanide (DLM), moxifloxacin (MFX), linezolid (LZD), and clofazimine (CFZ) replacing PA-824 as competing agents, at a concentration of 10 μg / mL. The cross-reactivity rate (CR%) was calculated as follows: CR = (IC50 - 1) / (1) 50 of PA-824 / IC 50 The result of (of analog) × 100% is shown in Table 1.

[0028] Table 1. Cross-reactivity test results of the anti-PA-824 antibody obtained in Example 1

[0029] Data show that both antibodies are highly specific to PA-824, and their cross-reactivity with other anti-tuberculosis drugs is less than 5%, meeting the requirements for low background interference in clinical testing.

[0030] Based on the aforementioned antibodies, a colloidal gold immunochromatographic assay (LFA) detection platform was constructed. The test strip structure is as follows: Figure 1 As shown, the sample pad 1 (Whatman CFSP203000), conjugate pad 2 (glass fiber membrane, Millipore GFCP203000), nitrocellulose membrane 3 (NC membrane, Merck Hi-Flow Plus HF180), and absorbent pad 4 (Whatman AP045) are sequentially adhered to a PVC base plate 5. The conjugate pad is coated with colloidal gold-labeled monoclonal antibody 3F7: A 30 nm colloidal gold solution (trisodium citrate reduction method) was prepared, the pH was adjusted to 8.2, and monoclonal antibody 3F7 (final concentration 15 μg / mL) was slowly added. The mixture was stirred at room temperature for 30 minutes, then 10% BSA was added to a final concentration of 1%, and stirring was continued for 10 minutes. The mixture was centrifuged at 12,000 rpm for 20 minutes, the supernatant was discarded, and the precipitate was resuspended in a buffer solution containing 1% BSA, 5% sucrose, and 0.5% Tween-20 to 1 / 10 of its original volume. The precipitate was then sprayed onto the conjugate pad (spray volume 3.0 μL / cm) and dried at 37°C for 2 hours. Nitrocellulose membranes were streaked with T-line 3-1 (detection line) and C-line 3-2 (control line): T-line 3-1 was coated with PA-824-OVA conjugate (1.0 mg / mL in PBS), and C-line 3-2 was coated with goat anti-mouse IgG (0.8 mg / mL in PBS). Both were streaked using a BioDot XYZ3000 streaking apparatus at a streaking rate of 1.0 μL / cm, and dried overnight at 37°C. After assembly, the membranes were cut into 4 mm wide strips and inserted into plastic cartridges.

[0031] The LFA assay is performed as follows: Add 50 μL of the human serum sample to the sample pad and incubate at room temperature for 5–10 minutes. If the sample contains PA-824, it will compete with the colloidal gold-antibody complex for binding to PA-824-OVA on the T-line, causing the T-line intensity to decrease with increasing PA-824 concentration; the C-line remains visible to verify the effectiveness of the chromatography. Read the results visually or use a portable colloidal gold reader (such as Getein 1100) for quantification. Method validation shows that the limit of detection (LoD, defined as the concentration at which signal / noise = 3) for this LFA is 0.25 μg / mL, and the linear range is 0.3–10 μg / mL (R0). 2 = 0.987), the intra-batch coefficient of variation (n=10) was 6.2%, and the inter-batch coefficient of variation (n=3 batches) was 9.8%.

[0032] As another preferred embodiment, an immunofluorescence quantitative assay (FIA) platform was constructed. Red fluorescent microspheres (Bangs Laboratories, Catalog No. FC02F) with a particle size of 200 nm and containing carboxyl groups on their surface were used. The labeling process was as follows: 10 mg / mL of fluorescent microspheres were placed in MES buffer (pH 5.2), 2 mg / mL of EDC and 1 mg / mL of NHS were added, and the mixture was activated at room temperature for 30 minutes; the microspheres were centrifuged (15,000 rpm, 10 minutes), the supernatant was discarded, and the precipitate was resuspended in PBS (pH 7.4). Monoclonal antibody 3F7 was added to a final concentration of 0.5 mg / mL, and the mixture was rotated and coupled at 4°C for 2 hours; 10% BSA was added to block unreacted sites, and after 1 hour, the mixture was centrifuged and washed three times. Finally, the microspheres were resuspended in PBS containing 1% BSA and 0.1% ProClin 300, and the concentration was adjusted to 0.1% w / v, which was the fluorescent microsphere-antibody working solution.

[0033] FIA assay procedure: Mix 50 μL of serum sample with 50 μL of fluorescent microsphere-antibody working solution and incubate at room temperature for 5 minutes; add to a 96-well black microplate coated with PA-824-OVA (1 μg / mL, 100 μL / well, overnight at 4°C), and react at 37°C with shaking (300 rpm) for 10 minutes; wash four times with 300 μL of PBST (containing 0.05% Tween-20); read the fluorescence intensity (FIU) using a fluorescence immunoassay analyzer (excitation wavelength 532 nm, emission wavelength 610 nm). Establish a standard curve using PA-824 standards (0–10 μg / mL) and fit using 4-PL. The detection limit of this FIA method is 0.1 μg / mL, and the linear range is 0.1–10 μg / mL (R0). 2= 0.993), intra-batch CV ≤8%, inter-batch CV ≤12%. Linear regression analysis of parallel detection results from 50 clinical serum samples by liquid chromatography-tandem mass spectrometry (LC-MS / MS) yielded the equation y = 0.96x + 0.08, R0. 2 =0.935, indicating that the two have good consistency.

[0034] To verify the applicability of the detection system of this invention in therapeutic drug monitoring (TDM), a clinical relevance study was conducted. Thirty MDR-TB patients receiving the BPaL regimen (bedaquilin + PA-824 + linezolid) were selected. Serum samples were collected 2 hours after administration, and PA-824 concentrations were measured using both the FIA ​​method and LC-MS / MS methods of this invention. Results showed that the concentration range measured by the FIA ​​method was 0.42–6.85 μg / mL, and by the LC-MS / MS method, it was 0.45–7.02 μg / mL. Among these, 25 cases (83.3%) were within the effective therapeutic window (0.5–5 μg / mL), 3 cases had too low a concentration (<0.5 μg / mL), and 2 cases had too high a concentration (>5 μg / mL). The FIA ​​method can accurately identify patients requiring dose adjustment, and the detection time is only 15 minutes, much faster than LC-MS / MS (>2 hours).

[0035] Example 2 The present invention provides the PA-824 hapten as shown in Formula III.

[0036] Formula III.

[0037] The preparation process of the PA-824 hapten, as shown in Formula III, is the same as in Example 1, except that ethyl 6-bromohexanoate is replaced with ethyl 3-bromopropionate (0.55 mL, excess). After the same post-treatment and hydrolysis steps, the target hapten pure product 2 (360 mg, yield 30%) is obtained. 1 H NMR (400 MHz, DMSO-d6): δ 11.08 (s, 1H), 8.01 (d, 2H), 7.37 (s,1H), 7.31 (d, 2H), 5.21 (m, 2H), 4.35 (t, J=6.0 Hz, 2H), 3.82 (m, 1H), 3.65(t, J=6.0 Hz, 2H), 2.78 (t, J=7.2 Hz, 2H), 2.52 (m, 1H); 13C NMR (100 MHz, DMSO-d6): δ 172.8, 158.2, 148.5, 138.7, 130.2, 129.8, 125.6, 122.4, 112.3, 108.9, 68.5, 55.8, 34.2, 28.6, 26.1.

[0038] Antigen 2 was prepared using the PA-824 hapten as shown in Formula III, following the preparation process described in Example 1. The conjugation ratio was determined by ultraviolet spectrophotometry: the average number of PA-824 hapten molecules conjugated per BSA molecule was calculated to be 11.7. 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 71.5 kDa, an increase of approximately 5.1 kDa compared to unmodified BSA (66.4 kDa), which is highly consistent with the theoretical increase (11.7 × 0.431 kDa ≈ 5.04 kDa).

[0039] Based on the above PA-824-BSA antigen 2, polyclonal antibody 2 and monoclonal antibody 2 were prepared, and the preparation process is as described in Example 1.

[0040] The performance of the obtained polyclonal and monoclonal antibodies was evaluated, and the evaluation process was the same as in Example 1. The results are shown in Table 2.

[0041] Table 2. Cross-reactivity test results of the anti-PA-824 antibody obtained in Example 2

[0042] Data show that both antibodies are highly specific to PA-824, and their cross-reactivity with other anti-tuberculosis drugs is less than 5%, meeting the requirements for low background interference in clinical testing.

[0043] Example 3 The present invention provides the PA-824 hapten as shown in Formula IV.

[0044] Formula IV.

[0045] The preparation process of the PA-824 hapten, as shown in Formula IV, was the same as in Example 1, except that ethyl 6-bromohexanoate was replaced with ethyl 9-bromononanoate (0.85 mL, excess). Following the same post-treatment and hydrolysis steps, the target hapten, purified product 3 (390 mg, yield 27%), was obtained. 1H NMR (400 MHz, DMSO-d6): δ 12.40 (s, 1H), 8.23 ​​(d, J=8.8 Hz, 2H), 7.59 (d, J=8.8 Hz, 2H), 7.36 (s, 1H), 5.19 (m, 2H), 4.33 (t, J=6.0 Hz, 2H),3.80 (m, 1H), 3.63 (t, J=6.0 Hz, 2H), 2.27 (t, J=7.2 Hz, 2H), 1.56–1.46 (m,4H), 1.28–1.18 (m, 8H); 13 C NMR (100 MHz, DMSO-d6): δ 173.2, 158.2, 148.5,138.7, 130.2, 129.8, 125.6, 122.4, 112.3, 108.9, 68.5, 55.8, 33.9, 29.1,28.8, 28.6, 28.4, 26.3, 24.6.

[0046] Antigen 3 was prepared using the PA-824 hapten as shown in Formula IV, following the preparation process described in Example 1. The conjugation ratio was determined by ultraviolet spectrophotometry: it was calculated that each BSA molecule was conjugated to an average of 10.5 PA-824 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 71.9 kDa, an increase of approximately 5.5 kDa compared to unmodified BSA (66.4 kDa), which is highly consistent with the theoretical increase (10.5 × 0.515 kDa ≈ 5.41 kDa).

[0047] Based on the above PA-824-BSA antigen 3, polyclonal antibody 3 and monoclonal antibody 3 were prepared, and the preparation process is as described in Example 1.

[0048] The performance of the obtained polyclonal and monoclonal antibodies was evaluated, and the evaluation process was the same as in Example 1. The results are shown in Table 3.

[0049] Table 3. Cross-reactivity test results of the anti-PA-824 antibody obtained in Example 3.

[0050] Data show that, except for a slightly higher response to clofazimine (CFZ), the cross-reactivity rates of both antibodies to PA-824 against other anti-tuberculosis drugs are both below 5%, which basically meets the requirements of clinical testing for low background interference.

[0051] Example 4

[0052] The present invention provides the PA-824 hapten as shown in Formula V.

[0053] Formula V.

[0054] The preparation process of the PA-824 hapten, as shown in Formula V, is as follows: PA-824 (1.0 g, 2.79 mmol) was dissolved in anhydrous DMF (15 mL), potassium tert-butoxide (0.28 g) was added, and the mixture was stirred at 0°C for 30 minutes; a DMF solution of N-Boc-6-aminohexyl bromide (1.2 g, excess) was added dropwise, and the mixture was allowed to react at room temperature for 20 hours. The post-treatment was the same as before, yielding a Boc protected intermediate (0.92 g). This intermediate was dissolved in dichloromethane (10 mL), trifluoroacetic acid (2 mL) was added, and the mixture was stirred at room temperature for 2 hours. The solvent was removed under reduced pressure, and the residue was precipitated with diethyl ether. The precipitate was filtered, purified by column chromatography, and further purified by chromatographic separation to obtain PA-824-(CH2)5-NH2 (550 mg, total yield 44%). 1 H NMR (400 MHz, D2O): δ8.07 (d, J=8.8 Hz, 2H), 7.44 (d, J=8.8 Hz,2H), 7.29 (d, 1H), 5.14 (m, 2H), 4.27 (t, J=6.0 Hz, 2H), 3.74 (d, 1H), 3.57(t, J=6.0 Hz, 2H), 2.85 (t, J=7.2 Hz, 2H), 1.62 (m, 2H), 1.52(m, 2H),1.25 (m,2H); 13 C NMR (100 MHz, D2O): δ 158.5, 148.8, 139.0, 130.5, 130.1, 125.9, 122.7,112.6, 109.2, 68.8, 56.1, 38.5, 29.3, 29.1, 26.5, 24.8.

[0055] Antigen 4 was prepared using the PA-824 hapten as shown in Formula V. The preparation process was as follows: 25 mg of hapten 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 the PA-824-BSA antigen 4 was obtained after purification. The coupling ratio was determined by ultraviolet spectrophotometry: it was calculated that an average of 13.5 PA-824 hapten molecules were coupled to each BSA molecule. 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 72.3 kDa, which was about 5.9 kDa higher than that of unmodified BSA (66.4 kDa), and was in high agreement with the theoretical increase (13.5 × 0.444 kDa ≈ 6.0 kDa).

[0056] Based on the above PA-824-BSA antigen 4, polyclonal antibody 4 and monoclonal antibody 4 were prepared, and the preparation process is as described in Example 1.

[0057] The performance of the obtained polyclonal and monoclonal antibodies was evaluated, and the evaluation process was the same as in Example 1. The results are shown in Table 4.

[0058] Table 4. Cross-reactivity test results of the anti-PA-824 antibody obtained in Example 4

[0059] Data show that both antibodies are highly specific to PA-824, and their cross-reactivity with other anti-tuberculosis drugs is less than 5%, meeting the requirements for low background interference in clinical testing.

[0060] Example 5 The present invention provides the PA-824 hapten as shown in Formula VI.

[0061] Formula VI.

[0062] The preparation process of the PA-824 hapten, as shown in Formula VI, is as follows: PA-824 (1.0 g, 2.34 mmol) was dissolved in anhydrous THF (20 mL), and n-butyllithium (1.0 mL) was added dropwise at -78 °C. After stirring for 30 minutes, a THF solution of 6-acetylthiohexyl bromide (0.88 g, excess) was added dropwise, and the reaction was carried out at room temperature for 18 hours. The acetyl-protected intermediate (0.88 g) was obtained after post-treatment. This intermediate was dissolved in methanol (10 mL) and concentrated ammonia (2 mL), stirred at room temperature for 4 hours, concentrated under reduced pressure, and the residue was recrystallized from ethyl acetate. After filtration, column purification and further purification by chromatography yielded PA-824-(CH2)5-SH (310 mg, total yield 24%).1 H NMR (400 MHz, DMSO-d6): 8.08 (d, J =8.8 Hz, 2H), 7.45 (d, J =8.8 Hz, 2H), 7.30 (s, 1H), 5.15 (m, 2H), 4.30 (t, J=6.0 Hz, 2H), 3.75 (d, 1H), 3.58(t, J =6.0 Hz, 2H), 2.78 (t, J=7.2 Hz, 2H), 1.55(m, 2H), 1.49(t, J=7.0 Hz,2H), 1.43 (m, 1H), 1.25 (m, 2H); 13 C NMR (100 MHz, DMSO-d6): δ 158.5, 148.8,139.0, 130.5, 130.1, 125.9, 122.7, 112.6, 109.2, 68.8, 56.1, 32.8, 29.0,28.9, 26.4, 24.7.

[0063] Antigen 5 was prepared using the PA-824 hapten as shown in Formula VI. The preparation process was as follows: 50 mg of BSA was activated with N-γ-maleimide butyryloxysulfonyl-succinimide ester (sulfo-GMBS) in PBS at pH 7.0 at room temperature for 1 hour to form maleimide-activated protein; then 25 mg of the obtained PA-824-(CH2)5-SH was added, and the reaction was carried out at 4°C in the dark for 12 hours. The thiol group underwent a Michael addition reaction with the maleimide ring to form a stable thioether bond. After the reaction, PA-824-BSA antigen 5 was obtained by dialysis purification. The coupling ratio was determined by ultraviolet spectrophotometry: the average number of PA-824 hapten molecules coupled to each BSA molecule was calculated to be 11.3. Furthermore, matrix-assisted laser desorption / ionization time-of-flight mass spectrometry (MALDI-TOFMS) analysis showed that the main peak molecular weight of the conjugate was approximately 71.1 kDa, which is about 4.7 kDa higher than that of unmodified BSA (66.4 kDa), and is in high agreement with the theoretical increase (12.3 × 0.416 kDa ≈ 4.7 kDa).

[0064] Based on the above PA-824-BSA antigen 5, polyclonal antibody 5 and monoclonal antibody 5 were prepared, and the preparation process is as described in Example 1.

[0065] The performance of the obtained polyclonal and monoclonal antibodies was evaluated, and the evaluation process was the same as in Example 1. The results are shown in Table 5.

[0066] Table 5. Cross-reactivity test results of the anti-PA-824 antibody obtained in Example 5.

[0067] Data show that both antibodies are highly specific to PA-824, and their cross-reactivity with other anti-tuberculosis drugs is less than 5%, meeting the requirements for low background interference in clinical testing.

[0068] 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 PA-824 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 A is one of -COOH, -NH2, and SH.

2. The method for preparing the PA-824 hapten as described in claim 1, characterized in that, The preparation steps are as follows: PA-824 is dissolved in anhydrous tetrahydrofuran or anhydrous N,N-dimethylformamide. Under inert gas protection, n-butyllithium or potassium tert-butoxide is added. The benzyl position of the benzyl side chain of PA-824 is deprotonated at -78℃ to 0℃ to generate a benzyl carbanion intermediate. Then, an appropriate derivative of ω-halocarboxylic acid ester, ω-haloamine hydrochloride or ω-halothiol is added dropwise to the reaction system, wherein the halogen is bromine or iodine, and the carbon chain length corresponds to n=2~8. The reaction temperature is raised to room temperature and stirred continuously for 12~24h. After the reaction is completed, the target hapten is obtained by water quenching, organic phase extraction and column chromatography purification. The active group A is deprotected and further purified to obtain the target hapten pure product.

3. The use of the PA-824 hapten as described in claim 1 in the preparation of PA-824 antigen.

4. A PA-824 antigen, characterized in that, The PA-824 antigen is obtained by conjugating the PA-824 hapten with a carrier protein as described in claim 1, wherein the carrier protein is one of bovine serum albumin, ovalbumin, or keyhole hemocyanin.

5. The use of the PA-824 antigen as described in claim 4 in the preparation of PA-824 antibody.

6. An antibody against PA-824, characterized in that, The antibody is prepared by immunizing animals with the PA-824 antigen as described in claim 4, and the antibody is a polyclonal antibody or a monoclonal antibody; the polyclonal antibody is obtained by immunizing rabbits with PA-824-bovine serum albumin conjugate as an immunogen; the monoclonal antibody is obtained by immunizing mice with PA-824-keyhole hemocyanin conjugate as an immunogen, followed by cell fusion, screening and subcloning to obtain hybridoma cell line 3F7, which is then secreted by this cell line.

7. The application of the PA-824 antigen as described in claim 4 and the anti-PA-824 antibody as described in claim 6 in a method for monitoring PA-824 therapeutic drugs, characterized in that, The PA-824 therapeutic drug monitoring method is a competitive immune detection method.

8. A kit for detecting PA-824, characterized in that, It includes the PA-824 antigen as described in claim 4 and the anti-PA-824 antibody as described in claim 6.

9. A colloidal gold immunochromatographic test strip for detecting PA-824, characterized in that, The test strip consists of a base plate, a sample pad, a conjugate pad, a nitrocellulose membrane, and an absorbent pad sequentially adhered to the base plate. The conjugate pad is coated with a colloidal gold-labeled antibody, which is the anti-PA-824 antibody as described in claim 6, with a colloidal gold particle size of 30 nm and a labeling density of 15 μg gold particles bound per microgram of antibody. The nitrocellulose membrane has T lines and C lines, wherein the T lines are coated with the PA-824 antigen as described in claim 4 at a coating concentration of 1.0 mg / mL and a streaking volume of 1.0 μL / cm; the C lines are coated with goat anti-rabbit IgG or goat anti-mouse IgG at a coating concentration of 0.8 mg / mL and a streaking volume of 1.0 μL / cm.

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