A kit for detecting the ratio of proBDNF to mature BDNF based on structure dynamics and its preparation method.

Abstract

The application belongs to the technical field of immune detection, and discloses a kit for proBDNF and mature BDNF ratio detection based on structural dynamics orientation and a preparation method thereof. The kit comprises a solid carrier and a detection reagent, and is configured with three antibodies determined based on brain-derived neurotrophic factor full-length conformation dynamics analysis: a first capture antibody targeting an inherent disordered region (SEQ ID NO: 1) of a proBDNF propeptide region; a second capture antibody, which is a neoepitope specific antibody, specifically binds to an N terminal (SEQ ID NO: 2) exposed by mature BDNF enzyme cutting, and the binding is strictly dependent on a free alpha-amino group of the first histidine; and a universal detection antibody combined with a Loop 4 region (SEQ ID NO: 3) of a mature domain. The application solves the problems of spatial steric hindrance and cross reaction in traditional immune detection through a structural biology strategy, and realizes accurate differentiation and ratio quantification of proBDNF and mature BDNF.

Description

Technical Field

[0001] This invention relates to the field of immunoassay technology, and in particular to a kit for detecting the ratio of proBDNF to mature BDNF based on structure dynamics and its preparation method. Background Technology

[0002] Brain-derived neurotrophic factor (BDNF), a core member of the neurotrophic factor family, plays a crucial role in nervous system development and kidney function. Its biosynthetic pathway is not straightforward: BDNF is initially synthesized as a precursor form (proBDNF) with a molecular weight of approximately 32 kDa. It then requires cleavage by intracellular or extracellular proteases to remove the N-terminal propeptide domain, transforming it into its mature form (mature BDNF) with a molecular weight of approximately 14 kDa.

[0003] Today, mounting evidence suggests that proBDNF and mature BDNF are not simply precursor and product, but rather exert diametrically opposed biological effects by binding to different receptors: mature BDNF specifically binds to the TrkB receptor, promoting neuronal survival and long-term potentiation; while proBDNF binds to the p75NTR receptor with high affinity, inducing apoptosis and long-term inhibition. This "yin-yang balance" mechanism maintains the body's physiological homeostasis.

[0004] Traditionally, it was believed that only mature BDNF was secreted into the extracellular matrix to exert its effects. However, groundbreaking research over the past decade has revised this established understanding: in fact, both proBDNF and mature BDNF can be released into the extracellular space in mixed forms and are widely present in bodily fluids such as blood and urine. This means that two forces simultaneously exist in the bodily fluid environment: one promoting growth and the other inducing apoptosis.

[0005] Based on the above findings, simply detecting the "total amount of BDNF" clearly masks the antagonistic relationship between the two functions and cannot meet the needs of accurate diagnosis. An imbalance in the ratio of proBDNF to mature BDNF often foreshadows pathological conditions such as depression, anxiety, Alzheimer's disease, and overactive bladder. Therefore, shifting the focus from "total amount" to the "ratio" of the two, used to assess the protease cleavage efficiency of BDNF precursors in subjects, has become a more crucial and precise biological indicator for assessing the progression of neurological, psychiatric, and urinary system diseases.

[0006] Existing immunoassay technologies face two major challenges: first, the "steric hindrance effect," where the prodomain of proBDNF randomly blocks antigen epitopes in mature regions, leading to a non-linear decrease in the detection signal; and second, "cross-reaction," where traditional antibodies cannot distinguish between the uncut internal sequence in proBDNF and the exposed N-terminal sequence in mature BDNF, making it impossible to accurately determine the ratio between the two.

[0007] No effective solutions have yet been proposed to address the problems in the relevant technologies. Summary of the Invention

[0008] This invention provides a kit and its preparation method for detecting the ratio of proBDNF to mature BDNF based on structure dynamics, in order to solve the problems of the above-mentioned technologies in the prior art.

[0009] The purpose of this invention is to provide a structure-dynamics-guided proBDNF to mature BDNF ratio detection kit. This invention overcomes the limitations of traditional blind antibody screening by employing "structural biology-guided epitope mapping" technology. Through a combination of software 3D reconstruction, predictive alignment error (PAE) matrix analysis, and kinetic simulation experiments, it identifies three key "golden antigen sites," thus solving the problems of steric hindrance and cross-reactivity from a physicochemical perspective.

[0010] I. Antigen Site Assessment and Screening Based on Structural Biology

[0011] This invention constructs a full-length BDNF structure (UniProt P23560) model based on the AlphaFold2 algorithm, and, combined with PAE matrix and molecular dynamics (MD) simulations, establishes the following core screening strategies and antigen sites:

[0012] 1) "Anti-occlusion" screening of general detection sites:

[0013] Structural Analysis: This invention constructs a high-precision full-length 3D model of BDNF. Unlike using only static structures, this invention deeply analyzes the PAE matrix and plots a "dynamic steric hindrance thermogram" by analyzing the interaction energy between the Prodomain (positions 19-128) and the Mature Domain (positions 129-247). The analysis shows that although the Loop 1 and Loop 2 regions of the Mature Domain have high immunogenicity, they are blocked by the Prodomain by up to 80% (i.e., "binding forbidden zones").

[0014] Screening Results: By performing molecular dynamics simulations on the protein model for up to 100 ns and calculating the accessible surface area (SASA), this invention found that Loop 4 (positions 216-224) near the C-terminus of the Mature Domain consistently has a SASA value >50% and a minimum distance >15 Å from the Prodomain. It is not located within the hydrophobic collapse coverage region of the propeptide domain, indicating it is a "constant exposure region." Simultaneously, the RMSF value of the backbone atoms in this region is less than 2.0 Å, demonstrating its rigid conformation in solution, which is beneficial for high-affinity antibody binding. Furthermore, the difference in dissociation constant (Kd1) between the universal detection antibody and the proBDNF antigen and the matureBDNF antigen is <3-fold (0.33 < Kd1 / Kd2 < 3.0), thus eliminating nonlinear detection bias caused by antigen conformational differences.

[0015] Sequence determination: SEQ ID NO: 3 (DSKKRIGWR) was selected as the target for the universal detection antibody. This region remains exposed in both proBDNF and mature BDNF, ensuring consistency in the signal responses of both.

[0016] 2) Confirmation of the conformation of the "new epitope" in mature BDNF:

[0017] Structural Analysis: For specific detection of mature BDNF, this invention focuses on evaluating the surface electrostatic potential changes before and after the cleavage of the restriction enzyme site (R128-H129). Simulation results show that in proBDNF, H129 is buried by the side chain of R128 and the surrounding negatively charged cavity; however, after cleavage, H129 becomes a new N-terminus, and its α-amino group is protonated, forming a unique positively charged pocket.

[0018] Screening criteria: Based on this structural feature, the present invention limits the antibodies used to specifically recognize this free N-terminal structure, and the binding energy must be highly dependent on the hydrogen bond network and charge characteristics of the free N-terminus.

[0019] Sequence determination: Select SEQ ID NO: 2 (HSDPARRGEL) and require the antibody to recognize the free α-amino group that depends on the first histidine to distinguish the uncleaved precursor.

[0020] 3) Screening for precursor-specific sites:

[0021] Structural analysis: Referring to the aforementioned scheme, by analyzing the degree of disorder of the Prodomain, this invention identifies the region at the very front of the Prodomain.

[0022] Sequence identification: SEQ ID NO: 1 (APMKEANIRG) was selected. This region was identified as a highly hydrophilic intrinsically disordered region (IDR) that extends significantly outward in spatial conformation and is highly susceptible to antibody capture.

[0023] II. Reagent Kit Construction Strategy

[0024] Using the antibody pairings obtained by SEQ ID NO: 1~3 above, the kits constructed in this invention are selected from conventional immunoassay platforms such as fluorescence immunochromatography, quantum dot immunoassay, ELISA, magnetic microparticle chemiluminescence reagents, or colloidal gold test strips.

[0025] Taking the colloidal gold lateral immunochromatography platform as an example, the construction strategy of this invention is as follows:

[0026] T1 line: coated with anti-SEQ ID NO: 1 antibody, utilizing the high exposure of IDR to achieve specific capture of proBDNF.

[0027] T2 line: Coated with a novel epitope antibody against SEQ ID NO: 2, which utilizes recognition dependence on the N-terminal free α-amino group to achieve specific capture of free mature BDNF.

[0028] Gold-labeled pad: Labeled with anti-SEQ ID NO: 3 antibody, utilizing the constant exposure characteristics of Loop 4 region to achieve binding to all molecules containing mature region sequences, unaffected by steric hindrance.

[0029] Process: The test strips are made by cutting, dicing, and assembling the film.

[0030] III. Beneficial Effects

[0031] Compared with the prior art, the technical solutions provided by the embodiments of the present invention may include the following beneficial effects:

[0032] 1) This invention solves the problems of steric hindrance and cross-reactivity in traditional immunoassay by using structural biology strategies, and achieves accurate differentiation and ratio quantification of proBDNF and mature BDNF.

[0033] 2) True Quantification: By selecting the Loop 4 exposure area as a universal detection site, this invention effectively avoids signal suppression caused by Prodomain occlusion, ensuring that proBDNF and mature BDNF have consistent colorimetric intensity at equimolar concentrations, thus solving the quantitative deviation caused by steric hindrance in traditional detection.

[0034] 3) Avoid cross-reaction: By using a "new epitope" antibody screening strategy based on conformational changes at enzyme cleavage sites (dependent on free α-amino), direct and specific capture of mature BDNF is achieved, avoiding non-specific binding of antibodies to uncleaved proBDNF. Compared with the traditional "subtract after measuring total value" scheme, the detection accuracy is significantly improved.

[0035] 4) Direct reading of the ratio and tolerance to the sample environment: The kit of this invention can be used with a reader to directly output the pro / mature ratio; at the same time, the antibody epitopes based on structure screening have strong rigidity and stability, and with the optimized buffer system, the detection deviation caused by sample pH fluctuations is effectively corrected, making it suitable for the detection of complex clinical samples.

[0036] It should be understood that the above general description and the following detailed description are exemplary and explanatory only, and are not intended to limit the invention. Attached Figure Description

[0037] The accompanying drawings, which are incorporated in and form part of this specification, illustrate embodiments consistent with the invention and, together with the description, serve to explain the principles of the invention.

[0038] Figure 1 This is a three-dimensional structural model of the BDNF protein illustrated according to an exemplary embodiment;

[0039] Figure 2 This is a PAE matrix heatmap of the BDNF protein according to an exemplary embodiment;

[0040] Figure 3 This is a photograph of the C-line, T1, and T2 lines after testing a real sample, according to an exemplary embodiment. Detailed Implementation

[0041] The following description and accompanying drawings fully illustrate specific embodiments described herein to enable those skilled in the art to practice them. Some portions and features of certain embodiments may be included in or replace portions and features of other embodiments. The scope of the embodiments herein includes the entire scope of the claims and all available equivalents thereof. The various embodiments described herein are presented in a progressive manner, with each embodiment focusing on its differences from other embodiments; similar or identical parts between embodiments can be referred to interchangeably.

[0042] Where there is no conflict, the embodiments and features in the embodiments of the present invention can be combined with each other.

[0043] Example 1: Establishment of an antibody screening and evaluation system based on structural biology

[0044] This embodiment describes how to construct a rational screening and evaluation system for antibody targets based on conformational stability using bioinformatics before formal antibody screening is implemented.

[0045] 1.1) Sequence and Structure Modeling: Based on the full-length human BDNF sequence (P23560), de novo prediction of the quaternary structure was performed using ColabFold (based on the AlphaFold2-Multimer algorithm) to construct a high-fidelity model, thus obtaining the three-dimensional structural model of the BDNF protein, such as... Figure 1 As shown, Figure 1 In the diagram, A, B, and C represent different viewing angles of the same model. Figure 1 The orange and yellow areas shown in A represent the Prodomain, which obscures the blue Mature BDNF region. Figure 1 The orange / brown spaced portion shown in B represents the signal peptide recognized by the BDNF protein used for cell transport, while the dark brown portion represents the prodomain of the BDNF protein. Figure 1 The green portion shown in C represents the key site where BDNF protein exerts its neurotrophic effect; the ESMFold algorithm is introduced for cross-validation to generate a PAE matrix (e.g., Figure 2 As shown in the figure, the rigid and flexible boundaries between structural domains are precisely defined.

[0046] 1.2) Quantification of spatial mapping of dynamic steric hindrance: The PyMOL system is used to perform interface topology analysis on the model. By calculating the buried surface area and the contact frequency of interface residues, a "dynamic steric hindrance thermodynamic analysis" is established to quantify the spatial shielding efficiency of the Prodomain on the MatureDomain, thereby delineating the "binding forbidden zone" of the antibody.

[0047] 1.3) Surface Accessibility Feature Mapping: For each candidate loop region, SASA and RMSF are calculated based on GROMACS kinetic simulation data. A scoring model based on "thermodynamic stability" and "conformational constancy" is established to optimize and rank potential epitopes.

[0048] 1.4) Optimal binding target locking

[0049] Based on the above multidimensional conformation evaluation system, three sets of characteristic targets were identified:

[0050] Universal anchoring target (SEQ ID NO: 3): DSKKRIGWR located in Loop 4 region. This region is conformationally independent and appears as a “constantly high exposure state” in SASA analysis, ensuring a linear response for full-form BDNF detection.

[0051] Mature-specific novel epitope (SEQ ID NO: 2): HSDPARRGEL located on the break surface. This is a "cleavage-dependent novel epitope," and its immunogenicity is strictly dependent on the protonation pocket of the free α-amino group at His129.

[0052] Precursor-specific capture target (SEQ ID NO: 1): APMKEANIRG located at the N-terminus. Identified as a highly hydrophilic IDR, its high conformational entropy enables sensitive capture of precursor molecules.

[0053] Example 2: Construction and screening of antibody libraries based on conformation specificity

[0054] This embodiment details how to obtain three monoclonal antibodies that meet the design requirements of "structure dynamics-guided" design through a specific positive and negative screening strategy.

[0055] 2.1) Antigen preparation

[0056] Immunogen: The polypeptides SEQ ID NO: 1 (APMKEANIRG), SEQ ID NO: 2 (HSDPARRGEL) and SEQ ID NO: 3 (DSKKRIGWR) were artificially synthesized, and cysteine ​​residues (Cys) were added to their ends and coupled with KLH protein as immunogens.

[0057] Antigen selection: The above three peptides are conjugated to BSA (for positive screening). Recombinant human proBDNF full-length protein (eukaryotic expression, maintaining native fold conformation, used for negative screening and cross-validation). Recombinant human mature BDNF protein (used for affinity validation).

[0058] 2.2) Immunization and Fusion: Following the conventional monoclonal antibody preparation process, mice were immunized with immunogens, and high-titer mouse spleen cells were selected and fused with myeloma cells to obtain a hybridoma cell bank.

[0059] 2.3) “Structure Locking” Screening Strategy

[0060] This invention implements a targeted screening process to eliminate non-specific antibodies that cannot distinguish conformations:

[0061] (1) Screening for antibodies against T2 line (novel anti-mature BDNF epitopes):

[0062] Initial screening (positive): Select positive wells that can bind to the polypeptide of SEQ ID NO: 2.

[0063] Secondary screening (negative elimination): Since the sequence of SEQ ID NO: 2 is also present in the full-length proBDNF protein (located at the R128-H129 junction, but in an internal, unexposed state or with the H129 α-amino group involved in peptide bond formation), specificity verification is required.

[0064] Methods: The supernatant of the antibody to be tested was pre-incubated with excess recombinant proBDNF protein (10 μg / mL) and the synthetic polypeptide of SEQ ID NO: 2 for 30 minutes.

[0065] Judgment criteria: If the signal is blocked by the SEQ ID NO: 2 peptide (signal decrease >80%), but not by the proBDNF protein (signal decrease <5%), then the antibody is determined to specifically recognize the "exposed free N-terminus cleaved," and the clone (mAb-Mat-Neo) is retained. If the signal is blocked by the proBDNF protein at the same time, it indicates that the antibody recognizes a side chain epitope (internal sequence), and it is discarded.

[0066] Results: A monoclonal antibody mAb-Mat-Neo was obtained that specifically recognizes mature BDNF and has no cross-reactivity with proBDNF.

[0067] (2) Screening for gold-labeled antibodies (universal test):

[0068] Screening objective: To find antibodies that are not affected by propeptide steric hindrance.

[0069] Methods: A double-antigen sandwich ELISA method was used, in which microplates were coated with equimolar concentrations of recombinant proBDNF and mature BDNF, respectively.

[0070] Judgment criteria: Calculate the binding affinity (EC50) of the antibody to the two antigens. Only clones with an EC50 (pro) and EC50 (mature) difference of less than 15% are retained, demonstrating that the recognized Loop 4 epitope is in a "constant exposure state" in both conformations.

[0071] Result: A universal monoclonal antibody, mAb-Uni-L4, was obtained.

[0072] (3) Screening for T1 line antibodies (anti-proBDNF):

[0073] Methods: Screen clones that bind to SEQ ID NO: 1 but do not bind to mature BDNF.

[0074] Result: The proBDNF-specific antibody mAb-Pro-IDR was obtained.

[0075] 2.4) Conclusion: Through the above-mentioned design and screening based on structure dynamics, the present invention successfully obtained three core antibodies (mAb-Pro-IDR, mAb-Mat-Neo, mAb-Uni-L4).

[0076] Example 3: Performance Comparison and Verification of Colloidal Gold Test Strips

[0077] This embodiment verifies the beneficial effects of the kit of the present invention in eliminating steric hindrance and avoiding cross-reactions by comparing it with the prior art (comparative reagent).

[0078] 3.1) Assembly of test strips

[0079] T1 line: Draw mAb-Pro-IDR (1 mg / mL).

[0080] T2 line: Draw mAb-Mat-Neo (1 mg / mL).

[0081] C line: Sheep anti-mouse IgG.

[0082] Bonding pad: mAb-Uni-L4 with spray-coated colloidal gold markings.

[0083] Comparison reagent group setup:

[0084] Comparative reagent group 1 (steric hindrance group): The gold-labeled antibody was replaced with a commercially available antibody that recognizes the Loop 2 region (which is shown to be obscured by the propeptide in the simulation). The remaining components are consistent with the present invention.

[0085] Comparative reagent group 2 (cross-reaction group): The T2 line antibody was replaced with a conventional anti-BDNF N-terminal antibody (recognizing the SEQ ID NO: 2 sequence, but not filtered out by proBDNF negative screening, i.e., recognizing the side chain). The remaining components are consistent with the present invention.

[0086] 3.2) Performance comparison test results: In order to intuitively demonstrate the technical advantages of the present invention, the laboratory constructed simulated urine samples with different ratios for testing, and the results are shown in Table 1 below.

[0087] Table 1. Comparison of detection results of different reagent kits on simulated urine samples.

[0088] Test Project Sample composition (simulated urine) The reagent kit of this invention (detection results) Comparative reagent group 1 (steric hindrance effect group) Comparative reagent group 2 (cross-reaction group) Results Analysis and Advantages Cross-reactivity test (specificity) Pure proBDNF (100 ng / mL) T1: ++ (strong) T2: - (negative) T1: + (weak) T2: - (negative) T1: ++ T2: + (False positive) Advantages of this invention: The T2 line shows no color development, proving that mAb-Mat-Neo strictly relies on the free N-terminus, completely solving the false positive problem caused by proBDNF. In contrast, control group 2 could not distinguish the precursor. Tinctuation shielding test (quantitative accuracy) Pure proBDNF (100 ng / mL) T1 signal value: 1520 (reference value) T1 signal value: 850 (signal suppression approximately 40%) T1 signal value: 1405 Advantages of this invention: The T1 signal of this invention is normal; in control group 1, the gold-labeled antibody (Loop 2) was blocked by the propeptide, resulting in missed detection or underestimation of proBDNF concentration. Ratio accuracy (core function) 1:1 mixture (50 ng / mL proBDNF + 50 ng / mL mature) The T1 / T2 signal ratio is approximately 1.22 (close to the theoretical value of 1.0). The T1 / T2 signal ratio is approximately 0.61 (significantly off). The T1 / T2 signal ratio is approximately 0.44 (significantly off). Advantages of this invention: This invention can accurately reflect the molar ratio of the two components. In comparison group 1, the ratio calculation was incorrect due to the weak color rendering of proBDNF; in comparison group 2, the proBDNF signal was superimposed on the T2 line, resulting in a significant error. Low sensitivity Low concentration mixture (2 ng / mL proBDNF + 2 ng / mL mature) Bands are visible at both T1 and T2. T1 band not visible (missed detection) Both T1 and T2 are visible. Advantages of this invention: Due to the selection of the Loop 4 site with a high SASA value, the antigen-antibody binding efficiency can be guaranteed even at low concentrations, and the sensitivity is better than that of the control group 1 with severe steric hindrance.

[0089] 3.3) Conclusion: Experimental data show that the kit of the present invention, through the "three-point structure locking" strategy, finally achieves accurate quantification of proBDNF and mature BDNF and their ratio in urine samples, which is significantly better than the existing technical solutions.

[0090] Example 4: Preparation process of colloidal gold test strips

[0091] This embodiment briefly describes the production process of the reagent kit.

[0092] 4.1) Preparation of colloidal gold universal antibody conjugates

[0093] Preparation of colloidal gold: Take 100 mL of 0.01% chloroauric acid solution, heat to boiling, quickly add 1.5 mL of 1% trisodium citrate solution, continue boiling for 15 minutes until the color changes, cool to room temperature, and obtain a 40 nm colloidal gold solution.

[0094] Conjugation: The pH of the colloidal gold solution was adjusted to 8.5 using 0.2 M K₂CO₃. The monoclonal antibody against SEQ ID NO:3 was slowly added with stirring, and the reaction was allowed to proceed for 30 minutes.

[0095] Blocking and stabilization: Add 1% BSA and 0.1% PEG2000 to a final concentration and stir for 30 minutes.

[0096] Centrifugation: Centrifuge at 10,000 rpm for 45 minutes and discard the supernatant. Resuspend the precipitate in Tris buffer (pH 8.2) containing 1% BSA, 5% sucrose, and 0.1% Tween 20, and adjust the OD.

[0097] Spraying: Spray the suspension onto the glass fiber cotton, dry at 37°C for 4 hours, and cut into strips to serve as bonding pads.

[0098] 4.2) Preparation of nitrocellulose membrane (NC membrane)

[0099] 4.2.1) Preparation of the film-drawing solution:

[0100] T1 coating solution: Capture antibody against SEQ ID NO: 1, concentration 1.5 mg / mL.

[0101] T2 coating solution: Capture antibody against the novel epitope of SEQ ID NO: 2, at a concentration of 1.5 mg / mL.

[0102] C. Dilation solution: Goat anti-mouse IgG antibody, concentration 1.0 mg / mL.

[0103] 4.2.2) Scribing: Use a scribing instrument to scribing the above solutions onto the NC membrane.

[0104] 4.2.3) Drying: Place at 37℃ and dry for 12 hours.

[0105] 4.3) Cutting and Assembly: On the PVC base plate, attach the sample pad, the prepared bonding pad, the scribed NC membrane, and the absorbent pad in sequence. Use a strip cutter to cut the assembly plate into test strips, pack them into plastic cartridges, and seal them in aluminum foil bags with desiccant inside.

[0106] Example 5: Urine Sample Detection and Data Processing Methods

[0107] This embodiment briefly describes the detection process of the reagent kit.

[0108] 5.1) Sample pretreatment: Take 100 μL of the urine sample to be tested and add 100 μL of sample processing solution (containing 0.2 M HEPES, pH 7.5, 1% CHAPS). Mix well and let stand for 1 minute.

[0109] 5.2) Detection procedure: Add the mixture dropwise to the sample well of the test strip. Lay flat and perform chromatography at room temperature for 10-15 minutes.

[0110] 5.3) Result interpretation and calculation: Use a colloidal gold reader to read the light reflection intensity of lines T1 and T2.

[0111] T1 line signal: represents proBDNF concentration.

[0112] T2 line signal: represents the concentration of free Mature BDNF.

[0113] Calculation: The instrument has a built-in standard curve that automatically converts the values ​​into concentrations (ng / mL), specifically:

[0114] Set the ratio R = V1 / V2, where V1 represents the molar concentration calculated by substituting the signal values ​​of the control line and the second detection line into the standard curve, and V2 represents the molar concentration calculated by substituting the signal values ​​of the control line and the first detection line into the standard curve.

[0115] Significance: The ratio R is used to assess the protease cleavage efficiency of BDNF precursors in the subject's body. The R value in normal human urine is usually >1.2; if R < 0.5, it suggests damage to renal podocytes or severe depression (precursor conversion is impaired).

[0116] Example 6: Evaluation of BDNF precursor conversion efficiency based on clinical urine samples

[0117] This embodiment aims to verify the performance of the kit in actual clinical sample testing, particularly its ability to distinguish between healthy and abnormal states by calculating the mature BDNF / proBDNF ratio (R value). (Note: This embodiment is only used to verify the detection capability of the kit and does not involve the diagnosis and treatment of diseases.)

[0118] 6.1) Sample Collection and Grouping

[0119] With ethical approval, 30 morning urine samples were collected and divided into three groups:

[0120] Group A (healthy control group): 10 cases, with no history of neuropsychiatric diseases or kidney disease, and normal physical examination indicators.

[0121] Group B (Major Depression Group): 10 cases, meeting the DSM5 diagnostic criteria, HAMD17 score >24, and not taking antidepressants.

[0122] Group C (nephropathy group): 10 cases, kidney podocyte injury.

[0123] All samples were centrifuged immediately after collection, and the supernatant was frozen and stored at -80°C for later testing.

[0124] 6.2) Testing procedure: Strictly follow the steps described in Example 5;

[0125] 6.3) Data recording and typical result display: Strictly follow the steps described in Example 5;

[0126] To visually demonstrate the differences in characteristics between different groups, this embodiment randomly selects two typical samples from each group (e.g., Figure 3 As shown, Figure 3 Each colloidal gold test strip consists of a C line, a T1 line, and a T2 line. The two colloidal gold test strips shown in area A represent examples of results for group A; the two colloidal gold test strips shown in area B represent examples of results for group B; and the two colloidal gold test strips shown in area C represent examples of results for group C. Their data are recorded in Table 2 below:

[0127] Table 2 Comparison of measured results for the three groups of samples

[0128] Sample number T1 line measurement (proBDNF) T2 line measurement (mature BDNF) The ratio R (T2 / T1) Result determination A-03 15.2 48.6 3.20 High conversion efficiency (normal) A-09 22.1 55.4 2.51 High conversion efficiency (normal) B-01 68.3 20.5 0.30 Conversion blocked (abnormal) B-04 55.4 25.1 0.45 Conversion blocked (abnormal) C-03 82.6 31.4 0.37 Conversion blocked (abnormal) C-08 75.1 28.9 0.38 Conversion blocked (abnormal)

[0129] 6.4) Statistical analysis of the overall sample

[0130] Statistical analysis was performed on all 30 collected samples:

[0131] Group A (healthy): The T1 line signal was low, while the T2 line signal was significant, with a mean ratio R of 2.85 ± 0.42. This is consistent with the structural biology prediction that "under normal physiological conditions, the enzyme cleavage sites are fully exposed, and the precursor is efficiently cleaved into the mature form."

[0132] Group B (Disease): Compared with Group A, the T1 line reading in Group B samples was significantly increased (P<0.01), while the T2 line reading was relatively decreased, resulting in a significant reversal of the ratio R (mean R value <0.5). This verifies that the kit of the present invention can sensitively capture the microscopic molecular event of "BDNF precursor conversion inhibition" under pathological conditions.

[0133] 6.5) Conclusion: Statistical results (P<0.001) show that the kit of this invention can effectively quantify the two conformational forms of BDNF in urine and their ratio. Through "structure-guided" antibody design, the kit successfully eliminated steric hindrance interference, providing a precise quantitative tool for the study of related metabolic pathways and the assessment of biological status.

[0134] Although the above embodiments are mainly illustrated using colloidal gold test strips as an example, those skilled in the art should understand that the antibody pairings obtained using the above SEQ ID NO: 1~3 can also be applied to conventional immunoassay platforms such as fluorescence immunochromatography, quantum dot immunoassay, ELISA, and preparation of magnetic microparticle chemiluminescence reagents, and the principle is the same.

[0135] In the embodiments of this invention, the physical positions of the first detection line (T1) and the second detection line (T2) on the chromatography strip can be interchanged. For example, they can be arranged sequentially along the chromatography direction as T1→T2→C, or they can be arranged as T2→T1→C. Those skilled in the art will understand that as long as the detection line is located between the conjugate pad and the absorbent pad, and the control line is located downstream of the detection line, different T-line arrangements do not affect the implementation of the structure-kinetics-guided antibody pairing strategy of this invention, and all fall within the protection scope of this invention.

[0136] This invention is not limited to the structures described above and shown in the accompanying drawings, and various modifications and changes can be made without departing from its scope. The scope of this invention is limited only by the appended claims.

Claims

1. A kit for detecting the ratio of proBDNF to mature BDNF based on structure dynamics-guided methods, characterized in that, The kit includes a solid-phase carrier and detection reagents, and is configured with three antibodies determined based on full-length conformational kinetic analysis of brain-derived neurotrophic factor. The three antibodies include: First capture antibody: specifically binds to the N-terminal intrinsic disordered region of the proBDNF propeptide domain, and the recognition epitope of the first capture antibody is SEQ ID NO: 1; The second capture antibody is a novel epitope-specific antibody that specifically binds to the N-terminal exposed interface of mature BDNF generated by protease cleavage. The recognition epitope of the second capture antibody is SEQ ID NO: 2, and the binding is strictly dependent on the electrostatic interaction provided by the free α-amino group of histidine at position 1 in SEQ ID NO:

2. The second capture antibody does not have an immune cross-reaction with proBDNF precursors that have not undergone enzymatic cleavage and whose α-amino group of histidine at position 1 participates in peptide bond formation. Universal detection antibody: Specifically binds to the Loop 4 region of the mature BDNF domain, and the recognition epitope of this universal detection antibody is SEQ ID NO: 3; the Loop 4 region is an exposed area with an accessible area of ​​more than 50% in both the proBDNF precursor and the mature BDNF spatial folding conformation, making the universal detection antibody have substantially equivalent binding affinity for proBDNF and mature BDNF; The sequence of SEQ ID NO: 1 is APMKEANIRG, the sequence of SEQ ID NO: 2 is HSDPARRGEL, and the sequence of SEQ ID NO: 3 is DSKKRIGWR.

2. The kit for detecting the ratio of proBDNF to mature BDNF based on structure dynamics-guided analysis according to claim 1, characterized in that, The application of the kit is selected from any one of enzyme-linked immunosorbent assay (ELISA), chemiluminescent immunoassay, lateral flow immunochromatography, or quantum dot fluorescence immunoassay.

3. The kit for detecting the ratio of proBDNF to mature BDNF based on structure dynamics-guided method according to claim 2, characterized in that, The kit is a lateral flow immunochromatographic test strip, and the lateral flow immunochromatographic test strip includes a base plate and a sample pad, a conjugate pad, a microporous membrane and an absorbent pad that are sequentially overlapped along the chromatography direction.

4. The kit for detecting the ratio of proBDNF to mature BDNF based on structure dynamics-guided analysis according to claim 3, characterized in that, The microporous membrane is provided with a first detection line, a second detection line, and a quality control line spaced apart along the chromatography direction. The first detection line and the second detection line are respectively fixed with the first capture antibody and the second capture antibody, which are used to spatially separate and capture proBDNF and mature BDNF. The quality control line is fixed with a secondary antibody or internal standard that specifically binds to the universal detection antibody; The conjugate pad adsorbs the universal detection antibody labeled with a tracer marker.

5. The kit for detecting the ratio of proBDNF to mature BDNF based on structure dynamics-guided analysis according to claim 4, characterized in that, The tracer label is selected from any one of colloidal gold particles, colloidal selenium particles, fluorescent microspheres, quantum dots, or latex microspheres.

6. The kit for detecting the ratio of proBDNF to mature BDNF based on structure dynamics-guided method according to claim 5, characterized in that, The tracer marker is a colloidal gold particle with an average particle size of 40nm±5nm. The kit is configured to achieve quantitative detection by reading the light reflection intensity or fluorescence intensity value of the control line, the first detection line and the second detection line.

7. The kit for detecting the ratio of proBDNF to mature BDNF based on structure dynamics-guided analysis according to claim 1, characterized in that, The Loop 4 epitope recognized by the universal detection antibody exhibits kinetic characteristics that combine spatial exposure stability with affinity consistency. Among them, the spatial exposure stability was verified by molecular dynamics simulation. The spatial distance between the epitope and the BDNF propeptide domain remained greater than 15 Å in the dynamic conformation and was not located within the hydrophobic collapse coverage region of the propeptide domain. In the reaction system of the kit, the affinity consistency is such that the dissociation constant of the universal detection antibody for proBDNF antigen differs from that for matureBDNF antigen by less than 3 times, in order to eliminate nonlinear detection bias caused by antigen conformation differences.

8. The kit for detecting the ratio of proBDNF to mature BDNF based on structure dynamics-guided method according to claim 1, characterized in that, The kit also includes a sample processing solution containing 0.1M~0.2M HEPES buffer, 0.5%~2.0% (w / v) of the zwitterionic surfactant CHAPS, 1%~3% (w / v) of bovine serum albumin, and 0.05% (w / v) of Proclin 300. The sample processing solution is configured to dissociate non-specific protein aggregates in the sample and maintain the free α-amino group of histidine at position 1 in SEQ ID NO: 2 in a protonated or deprotonated state that can be recognized by the second capture antibody.

9. The kit for detecting the ratio of proBDNF to mature BDNF based on structure dynamics-guided method according to claim 1, characterized in that, The detection and calculation of the kit are achieved by reading the light reflection intensity value or fluorescence signal value of the control line, the first detection line and the second detection line, and calculating the ratio R. The ratio R is used to evaluate the protease cleavage processing efficiency of BDNF precursor in the subject and to evaluate it as a biomarker for neuropsychiatric diseases or kidney pathology. The formula for calculating the ratio R is: ； In the formula, V1 represents the molar concentration calculated by substituting the signal values ​​of the control line and the second detection line into the standard curve, and V2 represents the molar concentration calculated by substituting the signal values ​​of the control line and the first detection line into the standard curve.

10. A method for preparing the kit for detecting the ratio of proBDNF to mature BDNF based on structure dynamics-guided assay as described in claim 6, characterized in that, The method includes: Structure-guided antibody screening: Immunogens were prepared based on the three characteristic polypeptide sequences of SEQ ID NO: 1~3, and high-affinity monoclonal or polyclonal antibodies against the first detection line, the second detection line and the gold standard were screened to obtain them respectively. Secondary antibodies that matched the host source of the gold standard antibody were selected as quality control antibodies. Preparation of gold-labeled conjugate: Anti-BDNF universal detection antibody was electrostatically coupled with 40nm colloidal gold particles under pH 8.0~9.0 conditions, and blocked with blocking solution containing PEG2000. The conjugate was then sprayed onto a glass fiber pad and vacuum dried to prepare the conjugate pad. Immobilization and assembly: Anti-proBDNF specific antibody, anti-mature BDNF novel epitope antibody and the quality control antibody are imprinted onto nitrocellulose membrane at a concentration of 0.5~2.0 mg / mL to form the first detection line, the second detection line and the quality control line in sequence. The test kit is obtained by assembling and cutting the components in sequence.

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