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CN-121559089-B - Kit for detecting ratio of proBDNF to material BDNF based on structural dynamics guidance and preparation method thereof

CN121559089BCN 121559089 BCN121559089 BCN 121559089BCN-121559089-B

Abstract

The invention belongs to the technical field of immunodetection, and discloses a kit for detecting the ratio of proBDNF to material BDNF based on structural dynamics guidance and a preparation method thereof, wherein the kit comprises a solid phase carrier and a detection reagent, and is provided with three antibodies determined based on brain-derived neurotrophic factor full-length conformational dynamics analysis, namely a first capture antibody targets an inherent disorder region (SEQ ID NO: 1) of a proBDNF pro-peptide region; the second capture antibody is a neoepitope specific antibody, specifically binds to the exposed N-terminus of the material BDNF enzyme (SEQ ID NO: 2) and binds to a free alpha-amino group that is strictly dependent on histidine 1, and the universal detection antibody binds to the Loop 4 region of the material domain (SEQ ID NO: 3). The invention solves the problems of steric hindrance and cross reaction in the traditional immunodetection through a structural biological strategy, and realizes the accurate distinction and ratio quantification of proBDNF and material BDNF.

Inventors

  • JING YIN
  • FENG YUJING
  • ZHAO LIMING

Assignees

  • 北京华瑞康源生物科技发展有限公司

Dates

Publication Date
20260512
Application Date
20260122

Claims (10)

  1. 1. A kit for detecting the ratio of proBDNF to material BDNF based on structural dynamics guidance, characterized in that the kit comprises a solid phase carrier and a detection reagent, and is configured with three antibodies determined based on full-length conformational dynamics analysis of brain-derived neurotrophic factors, and the three antibodies comprise: A first capture antibody which specifically binds to the N-terminal fixed unordered region of the proBDNF propeptide domain, wherein the recognition epitope of the first capture antibody is SEQ ID NO. 1; A second capture antibody which is a neoepitope specific antibody and specifically binds to an N-terminal exposed interface of the material BDNF generated by cleavage by protease, wherein the recognition epitope of the second capture antibody is SEQ ID NO. 2 and the binding is strictly dependent on electrostatic interaction provided by free alpha-amino group of 1 st histidine in SEQ ID NO. 2, and the second capture antibody has NO immunological cross reaction with proBDNF precursor which is not cleaved and is formed by the alpha-amino group of 1 st histidine participating in peptide bond; The universal detection antibody specifically binds with a Loop 4 region of a BDNF mature domain, and the recognition epitope of the universal detection antibody is SEQ ID NO 3, wherein the Loop 4 region is an exposed region with a contact area of more than 50% in a spatial folding conformation of a proBDNF precursor and a material BDNF, so that the universal detection antibody has substantially equivalent binding affinity to the proBDNF and the material BDNF; Wherein SEQ ID NO. 1 has the sequence APMKEANIRG, SEQ ID NO. 2, HSDPARRGEL, SEQ ID NO. 3 and DSKKRIGWR.
  2. 2. The kit for detecting the ratio of proBDNF to material BDNF based on structural dynamics guidance according to claim 1, wherein the application form of the kit is any one of enzyme-linked immunosorbent assay, chemiluminescent immunoassay, lateral flow immunochromatography or quantum dot fluorescent immunoassay.
  3. 3. The kit for detecting the ratio of proBDNF to material BDNF based on structural dynamics guidance according to claim 2, wherein the kit is a lateral flow immunochromatographic test strip, and the lateral flow immunochromatographic test strip comprises a bottom plate, and a sample pad, a binding pad, a microporous membrane and a water absorption pad which are sequentially overlapped along the chromatographic direction.
  4. 4. The kit for detecting the ratio of proBDNF to material BDNF based on structural dynamics guidance according to claim 3, wherein a first detection line, a second detection line and a quality control line are arranged on the microporous membrane at intervals along the chromatographic direction; the first detection line and the second detection line are respectively fixed with the first capture antibody and the second capture antibody and are used for spatially separating and capturing proBDNF and material BDNF; The quality control line is fixed with a secondary antibody or an internal standard substance which specifically binds to the universal detection antibody; the conjugate pad adsorbs the universal detection antibody labeled with a tracer label.
  5. 5. The kit for detecting the ratio of proBDNF to material BDNF based on structural dynamics guidance according to claim 4, wherein the tracer marker is any one of colloidal gold particles, colloidal selenium particles, fluorescent microspheres, quantum dots or latex microspheres.
  6. 6. The kit for detecting the ratio of proBDNF to material BDNF based on structural dynamics guidance according to claim 5, wherein the tracer marker is colloidal gold particles with an average particle size of 40nm + -5 nm, and the kit is configured to realize quantitative detection by reading light reflection intensity values or fluorescence intensity values of a quality control line, a first detection line and a second detection line.
  7. 7. The kit for detecting the ratio of proBDNF to material BDNF based on structural dynamics guidance according to claim 1, wherein the Loop 4 region epitope recognized by the universal detection antibody has the dynamic characteristics of combining space exposure stability with affinity consistency; The space exposure stability is verified by molecular dynamics simulation, and the space distance between the epitope and the BDNF propeptide domain is kept to be larger than 15A in a dynamic conformation and is not in the hydrophobic collapse coverage area of the propeptide domain; Affinity consistency in the reaction system of the kit, the dissociation constant of the universal detection antibody to the proBDNF antigen and the dissociation constant to the matureBDNF antigen are different by less than 3 times, so that nonlinear detection deviation caused by antigen conformational aberration is eliminated.
  8. 8. The kit for detecting the ratio of proBDNF to material BDNF based on structural dynamics guidance according to claim 1, wherein the kit further comprises a sample treatment liquid, wherein the sample treatment liquid comprises 0.1-0.2M HEPES buffer solution, 0.5-2.0% of zwitterionic surfactant CHAPS with mass-volume ratio concentration, 1-3% of bovine serum albumin with mass-volume ratio concentration and 0.05% of Proclin300 with mass-volume ratio concentration; Wherein the sample treatment fluid is configured to dissociate non-specific protein aggregates in the sample and maintain the free alpha-amino group of histidine 1 in SEQ ID NO. 2 in a protonated or deprotonated state recognizable by the second capture antibody.
  9. 9. The kit for detecting the ratio of pro BDNF to material BDNF based on structural dynamics guidance according to claim 1, wherein the detection and calculation of the kit are realized by reading the light reflection intensity values or fluorescence signal values of a quality control line, a first detection line and a second detection line, and calculating a ratio R, wherein the ratio R is used for evaluating the protease cleavage processing efficiency of BDNF precursors in a subject and is used as a biomarker for the evaluation of neuropsychiatric diseases or renal pathology; wherein, the calculation formula of the ratio R is: ; where V1 represents the molar concentration calculated from the signal values of the quality control line and the second detection line substituted into the standard curve, and V2 represents the molar concentration calculated from the signal values of the quality control line and the first detection line substituted into the standard curve.
  10. 10. A method of preparing the kit for structural kinetic-guided detection of the ratio of pro BDNF to material BDNF according to claim 6, characterized in that the method comprises: Preparing immunogens based on three sections of characteristic polypeptide sequences of SEQ ID NO. 1-3, screening to obtain high-affinity monoclonal antibodies or polyclonal antibodies respectively aiming at a first detection line, a second detection line and a gold-labeled antibody, and selecting secondary antibodies matched with a gold-labeled antibody host source as quality control antibodies; preparing a gold-labeled conjugate, namely performing electrostatic coupling on an anti-BDNF universal detection antibody and 40nm colloidal gold particles under the condition of pH 8.0-9.0, sealing by using a sealing liquid containing PEG2000, spraying on a glass fiber pad, and vacuum drying to prepare a conjugate pad; And (3) solid-phase and assembly, namely marking an anti-proBDNF specific antibody, an anti-information BDNF neoepitope antibody and the quality control antibody on a nitrocellulose membrane respectively at the concentration of 0.5-2.0 mg/mL, sequentially forming a first detection line, a second detection line and a quality control line, sequentially assembling and cutting all the components to obtain the paper box.

Description

Kit for detecting ratio of proBDNF to material BDNF based on structural dynamics guidance and preparation method thereof Technical Field The invention relates to the technical field of immunodetection, in particular to a kit for detecting the ratio of proBDNF to material BDNF based on structural dynamics guidance and a preparation method thereof. Background Brain-derived neurotrophic factor (BDNF) is a core member of the neurotrophic factor family and plays a vital role in nervous system development and kidney physiological function. On the biosynthetic pathway, BDNF is not a radical step in that it is initially synthesized in the form of a precursor having a molecular weight of about 32kDa (proBDNF) and subsequently cleaved by intracellular or extracellular proteases to remove the N-terminal pro-peptide domain (Prodomain) and can be converted to a mature form having a molecular weight of about 14kDa (material BDNF). Nowadays, more and more evidence shows that proBDNF and material BDNF are not purely precursor-to-product relationships, but exert diametrically opposite biological effects by binding to different receptors, namely material BDNF specifically binds to TrkB receptors, promoting neuronal survival and long-term enhancement, while proBDNF binds to p75NTR receptors with high affinity, inducing apoptosis and long-term inhibition. This "yin-yang balance" mechanism maintains the physiological homeostasis of the organism. The traditional view has long thought that only the material BDNF would be secreted into the extracellular matrix to function. However, over a decade of breakthrough studies have corrected this inherent experience in that in practice, both proBDNF and material BDNF can be released extracellularly in a mixed form and are widely present in body fluids such as blood, urine, etc. This means that there are two simultaneous forces in the humoral environment that promote growth and induce apoptosis. Based on the findings, simply detecting the BDNF total amount clearly covers up the oppositivity of the two functions, and cannot meet the requirement of accurate diagnosis. When the ratio of proBDNF to material BDNF is unbalanced, it is often predicted that pathological conditions such as depression, anxiety, alzheimer's disease and overactive bladder occur. Therefore, from focusing on "total amount" to focusing on "the ratio of both, the use of the ratio to evaluate the proteolytic cleavage efficiency of BDNF precursors in a subject has become a more critical and accurate biological indicator for assessing the progression of neurological, psychiatric and urinary diseases. The existing immunodetection technology faces two major problems, namely a steric effect, namely that an epitope of a mature region can be randomly shielded by a pro peptide domain (Prodomain) of proBDNF to cause nonlinear reduction of detection signals, and a cross reaction, wherein a traditional antibody cannot distinguish an uncleaved internal sequence in the proBDNF from an exposed N-terminal sequence in the material BDNF to cause the fact that the ratio of the two sequences cannot be accurately determined. For the problems in the related art, no effective solution has been proposed at present. Disclosure of Invention The invention provides a kit for detecting the ratio of proBDNF to material BDNF based on structural dynamics guidance and a preparation method thereof, which are used for solving the problems of the prior art. The invention aims to provide a proBDNF and material BDNF ratio detection kit based on structural dynamics guidance. The invention breaks through the limitation of the traditional blind screening antibody, adopts the technology of 'structure biology guided epitope mapping', locks three key 'gold antigen sites' by combining software three-dimensional reconstruction, predictive Alignment Error (PAE) matrix analysis and dynamic simulation test, and solves the problems of steric hindrance and cross reaction in physical and chemical principles. 1. Antigenic site evaluation and screening based on structural biology The invention builds a BDNF full-length structure (UniProt P23560) model based on AlphaFold algorithm, combines PAE matrix and Molecular Dynamics (MD) simulation, and establishes the following core screening strategy and antigen site: 1) "anti-occlusion" screening of universal detection sites: Structural analysis the invention constructs a high-precision full-length three-dimensional model of BDNF. Unlike the static structure, the PAE matrix is deeply analyzed, and the dynamic steric hindrance thermodynamic diagram is drawn by analyzing the interaction energy of Prodomain (19 th to 128 th bits) and Mature Domain (129 th to 247 th bits). Analysis shows that the Loop 1 and Loop 2 regions of the material Domain, while highly immunogenic, have a probability of being blocked by Prodomain as high as 80% (i.e. "binding exclusion zone"). Screening results show that the invention discovers that Loop 4 (216-2