Antibody for detecting soluble platelet-derived growth factor receptor beta, kit and application

Abstract

The invention discloses an antibody for detecting a soluble platelet-derived growth factor receptor beta, a kit and application. The antibody is prepared by coupling a soluble platelet-derived growth factor receptor beta-specific antigen epitope peptide with a carrier protein and then immunizing an animal, and the amino acid sequence of the soluble platelet-derived growth factor receptor beta-specific antigen epitope peptide is at least one of a sequence shown as SEQ ID NO.1 and a sequence shown as SEQ ID NO.2. The antibody can specifically detect the soluble platelet-derived growth factor receptor beta, and has the advantages of high sensitivity, good specificity, good repeatability and the like; when being used for auxiliary diagnosis or illness monitoring of blood-brain barrier dysfunction related diseases, the kit has good sensitivity, specificity and repeatability, meets clinical application requirements, provides a more accurate and reliable objective index for auxiliary diagnosis, illness monitoring and prognosis evaluation of related diseases, and has important significance and clinical application value.

Description

Technical Field

[0001] This application relates to the field of protein detection technology, and in particular to an antibody, kit, and application for detecting soluble platelet-derived growth factor receptor β. Background Technology

[0002] Pericytocyte damage in the blood-brain barrier (BBB) ​​microvascular unit has been shown to be an "upstream" driving event for a variety of cognitive impairment diseases, including Alzheimer's disease (AD), vascular cognitive impairment (VCI), and aneurysmal subarachnoid hemorrhage (aSAH).

[0003] Pericytes play a central role in the structural integrity and functional homeostasis of the blood-brain barrier (BBB) ​​by regulating tight junctions and transport systems among endothelial cells in brain microvessels. This regulatory process is highly dependent on the spatiotemporal precision activation of the platelet-derived growth factor-BB (PDGF-BB) signaling pathway by the PDGF receptor-β (PDGFRβ) on the pericyte membrane surface. PDGFRβ-mediated downstream cascades (such as PI3K / Akt and Ras / MAPK) not only drive the recruitment and encirclement of pericytes to endothelial cells but also inhibit non-selective leakage of substances across cellular and paracellular pathways. When the PDGF-BB / PDGFRβ axis is downregulated, mutated, or has impaired ligand-receptor binding, pericyte coverage is significantly reduced, leading to downregulation of tight junction proteins (claudin-5, occludin, ZO-1) and abnormal endothelial transcytosis, thereby disrupting the selective permeability of the BBB and interfering with the directional transport of metabolites and ions. The aforementioned microvascular dysfunction can lead to neuronal energy crisis, impaired synaptic plasticity, and disordered neural network oscillations through decoupling of the neurovascular unit (NVU), ultimately manifesting as cognitive decline, impaired memory encoding and consolidation, and accelerating the progression of neurodegenerative diseases. Therefore, in-depth analysis of the molecular and cellular mechanisms of the PDGF-BB / PDGFRβ signaling network holds promise for providing new targets for precise intervention in cognitive impairment related to BBB dysfunction.

[0004] Pericytes, the structural-functional hubs of neurovascular units, play a crucial role in maintaining selective permeability to the blood-brain barrier (BBB), ion homeostasis, and metabolite transport homeostasis. Pericytes limit uncontrolled extravasation of plasma components and safeguard the synaptic microenvironment (such as K+) by regulating the assembly of the endothelial tight junction complex and the efficiency of endocytosis. + Ca 2 +Precise control of pericyte concentrations (glucose and neurotransmitter concentrations) is crucial. Pericytes, whether absent or inactivated, can disrupt the ultrastructure of brain microvessels, leading to conditions such as basement membrane thickening, increased endothelial fenestration, and upregulation of vesicle transport. This, in turn, causes neurovascular decoupling, insufficient oxygen and glucose supply, and accumulation of metabolic waste, ultimately driving neuronal excitation-inhibition imbalance and progressive neurodegeneration. Furthermore, pericytes regulate precapillary sphincter tone through their α-smooth muscle actin (α-SMA) positive processes, participating in neurovascular coupling—a process essential for the rapid redistribution of energy substrates and oxygen in active brain regions. Pericyte dysfunction weakens this blood flow regulation, exacerbating the neuronal energy crisis. Therefore, elucidating the molecular networks governing pericyte homeostasis, particularly the pleiotropic regulation of the PDGF-BB / PDGFRβ signaling axis at the transcriptional, post-translational modification, and cell-matrix interaction levels, is a core scientific question for understanding the mechanisms of BBB-dependent cognitive decline and developing targeted intervention strategies.

[0005] Structural and functional instability of the blood-brain barrier (BBB) ​​has been established as a core pathological factor driving cognitive decline in various neurological diseases, including Alzheimer's disease (AD) and aneurysmal subarachnoid hemorrhage (aSAH). In the early stages of AD, increased BBB permeability is detectable in approximately 40% of patients, and its degree is positively correlated with the amount of blood-derived proteins (albumin, fibrinogen) deposited in the hippocampus and cortex. Longitudinal studies using high-resolution dynamic contrast-enhanced MRI have confirmed that BBB leakage can precede amyloid plaques by 6-10 months and is significantly positively correlated with the rate of decline in the Mini-Mental State Examination (MMSE). In aSAH individuals, BBB disruption induces delayed cerebral ischemia (DCI) and diffuse cortical microemboli, further leading to significant impairment of executive function, attention, and memory in 50-70% of survivors within one year.

[0006] The loss of BBB integrity triggers a cascade of damage mechanisms in the neurovascular unit, mainly including: (1) downregulation of endothelial tight junction proteins (claudin-5, occludin, ZO-1), leading to non-selective extravasation of macromolecules via the paracellular pathway; (2) pericellular degeneration and basement membrane thickening, reducing capillary perfusion reserve; (3) peripheral inflammatory mediators (IL-1β, TNF-α, C1q) enter the brain parenchyma, activating microglia and amplifying neuroinflammation. These pathological changes collectively cause impaired synaptic plasticity, neuronal energy crisis, and interruption of functional connectivity in the default mode network (DMN), ultimately manifesting as a comprehensive decline in the cognitive domain. Health economics models show that, for Alzheimer's disease alone, global direct healthcare expenditure reached US$1 trillion in 2023, and indirect care costs and productivity losses combined accounted for 1.3–1.8% of GDP in high-income countries. As the proportion of the population aged 65 and over is expected to double by 2050, the economic burden associated with dementia will increase exponentially, posing a severe and continuously expanding socioeconomic challenge.

[0007] Soluble platelet-derived growth factor receptor β (sPDGFRβ) is the soluble form of PDGFRβ released into body fluids after the extracellular segment is cleaved by proteases, with a molecular weight of approximately 110 kDa. Essentially, it is a soluble receptor for receptor tyrosine kinases. Its generation mechanism primarily involves brain microvascular pericytes releasing the full-length PDGFRβ from the membrane surface via cleavage by metalloproteinases such as ADAM10 / 17 and MMP-9 under hypoxic, Aβ40 / 42, or inflammatory stimulation. Recent studies have also found that selective precursor mRNA splicing can also produce soluble isoforms. Its biological function mainly involves competitively binding to PDGF-BB as a "decoy receptor," blocking its binding to membrane receptors, thereby inhibiting PDGFRβ-mediated pericyte migration, proliferation, and vascular stability signals. Therefore, elevated sPDGFRβ reflects pericyte-endothelial unit damage and may exacerbate blood-brain barrier disruption by weakening the PDGF-BB / PDGFRβ axis.

[0008] Given the current state of diagnosis regarding cognitive decline caused by BBB disruption and loss of brain capillary pericytes, clinical efforts have been focused on finding a stable and reliable biomarker that can objectively diagnose cognitive decline caused by blood-brain barrier dysfunction. Currently, brain imaging and peripheral blood protein markers are the most studied.

[0009] As research has progressed, scholars have discovered that sPDGFRβ can serve as an early biomarker for cognitive impairment in humans. For example, elevated sPDGFRβ levels in cerebrospinal fluid (CSF) indicate pericyte damage and BBB breakdown. Furthermore, sPDGFRβ concentrations are significantly elevated in the early CSF of patients with aneurysmal subarachnoid hemorrhage (aSAH). In the acute phase of aSAH patients with cognitive impairment, sPDGFRβ concentrations are higher than in patients without cognitive impairment, which may be related to more severe pericyte damage and pericyte migration with excessive fibrosis. One-way ANOVA showed significant differences in CSF sPDGFRβ concentrations among the mild AD, moderate-to-severe AD, and control groups. Post-hoc LSD tests showed that the CSF sPDGFRβ concentrations in both the mild AD and moderate-to-severe AD groups were significantly higher than those in the control group, with statistically significant differences. Compared with the mild AD group, the concentration of sPDGFRβ in the cerebrospinal fluid of the moderate to severe AD group was slightly higher.

[0010] Currently, research on sPDGFRβ (soluble platelet-derived growth factor receptor β) mainly focuses on cerebrospinal fluid, and its expression profile and dynamic changes in blood samples lack systematic explanation. Furthermore, previous studies have not stratified patients based on the severity of cognitive impairment, nor have they elucidated the dose-response relationship between peripheral blood sPDGFRβ levels and the degree of cognitive impairment. Given that sPDGFRβ can reflect disruption of the blood-brain barrier (BBB) ​​integrity and depletion of pericytes in brain microvessels, it holds promise as a novel peripheral biomarker for non-invasive, real-time monitoring of the pathological progression of cognitive decline.

[0011] In summary, precise quantification of peripheral blood sPDGFRβ not only provides an accessible and repeatable peripheral window for the early identification and dynamic monitoring of cognitive impairment, but also quantifies the core pathological processes of blood-brain barrier integrity disruption and cerebral microvascular pericyte depletion into operable clinical indicators at the molecular level. However, current research on sPDGFRβ-specific detection is relatively limited. Developing new sPDGFRβ-specific detection technologies is of great significance and clinical application value for the diagnosis of related diseases. Summary of the Invention

[0012] The purpose of this application is to provide a novel antibody, kit, and application for detecting soluble platelet-derived growth factor receptor β.

[0013] The following technical solution is adopted in this application:

[0014] The first aspect of this application discloses an antibody for detecting soluble platelet-derived growth factor receptor β, which is prepared by immunizing animals with a soluble platelet-derived growth factor receptor β-specific antigenic epitope peptide coupled with a carrier protein, wherein the amino acid sequence of the soluble platelet-derived growth factor receptor β-specific antigenic epitope peptide is at least one of the sequences shown in SEQ ID NO.1 and SEQ ID NO.2;

[0015] SEQ ID NO.1:

[0016] Tyr-Arg-Val-Ser-Glu-Leu-Thr-Leu-Val-Arg-Val-Lys-Val-Ala-Glu-Ala-Gly

[0017] SEQ ID NO.2:

[0018] Tyr-Leu-Glu-Leu-Gln-Val-Glu-Pro-Glu-Pro-Glu-Leu-Glu.

[0019] In one implementation of this application, the antibody is a polyclonal antibody prepared by immunizing animals with a soluble platelet-derived growth factor receptor β-specific antigenic epitope peptide coupled with a carrier protein.

[0020] It should be noted that the key to this application lies in the development of a novel soluble platelet-derived growth factor receptor β-specific antigenic epitope peptide, and based on this novel epitope peptide, a new antibody capable of specifically detecting soluble platelet-derived growth factor receptor β, namely the polyclonal antibody of this application, is obtained, thus providing a new scheme and approach for the detection of soluble platelet-derived growth factor receptor β. It is understood that the carrier protein, carrier protein conjugation, animal immunization, and post-immunization extraction of polyclonal antibodies in this application can all refer to existing technologies and are not specifically limited here.

[0021] The second aspect of this application discloses a kit for detecting soluble platelet-derived growth factor receptor β, the kit containing an antibody specifically for detecting soluble platelet-derived growth factor receptor β as described in this application.

[0022] In one implementation of this application, the kit includes a solid-phase support and the antibody of this application, which is directly or indirectly linked to the solid-phase support.

[0023] In one implementation of this application, the solid support is at least one of a microporous reaction plate, magnetic microspheres, and nitrocellulose membrane.

[0024] It should be noted that microporous reaction plates, magnetic microspheres, and nitrocellulose membranes are all solid-phase carriers conventionally used in this application. The key to this application lies in the development and preparation of a new antibody that specifically detects soluble platelet-derived growth factor receptor β. As for the specific type of solid-phase carrier used, it depends on the detection method used in the kit of this application, such as enzyme-linked immunosorbent assay (ELISA), chemiluminescence immunochromatography (CLC), colloidal gold immunochromatography (CICI), etc. Different specific detection methods can select the appropriate solid-phase carrier, and no specific limitation is made here.

[0025] In one implementation of this application, the kit further includes a marker for labeling the soluble platelet-derived growth factor receptor β antibody specifically for detection in this application.

[0026] In one implementation of this application, the marker is at least one of horseradish peroxidase (HRP), alkaline phosphatase (AP), a luminescent substance, a fluorescent substance, a dye, and colloidal gold.

[0027] It should be noted that different markers also depend on the specific detection method used. For example, horseradish peroxidase (HRP) is preferred for enzyme-linked immunosorbent assay (ELISA), and alkaline phosphatase (AP) is preferred for chemiluminescent immunoassay (CLIA).

[0028] In one implementation of this application, the kit further includes a soluble platelet-derived growth factor receptor β standard.

[0029] In one implementation of this application, the kit is at least one of an enzyme-linked immunosorbent assay kit, a chemiluminescence assay kit, a fluorescence immunochromatographic assay kit, and a colloidal gold immunoassay kit.

[0030] In one implementation of this application, the chemiluminescence assay kit is prepared using a combination of chemiluminescence immunoassay and a double-antibody sandwich method.

[0031] In one implementation of this application, the reagent kit further includes a magnetic microparticle suspension, a washing solution, and a chemiluminescent substrate.

[0032] In one implementation of this application, the chemiluminescent substrate includes at least one of luminol, isoluminol and its derivatives, and (goldenon)-1,2-dioxane and its derivatives.

[0033] The third aspect of this application discloses the antibody that specifically detects soluble platelet-derived growth factor receptor β, or the application of the kit of this application in the preparation of products for detecting diseases related to blood-brain barrier dysfunction.

[0034] It should be noted that the specific antibody or kit of this application can specifically detect soluble platelet-derived growth factor receptor β. According to existing research, soluble platelet-derived growth factor receptor β can serve as a biomarker for blood-brain barrier dysfunction-related diseases, such as cognitive impairment caused by blood-brain barrier dysfunction. Therefore, the antibody or kit of this application that specifically detects soluble platelet-derived growth factor receptor β can be used to prepare products for detecting blood-brain barrier dysfunction-related diseases, such as a detection or diagnostic kit for blood-brain barrier dysfunction-related diseases based on soluble platelet-derived growth factor receptor β assay.

[0035] The beneficial effects of this application are as follows:

[0036] This application describes an antibody that specifically detects soluble platelet-derived growth factor receptor β. It possesses advantages such as high sensitivity, good specificity, and good repeatability. When used for the auxiliary diagnosis or disease monitoring of blood-brain barrier dysfunction-related diseases, it exhibits good sensitivity, specificity, and repeatability, meeting clinical application requirements. It provides a more accurate and reliable objective indicator for the auxiliary diagnosis, disease monitoring, and prognostic assessment of blood-brain barrier dysfunction-related diseases, and has significant clinical application value for the diagnosis and treatment of these diseases. Attached Figure Description

[0037] Figure 1 This is a comparative analysis of sPDGFRβ levels between young and elderly individuals with normal CDR(0) cognition in the embodiments of this application;

[0038] Figure 2 This is a comparative analysis of sPDGFRβ levels in patients with different degrees of CDR (0, 0.5, 1) in the embodiments of this application. Detailed Implementation

[0039] Through a prospective cohort study, this application evaluated the longitudinal association between peripheral sPDGFRβ levels and BBB permeability, brain microvascular pericyte density, and the degree of cognitive impairment, and further explored its potential clinical value in disease progression monitoring, treatment response assessment, and long-term prognosis prediction.

[0040] Based on this research and understanding, this application develops a highly sensitive and specific sPDGFRβ assay kit, and uses a specific polyclonal antibody against sPDGFRβ to achieve accurate quantification of sPDGFRβ in serum / plasma samples.

[0041] Specifically, the specific antibody for detecting soluble platelet-derived growth factor receptor β in this application is prepared by immunizing animals with a soluble platelet-derived growth factor receptor β specific antigenic epitope peptide coupled with a carrier protein. The amino acid sequence of the soluble platelet-derived growth factor receptor β specific antigenic epitope peptide is at least one of the sequences shown in SEQ ID NO.1 and SEQ ID NO.2.

[0042] In one implementation of this application, when the specific antibody of this application or a kit based on the specific antibody of this application is used to prepare a product for diagnosing diseases related to blood-brain barrier dysfunction, the concentration of sPDGFRβ in the subject sample measured by the kit is compared with the control reference range. If the concentration of sPDGFRβ in the subject sample is higher than the concentration in the control reference range, the subject has the risk of cognitive impairment disease related to blood-brain barrier (BBB) ​​dysfunction.

[0043] In one implementation of this application, the specific antibody used in chemiluminescence detection exhibits high sensitivity, with a detection limit as low as 25 pg / mL, a wide linear range of 3-6 orders of magnitude, and the ability to directly measure samples with concentrations up to 18000 pg / mL without dilution. It also boasts high accuracy, with a recovery rate of 99.99% in recovery tests, high precision, a CV of less than 10%, and a short reaction time (results available in 30 minutes), significantly improving detection efficiency. Furthermore, the detection is highly automated, requiring no manual operation and reducing human error.

[0044] In summary, the specific antibody and kit of this application have the following advantages and positive effects compared with the prior art:

[0045] 1. This application uses sPDGFRβ as a biomarker in a kit for the auxiliary diagnosis or monitoring of cognitive impairment caused by blood-brain barrier dysfunction. This overcomes the shortcomings of current clinical diagnostic indicators for cognitive impairment caused by blood-brain barrier dysfunction, which are too subjective and lack objectivity. This provides a more accurate and reliable objective indicator for the auxiliary diagnosis, monitoring, and prognostic assessment of cognitive impairment caused by blood-brain barrier dysfunction.

[0046] 2. The reagent kit prepared in this application has good sensitivity, specificity and reproducibility, which meets the needs of clinical application.

[0047] 3. The sPDGFRβ polyclonal antibody prepared in this application can bind to sPDGFRβ in the sample with high specificity.

[0048] 4. The two sPDGFRβ specific antigenic epitope peptides screened in this application are characterized by hydrophilicity, strong antigenicity and ease of synthesis. The antigens (immunogens) prepared from them can produce highly specific polyclonal antibodies when used to immunize animals.

[0049] The present application will be further described in detail below through specific embodiments. The following embodiments are only for further illustration of the present application and should not be construed as limiting the present application.

[0050] Unless otherwise specified, the technical means used in the embodiments are conventional means well known to those skilled in the art.

[0051] Example

[0052] I. Synthesis of sPDGFRβ-specific antigenic epitope peptide

[0053] In this case, two sPDGFRβ-specific antigenic epitope peptides were developed, namely the sequences shown in SEQ ID NO.1 and SEQ ID NO.2.

[0054] SEQ ID NO.1:

[0055] Tyr-Arg-Val-Ser-Glu-Leu-Thr-Leu-Val-Arg-Val-Lys-Val-Ala-Glu-Ala-Gly

[0056] SEQ ID NO.2:

[0057] Tyr-Leu-Glu-Leu-Gln-Val-Glu-Pro-Glu-Pro-Glu-Leu-Glu

[0058] The synthesis of the two sPDGFRβ-specific antigenic epitope peptides is as follows:

[0059] 1. Main raw materials

[0060] HMP resin (P-hydroxymethylphenoxymethyl polyethylene resin, purchased from Sigma-Aldrich), Fmoc-AA (9-fluorenylmethoxycarbonyl-protected amino acid, purchased from Merck), NMP (N-methylpyrrolidone, purchased from Sigma-Aldrich), DCM (dichloromethane, purchased from Zhongyuan Chemical Co., Ltd.), MeoH (methanol, purchased from Zhongyuan Chemical Co., Ltd.), Piperidine (piperidine, purchased from Sigma-Aldrich), DMAP (dimethylaminopyridine, purchased from Sigma-Aldrich), HOBT (hydroxybenzotriazole, purchased from Sigma-Aldrich), DCC (dicyclohexylcarbodiimide, purchased from Sigma-Aldrich), TFA (trifluoroacetic acid, purchased from Sigma-Aldrich), EDT (1,2-ethylenedithiol, purchased from Sigma-Aldrich), thioanisole (purchased from Guangzhou Weiber Chemical Co., Ltd.), crystalline phenol (purchased from Sinopharm Chemical Reagent Co., Ltd.), acetonitrile (purchased from Sinopharm Chemical Reagent Co., Ltd.).

[0061] 2. Main Instruments

[0062] The automated peptide synthesizer, model 431A, was purchased from ABI Corporation; the rotary evaporator, model R-201, was purchased from Shanghai Shenshun Company; the high-performance liquid chromatograph, Waters 600, was purchased from Waters Corporation, USA; and the freeze dryer, model VFD-2000, was purchased from Beijing Boyikang Company.

[0063] 3. Synthesis methods and processes

[0064] Weigh 100 mg of HMP resin (equivalent to 1.0 meq), and place 0.1 mmol of HMP resin into the reaction chamber of an ABI 431A automated peptide synthesizer. The synthesizer automatically links specific amino acids in different sequences, achieving a coupling rate of 99%. The reaction is as follows:

[0065] (1) Activation of amino acids (HOBt / DCC method)

[0066]

[0067] Fmoc-protected amino acids

[0068]

[0069] (2) Connecting amino acids to resin (HO-Resin)

[0070]

[0071] (3) Removal of the Fmoc protecting group from amino acids

[0072]

[0073] (4) Activation of another amino acid (HOBt / DCC method)

[0074]

[0075] (5) Coupling

[0076]

[0077] (6) Repeat steps (3) to (5) until the synthesis is complete.

[0078] Peptide resins containing sPDGFRβ antigenic epitope peptide (1) and sPDGFRβ antigenic epitope peptide (2) were obtained. sPDGFRβ antigenic epitope peptide (1) and sPDGFRβ antigenic epitope peptide (2) are soluble platelet-derived growth factor receptor β-specific antigenic epitope peptides with sequences shown in SEQ ID NO.1 and SEQ ID NO.2, respectively.

[0079] (7) Peptide cleavage resin

[0080] The peptide chain was cleaved with TFA (trifluoroacetic acid), and EDT (2.5 vol%) and thioanisole (2.5 vol%) were used as scavengers. The reaction was carried out at room temperature for 3.0 hours. After removing the cleavage reagent, the peptide chain was extracted with diethyl ether to obtain crude sPDGFRβ antigenic epitope peptide (1) and sPDGFRβ antigenic epitope peptide (2).

[0081] 4. Purification of crude sPDGFRβ antigenic epitope peptides (1) and (2)

[0082] Separation and purification were performed using high performance liquid chromatography:

[0083] Conditions: Column: C8 10×100mm, purchased from Waters Corporation, USA

[0084] Chromatograph: Waters 600, Waters Corporation, USA

[0085] Mobile phase: A: 0.1% TFA (trifluoroacetic acid) aqueous solution

[0086] B: 0.1% TFA (trifluoroacetic acid) in 60% acetonitrile

[0087] Detection wavelength: 214nm

[0088] Flow rate: 4 mL / min

[0089] Elution gradient: 20-60% B, 30 minutes

[0090] HPLC (High Performance Liquid Chromatography) Analysis

[0091] Chromatographic column: C18 4.6×150mm, purchased from Waters Corporation, USA.

[0092] Mobile phase: A: 0.1% TFA (trifluoroacetic acid) aqueous solution

[0093] B: 0.1% TFA (trifluoroacetic acid) in acetonitrile

[0094] Detection wavelength: 214nm

[0095] Flow rate: 1 mL / min

[0096] Elution gradient: 0-60% B, 30 minutes

[0097] Peptide analysis results showed that the purity of the sPDGFRβ antigenic epitope peptide (1) and sPDGFRβ antigenic epitope peptide (2) prepared in this example was 96.5% and 96.9%, respectively, which can meet the requirements for subsequent use.

[0098] II. Preparation of sPDGFRβ-specific antibodies

[0099] 1. Preparation of antigens

[0100] The sPDGFRβ specific antigenic epitope peptide (1) or (2) was linked to the carrier protein KLH (keyhole hemocyanin) using the BDB (Bis-diazotized benzidine dichloride) method to prepare sPDGFRβ antigen (1) and sPDGFRβ antigen (2), respectively.

[0101] Take 20.0 mg of sPDGFRβ specific antigenic epitope peptide (1) or (2) and dissolve it in 0.2M borate buffer (pH 9.0); take 7.36 mL of KLH 6.25 mg / mL and cool it to 0℃; take 1 mL of BDB and put it into the mixture of ice and water to mix and protect from light; react on a shaker for 1-1.5 h; after the reaction is complete, adjust the pH to 9.0 with 0.2 mol / L NaOH; dialyze overnight and then aliquot and store at -20℃.

[0102] The borate buffer solution is prepared by mixing 80 mL of 0.05 mol / L borax with 20 mL of 0.2 mol / L boric acid.

[0103] 2. Preparation of polyclonal antibodies by immunizing animals

[0104] 2.1. Three-month-old New Zealand white rabbits weighing approximately 2 kg were selected as immunization animals. For the primary immunization, 1-2 mg of the prepared sPDGFRβ antigen (1) or (2) (immunogen) was mixed with an equal volume of Freund's complete adjuvant and thoroughly emulsified before being injected subcutaneously at multiple points on the rabbit's back. A booster immunization was administered every 2-3 weeks, with 1 mL of the antigen and incomplete Freund's adjuvant thoroughly emulsified before being injected subcutaneously at multiple points on the rabbit's back. On the 10th day after the final booster immunization, blood was excised from the carotid artery, and serum was separated.

[0105] 2.2. Antibody titer determination: The titer of sPDGFRβ polyclonal antibody (1) or (2) was determined by indirect ELISA. Specifically, the sPDGFRβ antigenic epitope peptide (1) or (2) was diluted to 2µg / mL with 0.05MCB buffer (pH 9.6), and coated into the microwells of the ELISA plate at 100µL / well. The plate was incubated overnight at 4°C (16-18 hours), washed 3 times, and 200µL of blocking buffer was added. The plate was then blocked at 37°C for two hours and patted dry before use. Rabbit immune serum was added to microplates at serially diluted ratios of 1:1000, 1:2000, 1:4000, 1:8000, 1:16000, 1:32000, 1:64000, 1:128000, and 1:256000. The plates were incubated at 37°C for 1 hour, washed three times, and then enzyme-labeled goat anti-rabbit IgG secondary antibody (1:5000) was added. The plates were incubated at 37°C for 1 hour, washed again, and then substrate was added for 15 minutes for color development. The absorbance of the serially diluted samples was measured using a microplate reader, with a cutoff value of 2.1 times (0.12) compared to the negative control, to determine the antibody titer.

[0106] The results showed that the titers of the sPDGFRβ polyclonal antibody (1) and sPDGFRβ polyclonal antibody (2) prepared in this example both reached 1:32000 or higher.

[0107] 2.3 Blood Collection and Serum Separation: Blood was collected via carotid artery cannulation, and serum was separated. Specifically, the blood was collected into a vacuum blood collection tube without anticoagulant. The blood collection tube was gently inverted 5 times, then placed upright in a test tube rack and allowed to stand at room temperature (approximately 25°C) for 60 minutes. The blood collection tube was then transferred to a 4°C refrigerator, allowed to stand for 60 minutes, centrifuged for 20 minutes, and the supernatant serum was collected.

[0108] 3. Isolation and purification of antibodies

[0109] The sPDGFRβ polyclonal antibody (1) and sPDGFRβ polyclonal antibody (2) to be loaded were dialyzed with a equilibration buffer (0.02M, PB, pH 8.0) until the pH was consistent. The column was then packed and connected to the protein chromatography system, and washed with the equilibration buffer until the pH was 8.0. The sPDGFRβ polyclonal antibody (1) and sPDGFRβ polyclonal antibody (2) to be purified were added to the column. After all the samples were injected, the column was washed with an elution buffer (0.05M, PB, pH 8.0) until the protein analyzer showed the beginning of elution. The protein solution was then collected. Collection was stopped when the peak gradually decreased. The concentration of sPDGFRβ polyclonal antibody (1) or sPDGFRβ polyclonal antibody (2) was measured and calculated using an L5S UV-Vis spectrophotometer (Boda Precision). The results showed that the concentration of sPDGFRβ polyclonal antibody (1) was 2 mg / mL and the concentration of sPDGFRβ polyclonal antibody (2) was 0.5 mg / mL. The purified sPDGFRβ polyclonal antibody (1) and sPDGFRβ polyclonal antibody (2) were aliquoted and stored at -20℃.

[0110] Experiment 1: Preparation of a chemiluminescent kit for sPDGFRβ

[0111] 1. Preparation of magnetic microparticle suspension (working solution of immunomagnetic beads coated with sPDGFRβ polyclonal antibody (1))

[0112] (1) Washing of magnetic beads

[0113] Add 1 mL of 0.1 M MES (pH 6.0) buffer to the coating tube, add 2 mg of JSR magnetic bead stock solution (Shenzhen Ruisi Biotechnology Co., Ltd.), and vortex for 1 min. Place the coating tube on a magnetic separator for 1 min, and discard the supernatant. Add 1 mL of 0.1 M MES (pH 6.0) buffer to the coating tube and vortex for 1 min. Repeat the above process twice.

[0114] (2) Activation of magnetic beads

[0115] Add 800 μL of 0.1M MES (pH 6.0) buffer to the coated tube, then add 100 μL of NHS (10 mg / mL) solution and vortex for 1 min. Add 100 μL of EDC (10 mg / mL) solution to the coated tube. Place the rotary mixer in a constant temperature environment of 25 ± 1 °C, place the coated tube on the rotary mixer, set the rotary mixer speed to 50 ± 1 RPM, and react for 30 min.

[0116] (3) Coating sPDGFRβ polyclonal antibody with activated magnetic beads (1)

[0117] Add 1 mL of 0.1 M MES (pH 6.0) buffer to the coating tube and vortex for 1 min. Add 20 μL of sPDGFRβ polyclonal antibody (1) (1 mg / mL) to the coating tube and vortex for 1 min. At room temperature, add 1 mL of 0.1 M MES (pH 6.0) buffer to the coating tube and vortex for 1 min. Place the rotary mixer in a constant temperature environment of 25 ± 1 °C, place the coating tube on the rotary mixer, set the rotary mixer speed to 50 ± 1 rpm, and react for 2 hours.

[0118] (4) Magnetic bead sealing

[0119] After placing the coated tube on a magnetic separator for 1 minute, discard the supernatant. Add 1 mL of 0.05 M TRIS (pH 7.4) buffer to the coated tube and vortex to mix. Repeat the operation twice. Place the rotary mixer in a constant temperature environment of 25 ± 1 °C, place the coated tube on the rotary mixer, set the rotary mixer speed to 50 ± 1 rpm, and react for 1 hour.

[0120] (5) Cleaning

[0121] After sealing, place the coated tube on a magnetic separator for 1 min, discard the supernatant, and add 1 mL of 0.05 M TRIS (pH 7.4) buffer to the coated tube and vortex for 1 min. Repeat this operation twice. Add 1 mL of 0.05 M TRIS (pH 7.4) buffer to the coated tube and vortex for 1 min.

[0122] (6) Preparation of magnetic microparticle suspension

[0123] The magnetic beads coated with sPDGFRβ polyclonal antibody (1) were diluted with magnetic bead diluent (0.05M TRIS (pH 7.4), 0.5% BSA, 0.9% NaCl) to obtain a final concentration of 2.0 μg / mL of coated antibody.

[0124] 2. Preparation of enzyme-labeled antibody (polyclonal antibody labeled with alkaline phosphatase sPDGFRβ (2))

[0125] (1) Take 250 μL of 0.1M MES (pH 4.5) and soak the ultrafiltration centrifuge tube (30KD) for 2 min.

[0126] (2) Add 250 μL of 0.1 M MES (pH 4.5) to the centrifuge column and bring the volume to 500 μL.

[0127] (3) Add 12.5 μL of AP enzyme to an ultrafiltration centrifuge tube, mix well, centrifuge at 13000 rpm for 15 min, and then discard the waste liquid.

[0128] (4) Add 200 μL of 0.1M MES (pH 4.5) to the ultrafiltration centrifuge tube, then centrifuge at 13000 rpm for 20 min and discard the waste liquid.

[0129] Add 50 μL EDC (10 mg / mL) and 6 μL NHS (10 mg / mL) to an ultrafiltration centrifuge tube, then add 100 μL 0.1M MES (pH 4.5) to a final volume of 250 μL. Mix well and place on a shaker to activate for 1.5 hours.

[0130] (5) Take 10 μL of sPDGFRβ polyclonal antibody (2) 0.5 mg / mL, mix well, centrifuge at 13000 rpm for 20 min and discard the waste liquid.

[0131] (6) Add 250 μL of 0.1 M PB (pH 9.0), mix well, centrifuge at 13000 rpm for 20 min and discard the waste liquid.

[0132] (7) Repeat the experimental steps (6) once.

[0133] (8) Add 0.1M PB (pH 9.0) to a final volume of 250μL and place on a shaker at 120rpm for 2h.

[0134] (9) Add 250 μL of 0.05 M TRIS (pH 8.0) and mix well. Place on a shaker at 120 rpm for 30 min. Take out about 500 μL of the coupled solution from the centrifuge column, add an equal amount of glycerol and mix well. Store at -20℃.

[0135] The concentration of alkaline phosphatase-labeled antibody was measured and calculated using an L5S UV-Vis spectrophotometer (Boda Precision Technology). The antibody was then diluted 1:1000 with 0.05M TRIS (pH 7.4) to obtain the enzyme-labeled antibody.

[0136] 3. Preparation of washing solution

[0137] It consists of 10 mM PBS (pH 7.2), 0.08% Tween-20 and 0.03% Proclin-300.

[0138] 4. Preparation of sPDGFRβ calibrators and quality control samples

[0139] Soluble platelet-derived growth factor receptor β recombinant protein (provided by Chengdu Cormorant Biotechnology, concentration 2 mg / mL) was serially diluted with calibrator diluent (containing 10 mM phosphate-buffered saline (PBS) (pH 7.2), 1% BSA, and 0.03% biological preservative Proclin-300) to prepare a series of calibrators (0.5–50 ng / mL) and two quality control samples of high and low concentrations.

[0140] 5. Preparation of luminescent substrates

[0141] It consists of the chemiluminescent substrate (4-chlorophenylmercapto)(10-methyl-9,10-dihydroacrylamide) disodium phosphate (APS-5) and a 0.3M Tris buffer containing 0.0003% glucosamine, 0.001% sodium sulfite, 0.1% sodium dodecyl sulfate (SDS), and 0.03% Tween 20.

[0142] 6. Components of the reagent kit

[0143] The kit mainly consists of reagent compartments (reagent 1, reagent 2), sPDGFRβ calibrator A (1.0mL×1 vial, concentration 300pg / mL), calibrator B (1.0mL×1 vial, concentration 15000pg / mL), quality control (1.0mL×1 vial, concentration 5000pg / mL), substrate solution (12.0mL / 25.0mL), concentrated washing solution (25×) (60.0mL×1 bottle / 60.0mL×2 bottles), and calibration / quality control information cards. Reagent 1 in the reagent compartment contains micromagnetic particles coated with sPDGFRβ polyclonal antibody (1), and reagent 2 contains alkaline phosphatase (AP) labeled sPDGFRβ polyclonal antibody (2). The kit is available in two packaging specifications: 50 doses / box and 100 doses / box.

[0144] The kit includes a substrate solution (the luminescent substrate solution), available in 12mL and 25mL vials for kits with 50 and 100 tests per kit, respectively. A concentrated washing buffer (the concentrated wash solution) should be diluted 25 times before use; similarly, one and two vials are available for kits with 50 and 100 tests per kit, respectively. An information card contains the calibration curve and quality control concentration information.

[0145] Experiment 2: Preparation of a chemiluminescent kit for sPDGFRβ

[0146] 1. Preparation of magnetic microparticle suspension reagent (working solution of immunomagnetic beads coated with sPDGFRβ monoclonal antibody)

[0147] (1) Washing of magnetic beads

[0148] Add 1 mL of 0.1 M MES (pH 6.0) buffer to the coating tube, add 2 mg of JSR magnetic bead stock solution (Shenzhen Ruisi Biotechnology Co., Ltd.), and vortex for 1 min. Place the coating tube on a magnetic separator for 1 min, and discard the supernatant. Add 1 mL of 0.1 M MES (pH 6.0) buffer to the coating tube and vortex for 1 min. Repeat the above process twice.

[0149] (2) Activation of magnetic beads

[0150] Add 800 μL of 0.1M MES (pH 6.0) buffer to the coated tube, then add 100 μL of NHS (10 mg / mL) solution and vortex for 1 min. Add 100 μL of EDC (10 mg / mL) solution to the coated tube. Place the rotary mixer in a constant temperature environment of 25 ± 1 °C, place the coated tube on the rotary mixer, set the rotary mixer speed to 50 ± 1 RPM, and react for 30 min.

[0151] (3) Coating sPDGFRβ monoclonal antibody with activated magnetic beads

[0152] Add 1 mL of 0.1 M MES (pH 6.0) buffer to the coating tube and vortex for 1 min. Add 40 μL of sPDGFRβ monoclonal antibody (0.5 mg / mL) (MAB1263, purchased from seeBio) to the coating tube and vortex for 1 min. At room temperature, add 1 mL of 0.1 M MES (pH 6.0) buffer to the coating tube and vortex for 1 min. Place the rotary mixer in a constant temperature environment of 25 ± 1 °C, place the coating tube on the rotary mixer, set the rotary mixer speed to 50 ± 1 rpm, and react for 2 hours.

[0153] (4) Magnetic bead sealing

[0154] After placing the coated tube on a magnetic separator for 1 minute, discard the supernatant. Add 1 mL of 0.05 M TRIS (pH 7.4) buffer to the coated tube and vortex to mix. Repeat the operation twice. Place the rotary mixer in a constant temperature environment of 25 ± 1 °C, place the coated tube on the rotary mixer, set the rotary mixer speed to 50 ± 1 rpm, and react for 1 hour.

[0155] (5) Cleaning

[0156] After sealing, place the coated tube on a magnetic separator for 1 min, discard the supernatant, and add 1 mL of 0.05 M TRIS (pH 7.4) buffer to the coated tube and vortex for 1 min. Repeat this operation twice. Add 1 mL of 0.05 M TRIS (pH 7.4) buffer to the coated tube and vortex for 1 min.

[0157] (6) Preparation of magnetic microparticle suspension reagent

[0158] The magnetic beads coated with sPDGFRβ monoclonal antibody were diluted with magnetic bead dilution buffer (0.05M TRIS (pH 7.4), 0.5% BSA, 0.9% NaCl) to obtain a final antibody concentration of 2.0 μg / mL.

[0159] 2. Preparation of enzyme-labeled antibody (polyclonal antibody labeled with alkaline phosphatase sPDGFRβ (2))

[0160] Same as Experiment 1.

[0161] 3. Preparation of sPDGFRβ calibrators / quality control samples, washing solution, and luminescent substrates

[0162] Same as Experiment 1.

[0163] 4. Components of the reagent kit

[0164] The kit mainly consists of reagent compartments (reagent 1, reagent 2), sPDGFRβ calibrators A and B (1.0 mL × 1 vial), quality control (1.0 mL × 1 vial), substrate solution (12.0 mL / 25.0 mL), concentrated washing solution (25 × 1) (60.0 mL × 1 bottle / 60.0 mL × 2 bottles), and calibration / quality control information cards. Reagent 1 in the reagent compartment contains micromagnetic particles coated with sPDGFRβ monoclonal antibody, and Reagent 2 contains alkaline phosphatase (AP) labeled sPDGFRβ polyclonal antibody (2). The kit is available in two packaging specifications: 50 doses / box and 100 doses / box.

[0165] Experiment 3: Preparation of ELISA assay kit

[0166] 1. Preparation of various buffer solutions and reagents:

[0167] 1.1 Coating buffer: 0.05M, pH 9.6, CB (carbonate buffer)

[0168] Na2CO3: 16.0 g, NaHCO3: 29.0 g, add deionized water to a final volume of 1000 mL.

[0169] 1.2, pH7.2, 10×PBS-Tween 20

[0170] Na2HPO4·12H2O: 58 g, KH2PO4: 4 g, NaCl: 100 g, KCl: 4 g, Tween 20: 20 mL, deionized water to a final volume of 1000 mL.

[0171] 1.3 Blocking buffer / antibody diluent:

[0172] 10×PBS-Tween 20: 100mL, BSA (bovine serum albumin): 10g, biological preservative (Proclin-300, purchased from Shanghai Xibao Company): 1mL, deionized water to a final volume of 1000mL.

[0173] 1.4 Enzyme label dilution buffer:

[0174] 10×PBS-Tween 20: 10 mL, FCS (fetal bovine serum): 20 mL, enzyme stabilizer (purchased from Shanghai Xibao Company, model ACE0070A): 1 g, biological preservative (Proclin-300, purchased from Shanghai Xibao Company): 1 mL, deionized water to a final volume of 1000 mL.

[0175] 1.5. Color developer A:

[0176] Citric acid: 35.5 g, urea peroxide: 10 g, Tween 20: 10 mL, deionized water to a final volume of 1000 mL.

[0177] 1.6. Color developer B:

[0178] Citric acid: 120 g, EDTA-2Na: 1 g, TMB·2HCl: 2 g, deionized water to a final volume of 1000 mL.

[0179] 1.7 Concentrated washing solution (pH 7.2, 25×PBS-Tween 20)

[0180] Na2HPO4·12H2O: 145 g, KH2PO4: 10 g, NaCl: 250 g, KCl: 10 g, Tween 20: 50 mL, deionized water to a final volume of 1000 mL.

[0181] 1.8 Termination solution: 2M H2SO4

[0182] Concentrated sulfuric acid (95-98%): 22.2 mL, deionized water: 177.3 mL. When preparing, slowly add the concentrated sulfuric acid dropwise to the deionized water and dilute to the final volume while shaking.

[0183] 2. Preparation of pre-coated plates

[0184] Dissolve the sPDGFRβ specific polyclonal antibody (1) in coating buffer to prepare a pre-coating solution. Add 100 μL of 0.1 μg / well to each well of an enzyme-labeled plate (purchased from Shenzhen Jincanhua Company), place at 4℃ for 18-24 hours, remove, discard the coating solution, wash, add 100 μL of blocking solution to each well, block at 4℃ for 16 hours, discard the blocking solution, air dry, pack into an aluminum foil bag, seal, and store at 4℃.

[0185] 3. Formulate by combining antibodies and enzyme markers

[0186] The conjugate antibody (sPDGFRβ specific polyclonal antibody (2)) and enzyme label (horseradish peroxidase labeled goat anti-rabbit IgG antibody, purchased from Beijing Zhongshan Jinqiao Company) were diluted with antibody diluent to the working concentration, which was determined by a checkerboard titration experiment.

[0187] 4. Preparation of sPDGFRβ calibrators and quality control samples

[0188] The recombinant sPDGFRβ (provided by Chengdu Cormorant Biotechnology, concentration 2 mg / mL) was serially diluted with sample diluent to prepare sPDGFRβ calibrators (25–18000 pg / mL) and two quality control samples with high and low concentrations.

[0189] 5. Components of the ELISA kit

[0190] The kit mainly consists of pre-coated plates (48 or 96 doses), one set of sPDGFRβ calibrators, sPDGFRβ quality control samples (high and low concentrations), binding antibody (10 mL), enzyme label (10 mL), chromogenic solution A (5 mL), chromogenic solution B (5 mL), concentrated washing buffer (20 mL), and stop solution (5 mL). Chromogenic solution A and chromogenic solution B are the prepared chromogenic reagents A and B, respectively.

[0191] Experiment 4: Preparation of sPDGFRβ-fluorescence chromatography assay kit

[0192] 1. Wrapped with padding

[0193] 1.1 Fluorescent microspheres labeled with sPDGFRβ polyclonal antibody (1)

[0194] 1.1.1 Activation of fluorescent microspheres:

[0195] (1) Take 500 μL of fluorescent microspheres (purchased from Bangs Laboratories, Inc.) with a content of 1 (w / v)% in aqueous dispersion, add initial wash buffer (50 mM MES aqueous solution, pH 6.5) to 1 mL, centrifuge at 16000 rpm for 20 minutes at 4 °C, remove the supernatant, disperse the precipitate in 1 mL of initial wash buffer, and sonicate (240 W) for 2 minutes.

[0196] (2) Repeat the above process twice;

[0197] (3) Add 375 μL (3 / 4 of the microspheres) of a mixture of 10 mg / mL carbodiimide solution and 10 mg / mL N-hydroxythiosuccinimide solution in a 1:3 ratio, shake for 15 minutes, and activate the fluorescent microspheres.

[0198] 1.1.2. Label sPDGFRβ polyclonal antibody with activated fluorescent microspheres (1):

[0199] (1) Disperse the precipitate in 1 mL of coupling buffer (50 mM MES aqueous solution, pH 6.0) and sonicate (240 W) for 2 minutes;

[0200] (2) Repeat the above process twice;

[0201] (3) Obtain 500 μL of buffer solution containing fluorescent microspheres;

[0202] (4) Add sPDGFRβ polyclonal antibody (1) to the activated fluorescent microspheres at a ratio of 15 mg antibody / g, and shake at room temperature for 2 hours;

[0203] (5) Add 1 mL of blocking buffer (0.5 (w / v)% BSA-0.05 M ethanolamine), continue shaking for 1 hour, then centrifuge at 16000 rpm for 20 minutes, repeat centrifugation 3 times, disperse the precipitate into 500 μL of final wash buffer (0.5 (w / v)% BSA-0.1 (v / v)% Tween-20 mM Tris solution), sonicate (240 W) for 2 minutes, and adjust the volume to 500 μL with the above final wash buffer.

[0204] 1.2, Covered with a bonding pad

[0205] The sPDGFRβ polyclonal antibody (1) labeled with fluorescent microspheres prepared above was diluted with microsphere diluent (0.5(w / v)%BSA-2(w / v)%S9-15%sucrose-0.5%PVP-40000-0.5%PEG20000-20mM Tris solution) at a ratio of 1:240 for microspheres to diluent to obtain working solution. Then, it was evenly sprayed onto the conjugate pad at a volume of 1200μL / 30cm using a micropipette (purchased from DRAGON). After that, it was dried in an oven at 37℃ and stored at 45% humidity for later use.

[0206] 2. Preparation of the reaction membrane

[0207] The sPDGFRβ polyclonal antibody (2) and goat anti-mouse IgG monoclonal antibody (purchased from Arista) were diluted to 0.5 mg / mL with 1% (w / v) PEG20000-5% (v / v) methanol-3% (w / v) sucrose 10mM PBS (pH 8.4) buffer. The detection line and control line spacing parameters of the gold spraying machine (purchased from Hangzhou Fenghang Company) were set to 8 mm, and the coating amount was set to 1.0 μL / cm. The sPDGFRβ polyclonal antibody (2) and goat anti-mouse IgG monoclonal antibody were sprayed onto the nitrocellulose membrane using the gold spraying machine. The membrane was dried in a 37°C oven and stored at 45% humidity for later use.

[0208] 3. Assembly and cutting of test strips

[0209] The sample pad, conjugate pad, NC reaction membrane, and absorbent filter paper are sequentially overlapped and pasted onto the base plate to obtain the test paper sheet, which is then cut into test paper strips with a width of 4mm.

[0210] 4. Preparation of sPDGFRβ fluorescent immunoassay card

[0211] Fix the cut test strips onto the plastic base card, and press the surface of the test strips firmly with the face card. The face card has sample application holes and observation windows at the positions of the sample pad and reaction membrane on the test strip. After assembling the test card, put it into an aluminum foil bag, add desiccant, seal and store. It can be stored for more than one year under dry conditions at room temperature.

[0212] 5. Composition of the sPDGFRβ-fluorescence chromatography assay kit (20T)

[0213] The kit mainly consists of a test card and an ID card. The test card is packaged for single use and consists of a test strip and a plastic card. The main components of the test strip are: nitrocellulose membrane, sample pad, absorbent pad, and PVC base. The detection area (T line) of the nitrocellulose membrane is coated with sPDGFRβ polyclonal antibody (2), and the control area (C line) is coated with goat anti-mouse IgG antibody (goat-derived). The sample pad is made of glass fiber and coated with fluorescent microsphere-labeled sPDGFRβ polyclonal antibody (1).

[0214] Test Experiment 1: Determination of sPDGFRβ using the chemiluminescence reagent kit in Experiment 1 and validation of kit performance.

[0215] 1. Plasma sample: Fresh plasma samples are preferred. They can be stored for 7 days at 2℃-8℃ and for one year at -20℃. When using the sample, remove it from the refrigerator, let it stand at room temperature, and then mix it well.

[0216] 2. The chemiluminescent immunoassay (CLIA) kit is used to determine the concentration of sPDGFRβ. The specific steps are as follows:

[0217] The specific steps are divided into testing procedures and calibration procedures.

[0218] Testing procedure:

[0219] (1) Concentrated cleaning solution (25×): Dilute the concentrated cleaning solution 25 times to the cleaning concentration to be used. For a 50-person batch, bring the volume to 1.5L, and for a 100-person batch, bring the volume to 3.0L.

[0220] (2) When loading the reagent kit onto the chemiluminescence analyzer (Shenzhen Yingkai Biotechnology, fully automated chemiluminescence immunoassay analyzer, model: Shinei1900) for the first time, the magnetic beads in reagent 1 in the reagent compartment need to be mixed well to resuspend the magnetic particles that have precipitated during storage and transportation.

[0221] (3) After the calibrator is taken out from 2℃~8℃, it should be equilibrated to room temperature. Before use, gently turn it over and mix it. After use, tighten the cap and put the calibrator back into storage at 2℃~8℃.

[0222] (4) Perform the calibration procedure: Place the calibrator in the sample rack, manually scan the reagent information in the reagent compartment, the main curve information on the calibration / quality control information card, and the QR code information of the calibrator, click calibration, select the location to place the calibrator, and the instrument will automatically perform the calibration procedure.

[0223] (5) Check the sample volume in the sample cup to ensure that the sample volume in the sample cup is above 300 μL before each test. If a blood collection tube is used, the liquid volume of the sample should be sufficient and not less than 300 μL.

[0224] (6) Sample injection: For information on sample injection, please refer to the instruction manual for the chemiluminescence analyzer.

[0225] (7) Click the instrument run button. The luminescence analyzer will perform the pre-set operation and detection (total detection time is about 25 minutes).

[0226] Calibration procedure:

[0227] (1) When using a new batch of reagents, the standard curve data must be entered and then calibration is performed; when the chemiluminescence analyzer is calibrated, each calibrator will be tested twice. After the test is successful, the sample can be tested directly without repeated calibration.

[0228] (2) Calibrator A and calibrator B need to be replicated for calibration experiments;

[0229] (3) Conduct the experiment according to the operation manual of the luminescence analyzer.

[0230] 3. Performance verification of the reagent kit in Experiment 1

[0231] 3.1 Linearity Range Verification

[0232] Samples close to the upper limit of the kit's linear range were serially diluted with calibrator diluent. Each diluted sample was measured twice, and the average concentration (yi) was calculated. A linear regression was performed with the dilution ratio (xi) as the independent variable and the corresponding average concentration (yi) as the dependent variable. The linear correlation coefficient (r) was calculated.

[0233] The test results are shown in Table 1. The sample dilution in Table 1 refers to the dilution of the calibrator mixed with the diluent. For example, 100% means the calibrator is undiluted, 33.3% means 100 μL of calibrator is mixed with 200 μL of diluent, and so on.

[0234] Table 1. Linearity range test results and calculations (unit: pg / mL)

[0235] Sample dilution Measurement 1 Measurement 2 mean 100% 17620 17891 17755.5 33.30% 6210 5932 6071 11.10% 2001 1913 1957 3.70% 700 682 691 1.20% 212 236 224 0.14% 27 33 30

[0236] The test results show that the kit prepared in Experiment 1 has a wide linear range in the range of 25 to 18000 pg / mL, with the linear equation formula: y=17761x+31.028 and a linear correlation coefficient of 0.9999. This indicates that the kit has a wide linear range.

[0237] 3.2 Minimum Detection Limit

[0238] The kit prepared in Experiment 1 was used to repeatedly test the zero-concentration reference sample 20 times. The average (M) and standard deviation (SD) of the 20 test concentrations were calculated. The limit of detection was then calculated as M + 2SD.

[0239] The test results showed that the limit of detection was 25 pg / mL, indicating that the kit has high sensitivity.

[0240] 3.3 Precision

[0241] Precision was validated on samples at two concentration levels, high and low, with each concentration tested 10 times using the kit from Example 1. The two concentrations were 300 pg / mL and 15000 pg / mL.

[0242] The mean and standard deviation (SD) of 10 measurements for each concentration sample were calculated. The coefficient of variation (CV) was calculated using the formula CV = SD / mean × 100% to evaluate the precision of the kit.

[0243] The test results are shown in Table 2 below.

[0244] Table 2 Precision Test Results

[0245] Test serial number Test concentration 300 pg / mL Test concentration 15000 pg / mL 1 310 15236 2 276 14788 3 298 14685 4 279 15022 5 288 15123 6 293 13998 7 301 14852 8 308 14269 9 293 15298 10 296 14009 mean 294.2 14728 Standard deviation SD 10.52 459.0 CV% 3.58% 3.12%

[0246] The test results show that the CV% of the kit prepared in Experiment 1 is less than 10% for both high and low concentration samples, indicating that the kit has high precision.

[0247] 3.4 Accuracy

[0248] The accuracy of the reagent kit was evaluated using a recovery test.

[0249] A high-concentration sample A is added to a low-concentration sample B, with a volume ratio of 1:9 between the added sample A and sample B. The concentrations of sample B and the mixed sample are then measured, and the recovery rate is calculated using the following formula.

[0250]

[0251] Where: R—recovery rate; V—volume of sample A added; V0—volume of sample B; C—detection concentration of sample B after adding sample A; C0—detection concentration of sample B; CS—concentration of sample A.

[0252] The recovery rate was calculated to be 98.21%, indicating that the kit has high accuracy.

[0253] Verification conclusion:

[0254] The validation results of the kit prepared in Experiment 1 showed that the kit has a wide linear range and can directly measure samples with concentrations up to 18,000 pg / mL without dilution; it has high sensitivity with a limit of detection of 25 pg / mL; it has high accuracy with a recovery rate of 98.21%; it has good precision with CV% of 3.58% and 3.12% for high and low concentrations, respectively; and the entire reaction time is short, with results available in 30 minutes, which greatly improves the detection efficiency.

[0255] Application Experiment 2: Determination of sPDGFRβ using the chemiluminescence kit from Experiment 2 and validation of kit performance.

[0256] 1. Sample collection: Same as application experiment 1.

[0257] 2. The concentration of sPDGFRβ was determined using a chemiluminescent immunoassay (CLIA) kit. The specific steps are the same as in application experiment 1.

[0258] 3. Reagent kit performance validation

[0259] 3.1 Linearity Range Verification

[0260] Same as application test 1

[0261] The test results are shown in Table 3 below.

[0262] Table 3. Linearity range test results and calculations (unit: pg / mL)

[0263] Sample dilution Measurement 1 Measurement 2 mean 100% 16629 17200 16914.5 33.30% 6311 6732 6521.5 11.10% 2429 1745 2087 3.70% 621 682 651.5 1.20% 199 246 222.5 0.14% 27 43 35

[0264] The test results show that the kit prepared in Experiment 2 has a wide linear range in the range of 25~18000 pg / mL, with the linear equation formula: y=16958x+181.63 and a linear correlation coefficient of 0.9972, indicating that the kit has a wide linear range.

[0265] 3.2 Minimum Detection Limit

[0266] Same as application test 1

[0267] The test results showed that the limit of detection was 25 pg / mL, indicating that the kit has high sensitivity.

[0268] 3.3 Precision

[0269] The validation method was the same as in application experiment 1. The two concentrations used for precision validation were 300 pg / mL and 15000 pg / mL.

[0270] The test results are shown in the table below.

[0271] Table 4 Precision Test Results

[0272] Serial Number Test concentration 300 pg / mL Test concentration 15000 pg / mL 1 311 15690 2 325 14360 3 310 14526 4 299 15963 5 310 15990 6 278 15015 7 303 14021 8 298 13200 9 301 14892 10 341 14360 mean 307.6 14801.7 Standard deviation SD 15.97 851.15 CV% 5.19% 5.75%

[0273] The test results show that the CV% of the kit prepared in Experiment 2 is less than 10% for both high and low concentration samples, indicating that the kit has high precision.

[0274] 3.4 Accuracy

[0275] The verification method is the same as that used in application experiment 1.

[0276] The recovery rate was calculated to be 96.01%, indicating that the kit has high accuracy.

[0277] Verification conclusion:

[0278] Experiment 2 validated the results, showing that the kit has a wide linear range, allowing direct measurement of samples with concentrations up to 18,000 pg / mL without dilution; high sensitivity, with a limit of detection of 25 pg / mL; high accuracy, with a recovery rate of 96.01%; good precision, with CV% of 5.19% for high concentration and 5.75% for low concentration; and a short reaction time, with results available in 30 minutes, greatly improving detection efficiency.

[0279] Application Experiment 3: Determination of sPDGFRβ using the enzyme-linked immunosorbent assay (ELISA) kit prepared in Experiment 3 and validation of kit performance.

[0280] 1. Sample collection: Same as application experiment 1.

[0281] 2. The sPDGFRβ concentration was determined using an enzyme-linked immunosorbent assay (ELISA) kit. The specific steps are as follows:

[0282] (1) Preparation of washing solution:

[0283] Dilute the 25x concentrated detergent solution with deionized water at a ratio of 1:25.

[0284] (2) Take out the kit prepared in Experiment 3 and bring it to room temperature. Prepare the samples, calibrators, and quality control materials. Dilute the clinical samples with sample diluent at a ratio of 1:20.

[0285] (3) Add 50 μL of diluted test sample / calibrator / quality control to each well of the microplate, then add 50 μL of binding antibody to the corresponding well, gently tap to mix, seal the microplate with sealing film, and incubate at 37°C for 30 minutes.

[0286] (4) Remove the reaction plate, discard the liquid in the plate, add 200-300 μL of washing solution to each well and wash 5 times, then pat dry;

[0287] (5) Add 100 μL of enzyme-labeled material to each well, seal the plate with sealing film, and incubate at 37°C for 30 minutes;

[0288] (6) Remove the reaction plate, discard the liquid in the plate, add 200-300 μL of washing solution to each well and wash 5 times, then pat dry;

[0289] (7) Add 50 μL of colorimetric reagent A and B solution to each well, mix thoroughly, and incubate at 37°C for 15 minutes;

[0290] (8) Add 1 drop (50 μL) of stop solution to each well as soon as possible and gently tap to mix;

[0291] (9) Measure the OD value of each well using an ELISA reader (with dual wavelengths of 450 / 630 nm);

[0292] (10) Plot the calibration curve and calculate the concentration of sPDGFRβ in the sample based on the calibration curve.

[0293] 3. Reagent kit performance validation

[0294] 3.1 Linearity Range Verification

[0295] The verification method is the same as that used in application experiment 1.

[0296] The test results are shown in Table 5 below.

[0297] Table 5. Linearity range test results and calculations (unit: ng / mL)

[0298] Sample dilution Measurement 1 Measurement 2 mean 100% 9.52 9.83 9.675 50% 4.51 4.9 4.705 25% 2.26 2.3 2.28 10% 0.97 1.23 1.1 5.00% 0.51 0.54 0.525 1% 0.28 0.32 0.3

[0299] The test results show that the kit prepared in Experiment 3 has a wide linear range in the range of 0.15 to 10 ng / mL, with the linear equation formula: y = 9.5115x + 0.0697 and a linear correlation coefficient of 0.9988. This indicates that the kit has a wide linear range.

[0300] 3.2 Minimum Detection Limit

[0301] The verification method is the same as that used in application experiment 1.

[0302] The test results showed that the limit of detection was 0.15 ng / mL, indicating that the kit has high sensitivity.

[0303] 3.2 Precision

[0304] The validation method was the same as in application test 1. The two concentrations used for precision validation were 2 ng / mL and 8 ng / mL.

[0305] The test results are shown in Table 6 below.

[0306] Table 6 Precision Test Results

[0307] Serial Number Test concentration 2ng / mL Test concentration 8 ng / mL 1 1.78 7.60 2 2.13 7.96 3 1.79 8.21 4 1.69 7.69 5 2.03 8.01 6 2.10 8.13 7 1.81 7.76 8 1.94 7.77 9 1.93 7.93 10 2.04 8.12 mean 1.92 7.94 Standard deviation SD 0.14 0.20 CV% 7.44% 2.51%

[0308] The test results show that the CV% of the kit prepared in Experiment 3 is less than 10% for both high and low concentration samples, indicating that the kit has high precision.

[0309] 3.4 Accuracy

[0310] The verification method is the same as that used in application experiment 1.

[0311] The recovery rate was calculated to be 97.65%, indicating that the kit has high accuracy.

[0312] Verification conclusion:

[0313] The validation results of the kit prepared in Experiment 3 showed that the kit had good linearity in the range of 0.15–10 ng / mL, reaching 0.999; high sensitivity, with a limit of detection of 0.15 ng / mL; recovery rate of 97.65%; high accuracy and precision; and CV% of both high and low concentrations less than 10%. It does not require complex equipment and can be used for batch detection, with more than 80 test results available simultaneously in 1.5 hours, demonstrating high detection efficiency.

[0314] Application Experiment 4: Validation of the performance of the fluorescence immunochromatographic kit used in Experiment 4 for the determination of sPDGFRβ.

[0315] 1. Sample collection: Same as application experiment 1.

[0316] 2. The concentration of sPDGFRβ was determined using a fluorescence immunochromatographic assay kit. The specific steps are as follows:

[0317] (1) Before testing, bring the reagents to room temperature (20±5℃), and conduct the test at room temperature;

[0318] (2) Turn on the fluorescence analyzer and adjust the instrument to the ready-to-detect state;

[0319] (3) Add 100 μL of each of the calibrator / quality control sample diluted 1:4 to the sample port of each test card and react for 15 min;

[0320] (4) After the reaction is complete, insert the test card into the fluorescence analyzer for scanning test, and calculate the sPDGFRβ concentration of each sample according to the built-in calibration curve of the analyzer.

[0321] 3. Reagent kit performance validation

[0322] 3.1 Linearity Range Verification

[0323] The testing method is the same as in application experiment 1.

[0324] The test results are shown in Table 7 below.

[0325] Table 7. Linearity Range Test Results and Calculations (Unit: ng / mL)

[0326] Sample dilution Measurement 1 Measurement 2 mean 100% 9.73 9.87 9.8 50% 3.99 4.98 4.485 25% 1.96 2.34 2.15 10% 1 1.2 1.1 5.00% 0.49 0.51 0.5 1% 0.23 0.31 0.27

[0327] The test results show that, within the range of 0.156–10 ng / mL, the linear equation of the kit in Experiment 4 is y = 9.6092x - 0.0081, and the linear correlation coefficient can reach 0.9962, indicating that the kit has a wide linear range.

[0328] 3.2 Minimum Detection Limit

[0329] The testing method is the same as in application experiment 1.

[0330] The test results showed that the limit of detection was 0.156 ng / mL.

[0331] 3.2 Precision

[0332] The test method was the same as in application test 1. The two concentrations used for precision verification were 2 ng / mL and 8 ng / mL.

[0333] The test results are shown in Table 8 below.

[0334] Table 8 Precision Test Results

[0335] Serial Number Test concentration 2ng / mL Test concentration 8 ng / mL 1 2.01 8.52 2 2.25 7.86 3 1.96 8.26 4 1.99 8.44 5 2.32 7.84 6 1.95 7.31 7 1.86 7.75 8 2.13 8.66 9 2.06 7.56 10 2.14 7.78 mean 2.07 8.00 Standard deviation SD 0.14 0.42 CV% 6.56% 5.29%

[0336] The test results show that the CV% of the kit prepared in Experiment 4 is less than 10% for both high and low concentration samples, indicating that the kit has high precision.

[0337] 3.4 Accuracy

[0338] The verification method is the same as that used in application experiment 1.

[0339] The recovery rate was calculated to be 96.34%, which meets the recovery rate requirement (90%–110%).

[0340] Verification conclusion:

[0341] The validation results of the kit prepared in Experiment 4 showed that the kit had good linearity in the range of 1.56 to 10 ng / mL, reaching 0.9962, with a sensitivity of 0.156 ng / mL, a recovery rate of 96.34%, and met the accuracy requirements. The precision was good, and the CV% of both high and low concentrations was less than 10%. It can be used for point-of-care diagnosis, is easy to operate, can be tested on demand, and results can be obtained in 15 minutes, making the detection rapid.

[0342] Test Comparison Example 1:

[0343] A chemiluminescence kit (comparative kit) was prepared using sPDGFRβ antibodies of AF385 and MAB1263 (purchased from seeBio) according to the method in Experiment 1, and its analytical performance was compared with that of the kit in Experiment 1.

[0344] The testing methods for each performance aspect are the same as in application test 1, and the comparison results are shown in Table 9 below.

[0345] Table 9 Performance comparison results between the comparison kit and the kit in Experiment 1

[0346]

[0347] In the comparison of various analytical performances, the kit prepared in Experiment 1 had a wider linear detection range and better performance in other indicators.

[0348] Clinical Trial 1: sPDGFRβ Assay Kit for the Auxiliary Diagnosis and Disease Monitoring of Cognitive Impairment Related to Blood-Brain Barrier (BBB) ​​Dysfunction

[0349] 1. Research Subjects

[0350] This study used serum sPDGFRβ as a biomarker for blood-brain barrier (BBB) ​​rupture to preliminarily explore the changes in serum sPDGFRβ in patients with BBB dysfunction and its relationship with the degree of cognitive impairment in the auxiliary diagnosis and disease monitoring.

[0351] This study and its procedures have been approved by the institutional review board, indicating compliance with all ethical requirements, and informed consent was obtained from all participants prior to enrollment. All participants underwent neurological and neuropsychological assessments using the Uniform Dataset (UDS). Assessments were based on T1-weighted MPRAGE scans, resting-state functional magnetic resonance imaging (rsfMRI) scans, and Clinical Dementia Rating Scale (CDR) scores, with the CDR assessment strictly following a standardized interview process. All participants underwent a comprehensive clinical interview, including history taking and physical examination, supplemented by consultations with relevant specialists. According to the study classification criteria, participants with a CDR score of 0 were considered to have no cognitive impairment, while those with a score higher than 0 were classified as having cognitive impairment. The inclusion criteria included 150 male and female participants aged 45 to 90 years with a total score of 0, 0.5 or 1 on the Clinical Dementia Rating Scale (CDR), corresponding to intact cognitive function, mild cognitive impairment and mild dementia, respectively. Among them, there were individuals with normal cognition, CDR 0 (n=20, young group; n=60, old group), CDR 0.5 (n=35) and CDR 1 (n=35) patients.

[0352] 2. Experimental Methods

[0353] The kit prepared in Example 1 of this invention was used to test the sPDGFRβ levels in serum samples from cognitively normal individuals with CDR0 (n=20, young group; n=60, elderly group), as well as patients with CDR 0.5 (n=35) and CDR 1 (n=35).

[0354] 3. Results

[0355] 3.1 Comparative analysis of sPDGFRβ levels between young and elderly individuals with normal cognition

[0356] like Figure 1 As shown, a comparative analysis of serum sPDGFRβ levels in 80 individuals with normal cognition and CDR (0) in the young and elderly groups revealed that, under CI (95%) conditions, the serum sPDGFRβ level in the young CDR (0) group (525.25 ± 16.13 pg / mL) was significantly lower than that in the elderly CDR (0) group (663.86 ± 8.22 pg / mL), with a significant difference (p < 0.001). This indicates that serum sPDGFRβ levels are significantly positively correlated with age, suggesting that sPDGFRβ, as a sensitive marker reflecting the integrity of the blood-brain barrier and pericyte damage in cerebral blood vessels, predicts that the process of cerebrovascular injury is age-related.

[0357] 3.2 Comparative analysis of sPDGFRβ levels in patients with different degrees of CDR (0, 0.5, 1)

[0358] like Figure 2 As shown, a comparative analysis of serum sPDGFRβ levels in 80 individuals with normal cognition (CDR 0), 35 individuals with mild cognitive impairment (CDR 0.5), and 35 individuals with mild dementia (CDR 1) revealed that, under CI (95%) conditions, their serum sPDGFRβ levels were 629.20 ± 9.95 pg / mL, 737.14 ± 8.07 pg / mL, and 791.09 ± 12.0 pg / mL, respectively. This indicates that sPDGFRβ levels vary among patients with different degrees of cognitive impairment, with higher severity patients exhibiting higher sPDGFRβ levels. This suggests that serum sPDGFRβ levels are of significant importance in the auxiliary diagnosis of CDR patients with different degrees of severity in the BBB.

[0359] The above description, in conjunction with specific embodiments, provides a further detailed explanation of this application and should not be construed as limiting the specific implementation of this application to these descriptions. Those skilled in the art to which this application pertains can make several simple deductions or substitutions without departing from the concept of this application.

Claims

1. An antibody for detecting soluble platelet-derived growth factor receptor β, characterized in that: The antibody is prepared by immunizing animals with a soluble platelet-derived growth factor receptor β-specific antigenic epitope peptide coupled with a carrier protein, wherein the amino acid sequence of the soluble platelet-derived growth factor receptor β-specific antigenic epitope peptide is at least one of the sequences shown in SEQ ID NO.1 and SEQ ID NO.

2. SEQ ID NO.1: Tyr-Arg-Val-Ser-Glu-Leu-Thr-Leu-Val-Arg-Val-Lys-Val-Ala-Glu-Ala-Gly SEQ ID NO.2: Tyr-Leu-Glu-Leu-Gln-Val-Glu-Pro-Glu-Pro-Glu-Leu-Glu.

2. The antibody according to claim 1, characterized in that: The antibody is a polyclonal antibody prepared by immunizing animals with the soluble platelet-derived growth factor receptor β-specific antigenic epitope peptide conjugated to a carrier protein.

3. A kit for detecting soluble platelet-derived growth factor receptor β, characterized in that: Contains the antibody as described in claim 1 or 2.

4. The reagent kit according to claim 3, characterized in that: It includes a solid support and the antibody, wherein the antibody is directly or indirectly attached to the solid support; Optionally, the solid support is at least one of a microporous reaction plate, magnetic microspheres, and nitrocellulose membrane.

5. The reagent kit according to claim 4, characterized in that: It also includes markers for labeling the antibodies; Optionally, the marker is at least one of horseradish peroxidase (HRP), alkaline phosphatase (AP), a luminescent substance, a fluorescent substance, a dye, and colloidal gold.

6. The reagent kit according to claim 4, characterized in that: It also includes soluble platelet-derived growth factor receptor β standards.

7. The kit according to any one of claims 3-6, characterized in that: The kit is at least one of the following: enzyme-linked immunosorbent assay kit, chemiluminescence assay kit, fluorescence immunochromatographic assay kit, and colloidal gold immunoassay kit.

8. The reagent kit according to claim 7, characterized in that: The chemiluminescence assay kit was prepared using a combination of chemiluminescence immunoassay and a double-antibody sandwich method.

9. The reagent kit according to claim 7, characterized in that: It also includes magnetic microparticle suspensions, washing solutions, and chemiluminescent substrates; Optionally, the chemiluminescent substrate includes at least one of luminol, isoluminol and its derivatives, (goldenane)-1,2-dioxane and its derivatives.

10. The use of the specific antibody of claim 1 or 2, or the kit of any one of claims 3-9, in the preparation of a product for detecting diseases related to blood-brain barrier dysfunction.

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