Specific antibody for detecting cathepsin S, kit and application thereof

Abstract

The application discloses a specific antibody for detecting cathepsin S, a kit and application. The specific antibody for detecting cathepsin S is prepared by immunizing animals after coupling a carrier protein to a specific antigen epitope peptide of cathepsin S, and the amino acid sequence of the antigen epitope peptide is at least one of the sequence shown in SEQ ID NO. 1 and the sequence shown in SEQ ID NO. 2. The specific antibody for detecting cathepsin S can specifically detect cathepsin S, has the advantages of high sensitivity, good specificity, good repeatability and the like, has good diagnostic sensitivity, specificity and repeatability when used for auxiliary diagnosis or curative effect evaluation of chronic obstructive pulmonary disease, meets the clinical application requirement, provides a more accurate and reliable objective index for auxiliary diagnosis, disease monitoring and prognosis evaluation of chronic obstructive pulmonary disease, and has important significance and clinical application value for diagnosis and treatment of chronic obstructive pulmonary disease.

Description

Technical Field

[0001] This application relates to the field of protein detection technology, and in particular to a specific antibody, kit, and application for detecting cathepsin S. Background Technology

[0002] Chronic obstructive pulmonary disease (COPD) is a preventable and treatable common disease worldwide, characterized by persistent airflow limitation and corresponding respiratory symptoms. Its pathological features are mainly chronic inflammatory changes in the airways, lung parenchyma, and pulmonary vessels, which are usually associated with exposure to harmful particles and gases. Multiple individual factors, such as genetic susceptibility, abnormal inflammatory response, and abnormal lung development, are involved in the pathogenesis. Therefore, studying its pathogenesis is crucial for prevention and treatment.

[0003] The pathogenesis of chronic obstructive pulmonary disease (COPD) is complex and involves multiple factors, mainly including five aspects. (1) Activation of inflammatory cells: COPD patients have a persistent chronic inflammatory response in their lungs, and various inflammatory cells such as macrophages, neutrophils, and T cells are activated. These cells release a large number of inflammatory mediators, such as interleukin (IL-8) and tumor necrosis factor (TNF-α), which lead to inflammatory damage to the airways and alveoli. The inflammatory response leads to damage and dysfunction of airway epithelial cells, destroying the barrier function of the airways, allowing more harmful substances to enter the lung tissue, further aggravating the inflammatory response and tissue damage. (2) Oxidative stress: Imbalance between oxidants and antioxidants. Environmental factors such as smoking and air pollution contain a large number of exogenous oxidants, while lung inflammation and structural cells also produce endogenous reactive oxygen species. The increase of these oxidants exceeds the body's antioxidant system's clearance capacity, leading to oxidative stress. Furthermore, oxidative stress can directly damage biochemical macromolecules such as proteins, lipids, and nucleic acids, leading to cell dysfunction or cell death. In addition, oxidative stress can also damage the extracellular matrix, causing protease-antiprotease imbalance, further aggravating inflammatory response and tissue damage. (3) Protease-antiprotease imbalance: Exposure to harmful particles and gases can stimulate lung cells to produce more proteases, such as neutrophil elastase and matrix metalloproteinases. These proteases can degrade structural proteins of the airway and alveoli, such as collagen and elastin, leading to airway wall destruction and emphysema, and increasing protease activity. Factors such as oxidative stress can also reduce the activity of antiproteases, such as the decrease in the activity of α1-antitrypsin, which makes the activity of proteases relatively increased, further aggravating tissue damage and airway remodeling and reducing the activity of antiproteases. (4) Cell senescence and apoptosis Cell senescence and apoptosis: Cell senescence exists in the lung tissue of COPD patients. Cell senescence leads to the gradual loss of cell function, affecting the repair and regeneration capacity of the airway and alveoli, making it difficult to recover from inflammatory damage and tissue destruction. Cell apoptosis in the airway mucosa and lung tissue is also related to the pathogenesis of COPD. Apoptosis may lead to a reduction in airway epithelial cells and destruction of alveolar structure, further aggravating airway obstruction and decreased lung function. (5) Airway remodeling: The airway structure of COPD patients undergoes remodeling, manifested as thickening of the airway wall, proliferation of smooth muscle cells, and thickening of the basement membrane. These structural changes lead to airway narrowing and airflow limitation, aggravating the symptoms of dyspnea. Inflammatory response and oxidative stress stimulate airway epithelial cells to secrete more mucus, and the accumulation of mucus will obstruct the airway, further aggravating airflow limitation and dyspnea. These mechanisms interact to jointly lead to the pathogenesis and progression of COPD. In-depth research on these mechanisms will help to better understand the pathophysiological process of COPD and provide a theoretical basis for the development of new diagnostic and treatment methods.

[0004] Catepsin S (CTSS) is a cysteine ​​protease belonging to the papain family. It is a single-chain protein containing 225 amino acids with a relative molecular mass of 24.8 kDa. It shares amino acid sequence homology with other cysteine ​​proteases. Its three-dimensional structure consists of two domains: (1) the L domain, which consists of three α-helices and a hydrophobic pocket; and (2) the R domain, which consists of an antiparallel β-sheet containing a hydrophobic nucleus and two α-helices located on the lateral side. The two domains form a narrow slit, which is the catalytic active site. Cysteine ​​25, histidine 159, and asparagine 175 in the structure constitute the catalytic triad, which is an important site for substrate binding. The thioanion in Cys25 acts as an affinity substance and is the key site for catalytic reaction, capable of attacking the substrate peptide. The N-terminus of CTSS has three grooves (S1, S2, and S3) that are related to the specific binding of the substrate, determining the specificity of the cysteine ​​protease inhibitor. C has a substrate-binding site, S1', which plays an important role in the specific binding of the enzyme to the conserved regions of class II molecules in the major histocompatibility complex.

[0005] CTSS is mainly expressed in the lungs, lymphocytes, and spleen. It maintains intracellular homeostasis by hydrolyzing harmful or damaged proteins and can interact with cytokines and other proteases, participating in immune and autoinflammatory responses. It is involved in the pathogenesis of various diseases, including emphysema, autoimmune diseases, cardiovascular diseases, and cancer. COPD is a heterogeneous disease characterized by persistent respiratory symptoms and airflow limitation, and its pathogenesis is not fully understood. Pulmonary function tests are the most important tests for diagnosing COPD and assessing its severity. They can also be used to evaluate treatment effectiveness. However, the effectiveness of pulmonary function tests is highly dependent on the experience of clinicians and the patient's cooperation. Currently, there are certain limitations in the clinical diagnosis, classification, assessment, and prognosis of COPD. Diagnosing COPD using blood biomarkers has become a research hotspot.

[0006] Given the current state of COPD diagnosis, clinicians have been trying to find a stable and reliable biomarker that can objectively diagnose and assess the efficacy of treatment for chronic obstructive pulmonary disease. Currently, brain imaging and peripheral blood protein markers are the most studied.

[0007] As research has progressed, some scholars have discovered that CTSS is expressed at elevated levels in the lung tissue of COPD patients, participating in the activation of inflammatory cells and the release of inflammatory mediators, thus promoting the occurrence and development of inflammatory responses.

[0008] CTSS can degrade extracellular matrix components, such as collagen and elastin, leading to the destruction and remodeling of lung tissue structure, thereby exacerbating airway obstruction. Simultaneously, CTSS participates in antigen processing and presentation, affecting the function of immune cells and the regulation of immune responses.

[0009] Studies have shown that serum CTSS levels in COPD patients are significantly higher than in healthy controls, and are positively correlated with disease severity and emphysema severity, suggesting that CTSS may serve as a potential diagnostic biomarker for COPD. In prognostic assessment, CTSS expression levels may be associated with disease progression and prognosis in COPD patients, helping to evaluate changes in their condition and treatment effectiveness.

[0010] In summary, CTSS plays a crucial role in the development and progression of COPD, and its research as a biomarker and therapeutic target provides new ideas and methods for the diagnosis and treatment of COPD. However, current research on the specific detection of cathepsin S is relatively limited. Developing new specific detection technologies for cathepsin S is of great significance and clinical application value for the diagnosis and treatment of COPD. Summary of the Invention

[0011] The purpose of this application is to provide a novel specific antibody, kit, and application for detecting cathepsin S.

[0012] The following technical solution is adopted in this application:

[0013] The first aspect of this application discloses a specific antibody for detecting cathepsin S, which is prepared by immunizing animals with a carrier protein coupled with a cathepsin S-specific antigenic epitope peptide, wherein the amino acid sequence of the cathepsin S-specific antigenic epitope peptide is at least one of the sequences shown in SEQ ID NO.1 and SEQ ID NO.2.

[0014] SEQ ID NO.1:

[0015] Cys-Ser-Ser-Ala-Val-Ala-Gln-Leu-His-Lys-Asp-Pro-Thr-Leu-Asp

[0016] SEQ ID NO.2:

[0017] Cys-Asn-Lys-Gly-Pro-Val-Ser-Val-Gly-Val-Asp-Ala-Arg-His-Pro.

[0018] In one implementation of this application, the specific antibody is a polyclonal antibody prepared by immunizing animals after conjugating a cathepsin S-specific antigen epitope peptide with a carrier protein.

[0019] It should be noted that the key to this application lies in the development of a novel specific antibody derived from a cathepsin S-specific antigenic epitope peptide, and based on this novel epitope peptide, a new specific antibody capable of specifically detecting cathepsin S, namely the polyclonal antibody of this application, thus providing a new scheme and approach for the detection of cathepsin S. It is understood that the carrier protein, carrier protein conjugation, animal immunization, and post-immunization extraction of the polyclonal antibody in this application can all refer to existing technologies and are not specifically limited here.

[0020] The second aspect of this application discloses a kit for detecting cathepsin S, the kit containing the specific antibody of this application.

[0021] In one implementation of this application, the kit includes a solid-phase carrier and a specific antibody of this application, wherein the specific antibody is directly or indirectly linked to the solid-phase carrier.

[0022] In one implementation of this application, the solid support is at least one of a microporous reaction plate, magnetic microspheres, and nitrocellulose membrane.

[0023] It should be noted that microporous reaction plates, magnetic microspheres, and nitrocellulose membranes are all solid-phase carriers commonly used in this application. The key to this application lies in the development and preparation of a novel cathepsin S-specific antibody. 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.

[0024] In one implementation of this application, the kit further includes a marker for labeling the specific antibody of this application.

[0025] 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.

[0026] 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).

[0027] In one implementation of this application, the kit further includes a cathepsin S calibrator.

[0028] In one implementation of this application, the cathepsin S calibrator is recombinantly expressed cathepsin S or cathepsin S extracted directly from the sample.

[0029] In one implementation of this application, the sample is a serum or plasma sample.

[0030] 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.

[0031] In one implementation of this application, the chemiluminescence assay kit is prepared using a combination of chemiluminescence immunoassay and a double-antibody sandwich method.

[0032] In one implementation of this application, the reagent kit further includes a magnetic microparticle suspension, a washing solution, and a chemiluminescent substrate.

[0033] 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.

[0034] The third aspect of this application discloses the use of the specific antibody of this application, or the kit of this application, in the preparation of products for detecting chronic obstructive pulmonary disease.

[0035] It should be noted that the specific antibody or kit of this application can specifically detect cathepsin S. According to existing research, cathepsin S can be used as a biomarker for chronic obstructive pulmonary disease. Therefore, the specific antibody or kit of this application can be used to prepare products for detecting chronic obstructive pulmonary disease, such as a chronic obstructive pulmonary disease detection or diagnostic kit based on cathepsin S assay.

[0036] The beneficial effects of this application are as follows:

[0037] This application provides a specific antibody for detecting cathepsin S, which can specifically detect cathepsin S and has the advantages of high sensitivity, good specificity, and good repeatability. When used for the auxiliary diagnosis or efficacy evaluation of chronic obstructive pulmonary disease (COPD), it has good diagnostic sensitivity, specificity, and repeatability, meeting the needs of clinical application. It provides a more accurate and reliable objective indicator for the auxiliary diagnosis, disease monitoring, and prognostic assessment of COPD, and has important significance and clinical application value for the diagnosis and treatment of COPD. Attached Figure Description

[0038] Figure 1 The results of serum CTSS levels in healthy controls and patients with chronic obstructive pulmonary disease, detected using specific antibodies in the embodiments of this application, are as follows:

[0039] Figure 2This is a graph showing the analysis results of the relationship between the severity (I-IV) of chronic obstructive pulmonary disease and the serum CTSS level of healthy controls, as analyzed by specific antibody detection in the embodiments of this application.

[0040] Figure 3 This is the ROC curve of serum CTSS detected by specific antibodies in the embodiments of this application for the diagnosis of chronic obstructive pulmonary disease. Detailed Implementation

[0041] CTSS plays a crucial role in the development and progression of COPD, and its research as a biomarker and therapeutic target provides new ideas and methods for the diagnosis and treatment of COPD. Based on this research and understanding, this application creatively develops a novel specific antibody for detecting cathepsin S, enabling the diagnosis, assessment, and prognostic detection of COPD.

[0042] Specifically, the specific antibody for detecting cathepsin S in this application is prepared by immunizing animals with a carrier protein conjugated to a cathepsin S-specific antigenic epitope peptide, and the amino acid sequence of the cathepsin S-specific antigenic epitope peptide is at least one of the sequences shown in SEQ ID NO. 1 and SEQ ID NO. 2.

[0043] 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 chronic obstructive pulmonary disease, the CTSS concentration of the subject sample measured by the kit is compared with the control reference range (i.e., the concentration level of healthy subjects). If the CTSS concentration of the subject sample is higher than the concentration of the control reference range, the subject has the risk of chronic obstructive pulmonary disease, and the severity of the disease is positively correlated with the CTSS concentration level in the sample, that is, the higher the CTSS concentration of the subject sample, the more severe the disease.

[0044] The concentrations of CTSS in patient samples before and after treatment were compared using the kit. If the concentration of CTSS in the patient samples did not change or showed an increasing trend as treatment progressed, it indicated that the drug treatment was ineffective. If the concentration of CTSS in the patient samples showed a decreasing trend as treatment progressed, it indicated that the drug treatment was effective.

[0045] In one implementation of this application, the specific antibody used in chemiluminescence detection exhibits high sensitivity with a detection limit of 0.335 ng / mL; a wide linear range of 3-6 orders of magnitude, allowing direct measurement of samples with concentrations up to 50 ng / mL without dilution; high accuracy with a recovery rate of 97.02% in recovery tests; high precision with a CV of less than 10%; short reaction time with results available in 30 minutes, significantly improving detection efficiency; and a high degree of automation, eliminating the need for manual operation and reducing human error.

[0046] In summary, the specific antibody and kit of this application have the following advantages and positive effects compared with the prior art:

[0047] 1. This application uses CTSS as a biomarker to prepare a kit for the auxiliary diagnosis or efficacy evaluation of chronic obstructive pulmonary disease (COPD), which overcomes the shortcomings of the indicators currently used in the clinical diagnosis of COPD, which are too subjective and lack objectivity. This provides a more accurate and reliable objective indicator for the auxiliary diagnosis, disease monitoring and prognosis assessment of COPD.

[0048] 2. The reagent kit prepared in this application must have good diagnostic sensitivity, specificity and reproducibility, and meet the needs of clinical application.

[0049] 3. The CTSS polyclonal antibody prepared in this application can bind to CTSS in the sample with high specificity.

[0050] 4. The two CTSS-specific antigenic epitope peptides screened in this application are hydrophilic, highly antigenic, and easy to synthesize. The antigens (immunogens) prepared from them can produce highly specific polyclonal antibodies when used to immunize animals.

[0051] 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.

[0052] Unless otherwise specified, the technical means used in the embodiments are conventional means well known to those skilled in the art.

[0053] Example

[0054] I. Synthesis of CTSS-specific antigenic epitope peptides

[0055] In this case, two CTSS-specific antigenic epitope peptides were developed, namely the sequences shown in SEQ ID NO.1 and SEQ ID NO.2.

[0056] SEQ ID NO.1:

[0057] Cys-Ser-Ser-Ala-Val-Ala-Gln-Leu-His-Lys-Asp-Pro-Thr-Leu-Asp

[0058] SEQ ID NO.2:

[0059] Cys-Asn-Lys-Gly-Pro-Val-Ser-Val-Gly-Val-Asp-Ala-Arg-His-Pro

[0060] The synthesis methods of the two CTSS-specific antigenic epitope peptides are as follows:

[0061] 1. Main raw materials

[0062] 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.).

[0063] 2. Main Instruments

[0064] 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.

[0065] 3. Synthesis methods and processes

[0066] 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:

[0067] (1) Activation of amino acids (HOBt / DCC method)

[0068]

[0069] Fmoc-protected amino acids

[0070]

[0071] (2) Connecting amino acids to resin

[0072]

[0073] (3) Removal of the Fmoc protecting group from amino acids

[0074]

[0075] (4) Activation of another amino acid (HOBt / DCC method)

[0076]

[0077] (5) Coupling

[0078]

[0079] (6) Repeat steps (3) to (5) until the synthesis is complete.

[0080] Peptide resins containing CTSS antigenic epitope peptide (1) and CTSS antigenic epitope peptide (2) were obtained. CTSS antigenic epitope peptide (1) and CTSS antigenic epitope peptide (2) are cathepsin S-specific antigenic epitope peptides with sequences shown in SEQ ID NO.1 and SEQ ID NO.2, respectively.

[0081] (7) Peptide cleavage resin

[0082] 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 CTSS antigenic epitope peptide (1) and crude CTSS antigenic epitope peptide (2).

[0083] 4. Purification of crude CTSS antigenic epitope peptides (1) and (2)

[0084] Separation and purification were performed using high performance liquid chromatography:

[0085] Conditions: Column: C8 10×100mm, purchased from Waters Corporation, USA

[0086] Chromatograph: Waters 600, Waters Corporation, USA

[0087] Mobile phase: A: 0.1% TFA (trifluoroacetic acid) aqueous solution

[0088] B: 0.1% TFA (trifluoroacetic acid) in 60% acetonitrile

[0089] Detection wavelength: 214nm

[0090] Flow rate: 4 mL / min

[0091] Elution gradient: 20-60% B, 30 minutes

[0092] HPLC (High Performance Liquid Chromatography) Analysis

[0093] Column: C18 4.6×150mm, purchased from Waters Corporation, USA

[0094] Mobile phase: A: 0.1% TFA (trifluoroacetic acid) aqueous solution

[0095] B: 0.1% TFA (trifluoroacetic acid) in acetonitrile

[0096] Detection wavelength: 214nm

[0097] Flow rate: 1 mL / min

[0098] Elution gradient: 0-60% B, 30 minutes

[0099] Peptide analysis results showed that the purity of CTSS antigenic epitope peptide (1) and CTSS antigenic epitope peptide (2) prepared in this example was 98.2% and 97.5%, respectively, which can meet the requirements for subsequent use.

[0100] II. Preparation of CTSS-specific antibodies

[0101] 1. Preparation of antigens

[0102] CTSS antigen (1) and CTSS antigen (2) were prepared by linking CTSS-specific antigen epitope peptide (1) or (2) to carrier protein KLH (keyhole hemocyanin) using the BDB (Bis-diazotized benzidine dichloride) method.

[0103] Take 20.0 mg of CTSS-specific antigenic epitope peptide (1) or (2) and dissolve it in 0.2 M 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 a 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℃.

[0104] 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.

[0105] 2. Preparation of polyclonal antibodies by immunizing animals

[0106] 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 CTSS 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 fully emulsified and 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.

[0107] 2.2. Antibody titer determination: The titer of CTSS polyclonal antibody (1) or (2) was determined by indirect ELISA. Specifically, the CTSS antigen 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 (16-18 hours) at 4°C, washed 3 times, and then 200µL of blocking buffer was added. The plate was blocked at 37°C for two hours and then dried 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.

[0108] The results showed that the titers of the CTSS polyclonal antibody (1) and CTSS polyclonal antibody (2) prepared in this example both reached 1:64000.

[0109] 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.

[0110] 3. Isolation and purification of antibodies

[0111] First, dialyze the CTSS polyclonal antibody (1) and CTSS polyclonal antibody (2) to be loaded with a balancing buffer (0.02M, PB, pH 8.0) until the pH is consistent. Then, pack the column and connect it to the protein chromatography system, and wash it with the balancing buffer until the pH is 8.0. Next, add the CTSS polyclonal antibody (1) and CTSS polyclonal antibody (2) to be purified to the column. After all the sample has been injected, wash with an elution buffer (0.05M, PB, pH 8.0) until the protein analyzer shows the beginning of elution. Then, start collecting the protein solution. Stop collecting when the peak declines gradually. The concentrations of CTSS polyclonal antibody (1) or CTSS polyclonal antibody (2) were measured and calculated using an L5S UV-Vis spectrophotometer (Boda Precision Technology). The concentrations of antibody (1) and antibody (2) were 1.0 mg / mL and 0.5 mg / mL, respectively. The purified CTSS polyclonal antibody (1) and CTSS polyclonal antibody (2) were then aliquoted and stored at -20°C.

[0112] Experiment 1: Preparation of the CTSS chemiluminescence reagent kit

[0113] 1. Preparation of magnetic microparticle suspension (working solution of CTSS polyclonal antibody (1) immunomagnetic beads)

[0114] (1) Washing of magnetic beads

[0115] 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.

[0116] (2) Activation of magnetic beads

[0117] 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.

[0118] (3) Coat CTSS polyclonal antibody with activated magnetic beads (1).

[0119] 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 CTSS polyclonal antibody (1) (1.0 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.

[0120] (4) Magnetic bead sealing

[0121] 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.

[0122] (5) Cleaning

[0123] After sealing, place the coated tube on a magnetic separator for 1 minute, discard the supernatant, and add 1 mL of 0.05 M TRIS (pH 7.4) buffer to the coated tube and vortex for 1 minute. Repeat this operation twice. Add 1 mL of 0.05 M TRIS (pH 7.4) buffer to the coated tube and vortex for 1 minute.

[0124] (6) Preparation of magnetic microparticle suspension

[0125] The magnetic beads coated with CTSS 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.

[0126] 2. Preparation of enzyme-labeled antibody (polyclonal antibody labeled with alkaline phosphatase CTSS (2))

[0127] (1) Take 250 μL of 0.1M MES (pH 4.5) and soak the ultrafiltration centrifuge tube (30KD) for 2 min.

[0128] (2) Add 250 μL of 0.1 M MES (pH 4.5) to the centrifuge column and bring the volume to 500 μL.

[0129] (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.

[0130] (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.

[0131] 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.

[0132] (5) Take 10 μL of CTSS polyclonal antibody (2) 0.5 mg / mL, mix well, centrifuge at 13000 rpm for 20 min and discard the waste liquid.

[0133] (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.

[0134] (7) Repeat the experimental steps (6) once.

[0135] (8) Add 0.1M PB (pH 9.0) to a final volume of 250μL and place on a shaker at 120rpm for 2h.

[0136] (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℃.

[0137] 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.

[0138] 3. Preparation of washing solution

[0139] It consists of 10 mM PBS (pH 7.2), 0.08% Tween-20 and 0.03% Proclin-300.

[0140] 4. Preparation of CTSS calibrators and quality control samples

[0141] Recombinant cathepsin S protein (purchased from Abcam, concentration 2 mg / mL) was serially diluted with calibrator solution (containing 10 mM phosphate buffer (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.

[0142] 5. Preparation of luminescent substrates

[0143] 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.

[0144] 6. Components of the reagent kit

[0145] The kit mainly consists of reagent compartments (Reagent 1, Reagent 2), CTSS calibrator A (1.0mL×1 vial, concentration: 3ng / mL), CTSS calibrator B (1.0mL×1 vial, concentration: 40ng / mL), quality control (1.0mL×1 vial, concentration: 30ng / 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 magnetic microparticles coated with CTSS polyclonal antibody (1), and Reagent 2 contains alkaline phosphatase (AP) labeled CTSS polyclonal antibody (2). The kit is available in two packaging specifications: 50 doses / box and 100 doses / box. The substrate solution is the luminescent substrate solution, with 12mL and 25mL corresponding to the 50-dose / box and 100-dose / box kit specifications, respectively. The concentrated cleaning solution is the same as the concentrated washing solution. It should be diluted 25 times before use. Similarly, one bottle and two bottles correspond to the 50-test / box and 100-test / box kit specifications, respectively. The information card contains the calibration curve and quality control concentration information.

[0146] Experiment 2: Preparation of CTSS Chemiluminescence Reagent Kit

[0147] 1. Preparation of magnetic microparticle suspension reagent (working solution of CTSS monoclonal antibody-coated immunomagnetic beads)

[0148] (1) Washing of magnetic beads

[0149] 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.

[0150] (2) Activation of magnetic beads

[0151] Add 800 μL of 0.1 M MES (pH 6.0) buffer to the coating 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 coating tube. 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 30 min.

[0152] (3) Coating CTSS monoclonal antibody with activated magnetic beads.

[0153] Add 1 mL of 0.1 M MES (pH 6.0) buffer to the coating tube and vortex for 1 min. Add 50 μL of CTSS monoclonal antibody (0.46 mg / mL) (provided by Guyan Industrial Co., Ltd., catalog number GY-L1609) 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.

[0154] (4) Magnetic bead sealing

[0155] 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.

[0156] (5) Cleaning

[0157] After sealing, place the coated tube on a magnetic separator for 1 minute, discard the supernatant, and add 1 mL of 0.05 M TRIS (pH 7.4) buffer to the coated tube and vortex for 1 minute. Repeat this operation twice. Add 1 mL of 0.05 M TRIS (pH 7.4) buffer to the coated tube and vortex for 1 minute.

[0158] (6) Preparation of magnetic microparticle suspension reagent

[0159] The magnetic beads coated with CTSS 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 2.3 μg / mL.

[0160] 2. Preparation of enzyme-labeled antibody (polyclonal antibody labeled with alkaline phosphatase CTSS (2))

[0161] Same as Experiment 1.

[0162] 3. Preparation of CTSS calibrators / quality control samples, washing solutions, and luminescent substrates.

[0163] Same as Experiment 1.

[0164] 4. Components of the reagent kit

[0165] The kit mainly consists of reagent compartments (reagent 1, reagent 2), CTSS calibrator A (1.0mL×1 vial, concentration: 3ng / mL), CTSS calibrator B (1.0mL×1 vial, concentration: 40ng / mL), quality control (1.0mL×1 vial, concentration: 30ng / mL), substrate solution (12.0mL / 25.0mL), concentrated washing solution (25×) (60.0mL×1 bottle / 60.0mL×2 bottles) and calibration / quality control information card. Reagent 1 in the reagent compartment contains micromagnetic particles coated with CTSS monoclonal antibody (provided by Guyan Industrial, catalog number GY-L1609), and reagent 2 contains alkaline phosphatase (AP) labeled CTSS polyclonal antibody (2). The kit is available in two packaging specifications: 50 doses / box and 100 doses / box. The substrate solution is the luminescent substrate solution, available in 12mL and 25mL vials for kits of 50 and 100 tests per kit, respectively. The concentrated washing buffer is a concentrated wash solution, diluted 25 times before use; similarly, one and two vials are available for kits of 50 and 100 tests per kit, respectively. The information card contains the calibration curve and quality control concentration information.

[0166] Experiment 3: Preparation of ELISA assay kit

[0167] 1. Preparation of various buffer solutions and reagents:

[0168] 1.1 Coating buffer: 0.05M, pH 9.6, CB (carbonate buffer):

[0169] Na2CO3: 16.0 g, NaHCO3: 29.0 g, add deionized water to a final volume of 1000 mL.

[0170] 1.2, pH7.2, 10×PBS-Tween 20:

[0171] 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.

[0172] 1.3 Blocking buffer / antibody diluent:

[0173] 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.

[0174] 1.4 Enzyme label dilution buffer:

[0175] 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.

[0176] 1.5. Color developer A:

[0177] Citric acid: 35.5 g, urea peroxide: 10 g, Tween 20: 10 mL, deionized water to a final volume of 1000 mL.

[0178] 1.6. Color developer B:

[0179] Citric acid: 120 g, EDTA-2Na: 1 g, TMB·2HCl: 2 g, deionized water to a final volume of 1000 mL.

[0180] 1.7 Concentrated washing solution (pH 7.2, 25×PBS-Tween 20):

[0181] 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.

[0182] 1.8 Termination solution: 2M H2SO4

[0183] 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.

[0184] 2. Preparation of pre-coated plates

[0185] Dissolve CTSS-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℃.

[0186] 3. Formulate by combining antibodies and enzyme markers

[0187] The antibody (CTSS-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 square matrix titration experiment.

[0188] 4. Preparation of CTSS calibrators and quality control samples

[0189] The recombinant CTSS (purchased from Abcam, concentration 2 mg / mL) was serially diluted with sample diluent to prepare CTSS calibrators (0.5–50 ng / mL) and two quality control samples at high and low concentrations.

[0190] 5. Components of the ELISA kit

[0191] The kit mainly consists of pre-coated plates (48 or 96 doses), one set of CTSS calibrators, CTSS 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 wash buffer (20 mL), and stop solution (5 mL). Chromogenic solution A and chromogenic solution B are the prepared chromogenic reagents A and B, respectively.

[0192] Experiment 4: Preparation of CTSS-Fluorescence Chromatography Assay Kit

[0193] 1. Wrapped with padding

[0194] 1.1 Fluorescent microspheres labeled with CTSS polyclonal antibody (1)

[0195] 1.1.1 Activation of fluorescent microspheres:

[0196] (1) Take 500 μL of fluorescent microspheres (purchased from Bangs Laboratories, Inc.) with a content of 1 (w / v)% in aqueous dispersion, add 1 mL of initial wash buffer (50 mM MES aqueous solution, pH 6.5), 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.

[0197] (2) Repeat the above process twice;

[0198] (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.

[0199] 1.1.2. Label CTSS polyclonal antibody with activated fluorescent microspheres (1):

[0200] (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;

[0201] (2) Repeat the above process twice;

[0202] (3) Obtain 500 μL of buffer solution containing fluorescent microspheres;

[0203] (4) Add CTSS polyclonal antibody (1) to the activated fluorescent microspheres at a ratio of 15 mg antibody / g, and shake at room temperature for 2 hours;

[0204] (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.

[0205] 1.2, Covered with a bonding pad

[0206] The CTSS 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.

[0207] 2. Preparation of the reaction membrane

[0208] CTSS 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. CTSS polyclonal antibody (2) and goat anti-mouse IgG monoclonal antibody were sprayed onto nitrocellulose membranes using the gold spraying machine. The membranes were dried in an oven at 37°C and stored at 45% humidity for later use.

[0209] 3. Assembly and cutting of test strips

[0210] 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.

[0211] 4. Preparation of CTSS Fluorescent Immunoassay Card

[0212] 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.

[0213] 5. Composition of the CTSS-Fluorescence Chromatography Assay Kit (20T)

[0214] 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 CTSS 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 CTSS polyclonal antibody labeled with fluorescent microspheres (1).

[0215] Test Experiment 1: Measurement of CTSS using the chemiluminescence reagent kit from Experiment 1 and validation of kit performance.

[0216] 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.

[0217] 2. The chemiluminescent immunoassay (CLIA) kit is used to determine the CTSS concentration. The specific steps are as follows:

[0218] The specific steps are divided into testing procedures and calibration procedures.

[0219] Testing procedure:

[0220] (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.

[0221] (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.

[0222] (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℃.

[0223] (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.

[0224] (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.

[0225] (6) Sample injection: For information on sample injection, please refer to the instruction manual for the chemiluminescence analyzer.

[0226] (7) Click the instrument run button. The luminescence analyzer will perform the pre-set operation and detection (total detection time is about 25 minutes).

[0227] Calibration procedure:

[0228] (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.

[0229] (2) Calibrator A and calibrator B need to be replicated for calibration experiments;

[0230] (3) Conduct the experiment according to the operation manual of the luminescence analyzer.

[0231] 3. Performance verification of the reagent kit in Experiment 1

[0232] 3.1 Linearity Range Verification

[0233] 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.

[0234] 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, 50% means 150 μL of calibrator is mixed with 150 μL of diluent, and so on.

[0235] Table 1. Linearity range test results and calculations (unit: ng / mL)

[0236] 100% 48.21 49.37 48.79 50% 23.97 24.22 24.10 20% 11.12 10.38 10.75 10% 5.22 5.98 5.60 5% 2.71 2.52 2.62 1% 0.52 0.57 0.55

[0237] The test results show that the kit prepared in Experiment 1 has a wide linear range in the range of 0.5 to 50 ng / mL, with the linear equation formula: y = 48.2636x + 0.4399 and a linear correlation coefficient of 0.9965, indicating that the kit has a wide linear range.

[0238] 3.2 Minimum Detection Limit

[0239] 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.

[0240] The test results showed that the limit of detection was 0.335 ng / mL, indicating that the kit has high sensitivity.

[0241] 3.3 Precision

[0242] 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 5 ng / mL and 40 ng / mL.

[0243] 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 / ×100% to evaluate the precision of the kit.

[0244] The test results are shown in Table 2 below.

[0245] Table 2 Precision Test Results

[0246] 1 5.32 39.72 2 5.43 38.76 3 4.75 42.46 4 4.89 37.65 5 5.65 42.96 6 5.71 41.86 7 5.24 37.57 8 4.75 41.56 9 5.49 42.33 10 5.32 36.98 mean 5.26 40.19 Standard deviation SD 0.35 2.31 CV% 6.65% 5.75%

[0247] 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.

[0248] 3.4 Accuracy

[0249] The accuracy of the reagent kit was evaluated using a recovery test.

[0250] 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.

[0251]

[0252] Where: R—recovery rate;

[0253] V — Volume of sample A added;

[0254] V0 – Volume of sample B;

[0255] C – The concentration of sample B after adding sample A;

[0256] C0—Detection concentration of sample B;

[0257] CS — Concentration of sample A.

[0258] The recovery rate was calculated to be 97.02%, indicating that the kit has high accuracy.

[0259] Verification conclusion:

[0260] 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 50 ng / mL without dilution; it has high sensitivity with a limit of detection of 0.335 ng / mL; it has high accuracy with a recovery rate of 97.02%; it has good precision with CV% of 6.65% for high concentration and 5.75% for low concentration; and the entire reaction time is short, with results available in 30 minutes, which greatly improves the detection efficiency.

[0261] Application Experiment 2: Measurement of CTSS using the chemiluminescence reagent kit from Experiment 2 and validation of kit performance.

[0262] 1. Sample collection: Same as application experiment 1.

[0263] 2. The concentration of CTSS was determined using a chemiluminescent immunoassay (CLIA) kit. The specific steps are the same as in application experiment 1.

[0264] 3. Reagent kit performance validation

[0265] 3.1 Linearity Range Verification

[0266] Same as application test 1

[0267] The test results are shown in Table 3 below.

[0268] Table 3. Linearity range test results and calculations (unit: ng / mL)

[0269] 100% 47.1 49.37 48.24 50% 27.97 24.22 26.10 20% 16.12 13.38 14.75 10% 8.22 7.98 8.10 5% 3.71 2.52 3.12 1% 0.42 0.57 0.50

[0270] The test results show that the kit prepared in Experiment 2 has a wide linear range in the range of 0.5 to 50 ng / mL, with the linear equation formula: y = 46.7767x + 2.3009 and a linear correlation coefficient of 0.9940, indicating that the kit has a wide linear range.

[0271] 3.2 Minimum Detection Limit

[0272] Same as application test 1

[0273] The test results showed that the limit of detection was 0.628 ng / mL, indicating that the kit has high sensitivity.

[0274] 3.3 Precision

[0275] The validation method was the same as in application test 1. The two concentrations used for precision validation were 5 ng / mL and 40 ng / mL.

[0276] The test results are shown in Table 4 below.

[0277] Table 4 Precision Test Results

[0278] 1 5.72 40.36 2 4.95 43.69 3 6.01 45.21 4 6.56 40.25 5 5.32 43.39 6 6.09 39.12 7 5.25 40.36 8 5.26 37.12 9 5.25 38.39 10 5.23 42.32 mean 5.56 41.02 Standard deviation SD 0.51 2.56 CV% 9.17% 6.24%

[0279] 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.

[0280] 3.4 Accuracy

[0281] The verification method is the same as that used in application experiment 1.

[0282] The recovery rate was calculated to be 94.32%, indicating that the kit has high accuracy.

[0283] Verification conclusion:

[0284] Experiment 2 validated the results, showing that the kit has a wide linear range, allowing direct measurement of samples with concentrations up to 50 ng / mL without dilution; it has high sensitivity, with a limit of detection of 0.628 ng / mL; high accuracy, with a recovery rate of 94.32%; good precision, with CV% of 9.17% for high concentration and 6.24% for low concentration; and a short reaction time, with results available in 30 minutes, greatly improving detection efficiency.

[0285] Application Experiment 3: Measurement of CTSS using the enzyme-linked immunosorbent assay (ELISA) kit prepared in Experiment 3 and validation of kit performance.

[0286] 1. Sample collection: Same as application experiment 1.

[0287] 2. The CTSS concentration was determined using an enzyme-linked immunosorbent assay (ELISA) kit. The specific steps are as follows:

[0288] (1) Preparation of washing solution:

[0289] Dilute the 25x concentrated detergent solution with deionized water at a ratio of 1:25.

[0290] (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.

[0291] (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.

[0292] (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;

[0293] (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;

[0294] (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;

[0295] (7) Add 50 μL of colorimetric reagent A and B solution to each well, mix thoroughly, and incubate at 37°C for 15 minutes;

[0296] (8) Add 1 drop (50 μL) of stop solution to each well as soon as possible and gently tap to mix;

[0297] (9) Measure the OD value of each well using an ELISA reader (with dual wavelengths of 450 / 630 nm);

[0298] (10) Plot the calibration curve and calculate the CTSS concentration in the sample based on the calibration curve.

[0299] 3. Reagent kit performance validation

[0300] 3.1 Linearity Range Verification

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

[0302] The test results are shown in Table 5 below.

[0303] Table 5. Linearity range test results and calculations (unit: ng / mL)

[0304] 100% 50.03 48.39 49.21 50% 26.45 25.77 26.11 20% 11.23 12.52 11.88 10% 6.03 6.11 6.07 5% 3.12 2.77 2.95 1% 0.62 0.57 0.60

[0305] The test results show that the kit prepared in Experiment 3 has a wide linear range in the range of 0.5 to 50 ng / mL, with the linear equation formula: y = 48.7428x + 1.0264 and a linear correlation coefficient of 0.9991. This indicates that the kit has a wide linear range.

[0306] 3.2 Minimum Detection Limit

[0307] The verification method is the same as that used in application experiment 1.

[0308] The test results showed that the limit of detection was 0.407 ng / mL, indicating that the kit has high sensitivity.

[0309] 3.2 Precision

[0310] The validation method was the same as in application test 1. The two concentrations used for precision validation were 5.0 ng / mL and 40.0 ng / mL.

[0311] The test results are shown in Table 6 below.

[0312] Table 6 Precision Test Results

[0313] 1 5.06 38.07 2 4.52 38.56 3 5.53 39.42 4 4.52 40 5 5.67 44.13 6 4.46 42.18 7 5.47 45.6 8 4.66 40.61 9 4.85 36.47 10 5.45 44.63 mean 5.02 40.97 Standard deviation SD 0.48 3.06 CV% 9.56% 7.47%

[0314] 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.

[0315] 3.4 Accuracy

[0316] The verification method is the same as that used in application experiment 1.

[0317] The recovery rate was calculated to be 95.13%, indicating that the kit has high accuracy.

[0318] Verification conclusion:

[0319] The validation results of the kit prepared in Experiment 3 showed that the kit had good linearity in the range of 0.5–50 ng / mL, reaching 0.9991; high sensitivity, with a limit of detection of 0.407 ng / mL; recovery rate of 95.13%; high accuracy and precision; and CV% of both high and low concentrations less than 10%. It does not require complex equipment, can be used for batch detection, and can simultaneously produce more than 80 test results in 1.5 hours, with high detection efficiency.

[0320] Application Experiment 4: Validation of the fluorescence immunochromatographic kit used in Experiment 4 for CTSS assay and kit performance.

[0321] 1. Sample collection: Same as application experiment 1.

[0322] 2. The concentration of CTSS was determined using a fluorescence immunochromatographic assay kit. The specific steps are as follows:

[0323] (1) Before testing, bring the reagents to room temperature (20±5℃), and conduct the test at room temperature;

[0324] (2) Turn on the fluorescence analyzer and adjust the instrument to the ready-to-detect state;

[0325] (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;

[0326] (4) After the reaction is complete, insert the test card into the fluorescence analyzer for scanning and testing. Calculate the CTSS concentration of each sample based on the built-in calibration curve of the analyzer.

[0327] 3. Reagent kit performance validation

[0328] 3.1 Linearity Range Verification

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

[0330] The test results are shown in Table 7 below.

[0331] Table 7. Linearity Range Test Results and Calculations (Unit: ng / mL)

[0332] 100% 46.86 47.96 47.41 50% 26.53 28.52 27.53 20% 9.63 10.05 9.84 10% 5.77 4.92 5.35 5% 3.23 3.09 3.16 1% 0.63 0.72 0.68

[0333] The test results show that, within the range of 0.5–50 ng / mL, the linear equation of the kit in Experiment 4 is: y = 47.8128x + 0.8397, and the linear correlation coefficient can reach 0.9970, indicating that the kit has a wide linear range.

[0334] 3.2 Minimum Detection Limit

[0335] The testing method is the same as in application experiment 1.

[0336] The test results showed that the limit of detection was 0.452 ng / mL.

[0337] 3.2 Precision

[0338] The test method was the same as in application test 1. The two concentrations used for precision verification were 5.0 ng / mL and 40.0 ng / mL.

[0339] The test results are shown in Table 8 below.

[0340] Table 8 Precision Test Results

[0341] 1 5.16 34.81 2 5.74 40.41 3 5.67 35.15 4 5.39 36.19 5 4.68 42.14 6 4.76 35.58 7 4.51 44.62 8 5.37 34.81 9 4.78 42.21 10 4.58 37.35 mean 5.06 38.33 Standard deviation SD 0.46 3.67 CV% 9.09% 9.57%

[0342] 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.

[0343] 3.4 Accuracy

[0344] The verification method is the same as that used in application experiment 1.

[0345] The recovery rate was calculated to be 95.3%, which meets the recovery rate requirement (90%–110%).

[0346] Verification conclusion:

[0347] The validation results of the kit prepared in Experiment 4 showed that the kit had good linearity in the range of 0.5–50.0 ng / mL, reaching 0.9970, with a limit of detection of 0.452 ng / mL, a recovery rate of 95.3%, 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 diagnostics, is easy to operate, can be tested on demand, and results can be obtained in 15 minutes, making the detection rapid.

[0348] Test Comparison Example 1:

[0349] The CTSS antibodies for ANT-485 and DF8246 (purchased from Guyan Company) were used to prepare a chemiluminescence kit (comparative kit) according to the method in Experiment 1, and the analytical performance was compared with that of the kit in Experiment 1.

[0350] 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.

[0351] Table 9 Performance comparison results between the comparison kit and the kit in Experiment 1

[0352]

[0353] Comparison of various analytical performance indicators showed that the kit prepared in Experiment 1 had better sensitivity and other indicators.

[0354] Clinical Trial 1: CTSS Measurement Kit for the Auxiliary Diagnosis and Disease Monitoring of Chronic Obstructive Pulmonary Disease

[0355] 1. Research Subjects

[0356] Chronic obstructive pulmonary disease (COPD) group: 89 patients with COPD were selected as the study group. Their ages were (66±5.4) years, years of education were (11.8±5.6), and BMI was (23.25±1.4). All met the diagnostic criteria for COPD (GOLD 2025) guidelines; in an appropriate clinical context, the presence of incompletely reversible airflow limitation (i.e., FEV1 / FVC < 0.7 after inhalation of a bronchodilator) on spirometry was sufficient for diagnosis. Based on the severity of airflow limitation, COPD patients were further divided into grades I to IV. Grade I consisted of 20 patients, all with mild COPD, with data showing FEV1 / FVC < 70% but FEV1 ≥ 80% of predicted value; Grade II consisted of 33 patients, all with moderate COPD, with data showing 50% ≤ FEV1 < 80% of predicted value; Grade III consisted of 26 patients, all with severe COPD, with data showing 30% ≤ FEV1 < 50% of predicted value; and Grade IV consisted of 10 patients, all with very severe COPD, with data showing FEV1 < 30% of predicted value, often accompanied by symptoms of chronic respiratory failure.

[0357] Healthy control group: 67 healthy individuals served as the control group, including 35 males and 32 females, with an average age of (67±8.2) years, years of education of (12.3±6.2) years, and a BMI of (23.04±2.8).

[0358] There were no statistically significant differences in general characteristics between the two groups, and all participants gave informed consent to this study.

[0359] 2. Experimental Methods

[0360] 2.1 The CTSS levels in serum samples from the healthy control group and COPD patients were tested using the kit prepared in Experiment 1.

[0361] 2.2 The serum CTSS levels of enrolled COPD patients with different severities (I-IV) were compared with those of healthy controls using the kits prepared in Experiment 1.

[0362] 2.3 Receiver Operating Curve (ROC Curve): ROC curves were generated based on the CTSS levels of patients and healthy controls to analyze the value of serum CTSS in the auxiliary diagnosis of chronic obstructive pulmonary disease.

[0363] 3. Results

[0364] 3.1 Comparative analysis of CTSS levels between patients with chronic obstructive pulmonary disease (COPD) and healthy controls

[0365] The results are as follows Figure 1 As shown, a comparative analysis of serum CTSS levels in 89 COPD patients and 67 healthy controls revealed that, under CI (95%) conditions, the serum CTSS level in COPD patients (24.04 ± 0.53 ng / mL) was significantly higher than that in the control group (10.18 ± 0.49 ng / mL), with a statistically significant difference (p < 0.001). This indicates that serum CTSS levels have good value in the auxiliary diagnosis of COPD patients.

[0366] 3.2 Comparative analysis of CTSS levels between patients with chronic obstructive pulmonary disease (COPD) of different severities (grades I-IV) and healthy controls.

[0367] The results are as follows Figure 2As shown, a comparative analysis of serum CTSS levels in 20 patients with mild COPD (Grade I), 33 patients with moderate COPD (Grade II), 26 patients with severe COPD (Grade III), 10 patients with very severe COPD (Grade IV), and 67 healthy controls revealed that, under CI (95%) conditions, the serum CTSS levels in patients with (Grade I–IV) COPD were 17.60 ± 0.19 ng / ml, 22.55 ± 0.35 ng / ml, 27.31 ± 0.34 ng / ml, and 33.03 ± 0.55 ng / ml, respectively, while the serum CTSS level in the control group was 10.18 ± 0.49 ng / ml.

[0368] This indicates that CTSS levels vary among COPD patients of different severities, with higher CTSS levels observed in more severe COPD patients. Furthermore, all levels were higher than those in the healthy control group. This suggests that serum CTSS levels are of significant importance in the auxiliary diagnosis of COPD patients with different severities.

[0369] 3.3. Diagnostic sensitivity and specificity of ROC curve analysis for determining CTSS levels in patients with chronic obstructive pulmonary disease (COPD).

[0370] The results are as follows Figure 3 As shown in the figure, receiver operating characteristic (ROC) curves for the diagnosis of chronic obstructive pulmonary disease (COPD) were plotted. The efficacy of serum CTSS levels in distinguishing between migraine patients and healthy individuals was 88%, and the specificity was 89%. The area under the ROC curve (AUC) was 0.91 (95% CI = 0.84–0.97), P < 0.001. This indicates that serum CTSS has good value in the auxiliary diagnosis of COPD.

[0371] 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. A cathepsin S specific antigenic epitope peptide, characterized by: The amino acid sequence of the cathepsin S-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: Cys-Ser-Ser-Ala-Val-Ala-Gln-Leu-His-Lys-Asp-Pro-Thr-Leu-Asp; SEQ ID NO.2: Cys-Asn-Lys-Gly-Pro-Val-Ser-Val-Gly-Val-Asp-Ala-Arg-His-Pro.

2. A kit for detecting cathepsin S, characterized in that: A polyclonal antibody containing a specific detection of cathepsin S, said polyclonal antibody being prepared from the cathepsin S-specific antigenic epitope peptide of claim 1; The method for preparing the polyclonal antibody includes using the conjugation product of the cathepsin S-specific antigenic epitope peptide and the carrier protein as an immunogen, using New Zealand white rabbits as immunization animals, administering multiple subcutaneous injections at the back of the New Zealand white rabbits, and performing booster immunizations every 2-3 weeks. After immunization, serum is separated from the blood, and the polyclonal antibody is isolated and purified from the serum.

3. The kit of claim 2, wherein: It includes a solid-phase support and the polyclonal antibody, wherein the polyclonal antibody is directly or indirectly attached to the solid-phase support.

4. The kit of claim 3, wherein: It also includes markers for labeling the polyclonal antibody.

5. The kit of claim 3, wherein: It also includes cathepsin S calibrator.

6. The kit according to any one of claims 2 to 5, 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.

7. The kit of claim 6, wherein: The chemiluminescence assay kit was prepared using a combination of chemiluminescence immunoassay and a double-antibody sandwich method.

8. The kit of claim 7, wherein: It also includes magnetic microparticle suspensions, washing solutions, and chemiluminescent substrates.

9. The use of the kit according to any one of claims 2-8 in the preparation of a product for detecting chronic obstructive pulmonary disease.

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