Antibody for detecting type II collagen C-terminal peptide, kit and application

Abstract

The invention discloses an antibody for detecting type II collagen C-terminal peptide, a kit and application. The antibody for detecting the type II collagen C-terminal peptide is prepared by coupling a specific antigen epitope peptide of the type II collagen C-terminal peptide with a carrier protein and then immunizing an animal, and the amino acid sequence of the specific antigen epitope peptide is at least one of a sequence as shown in SEQ ID NO.1 and a sequence as shown in SEQ ID NO.2. The antibody provided by the invention can specifically detect the type II collagen C-terminal peptide, and has the advantages of high sensitivity, good specificity, good repeatability and the like; when being used for auxiliary diagnosis or curative effect evaluation of osteoarthritis, the kit has good diagnosis sensitivity, specificity and repeatability, meets clinical application requirements, provides a more accurate and reliable objective index for auxiliary diagnosis, disease 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 type II collagen C-terminal peptide. Background Technology

[0002] Osteoarthritis (OA) is a chronic joint disease characterized by degenerative changes in articular cartilage. Type II collagen is a major structural component of cartilage, and its degradation product, type II collagen C-terminal peptide (also known as type II collagen cross-linked carboxyl-terminal peptide, abbreviated as CTX-II), has received widespread attention in recent years as a specific biomarker of cartilage destruction in the early diagnosis, disease assessment, and treatment monitoring of OA.

[0003] Type II collagen is mainly found in tissues such as articular cartilage, vitreous humor, and intervertebral discs, accounting for more than 90% of the total collagen in the cartilage matrix. It is crucial for maintaining the structural integrity and mechanical properties of cartilage. In degenerative joint diseases such as osteoarthritis (OA), type II collagen is specifically cleaved by matrix metalloproteinases (such as MMP-13), releasing its C-terminal cross-linked peptide, CTX-II. This fragment can enter the bloodstream and be excreted in urine, becoming a detectable cartilage degradation product.

[0004] The pathogenesis of osteoarthritis involves localized stress concentration in articular cartilage under conditions of mechanical overload, obesity, trauma, or genetics, inducing type II collagen microcracks. Chondrocytes sense mechanical signals through Piezo channels and immediately release PGE2, NO, and ROS, activating NF-κB, Wnt / β-catenin, and MAPK signaling, and upregulating MMP-13, ADAMTS-4 / 5, and cathepsin. K leads to the breakdown of type II collagen to generate CTX-II and the degradation and loss of aggregates and glycans. At the same time, IL-1β, TNF-α, leptin and complement fragments promote the infiltration of synovial M1 macrophages, forming an inflammatory positive feedback. ROS and DNA methylation inhibit COL2A1 and ACAN transcription, resulting in matrix synthesis being consistently lower than degradation. The number of chondrocytes is sharply reduced in p16^INK4a-mediated senescence, Runx2-driven hypertrophy and caspase-3-dependent apoptosis. Subchondral bone microfissures recruit osteoclasts, causing bone resorption. Subsequently, TGF-β imbalance induces osteoblast hypermineralization, trabecular bone thickening and sclerosis lose their shock-absorbing function, and VEGF-A and NGF mediate the ingrowth of blood vessels and sensory nerves into non-calcified cartilage. Ultimately, this leads to pain, cartilage thinning, osteophyte formation and joint space narrowing, resulting in the destruction of the entire joint structure.

[0005] CTX-II is the shortest and most immunologically active degradation fragment released from the C-terminus of the triple helix region of type II collagen fibers by proteases such as MMP-13 or Cathepsin K. Its core peptide is EKGPDPL (7 amino acids), which can be further elongated to EKGPDPLQ (8 amino acids) under the action of Cathepsin K. This fragment forms a stable dimer structure of ≈3.2 kDa through covalent cross-linking mediated by pyridinoline (PYD) or deoxypyridinoline (DPD). Its conformation is an antiparallel β-hairpin morphology, with the PYD ring acting as the hinge and Pro-5 and Asp-4 forming rigid nodes, ensuring that the EKGPDP epitope is always exposed to the solvent, thus ensuring high affinity binding to antibodies (such as 12A1) (Kd≈0.8 nM). Crystal structure analysis shows that the antibody light chain Asn-92 forms a double hydrogen bond with Asp-4, and the heavy chain Tyr-104 forms a π-π stack with Pro-5. Any truncation / extension of the N-terminus or C-terminus will significantly reduce affinity, which explains the fundamental reason for the numerical differences between different detection platforms.

[0006] At the functional level, CTX-II is the endpoint messenger of cartilage degradation, and its release marks the structural destruction of the type II collagen network. Due to its small molecular weight (≈1.6 kDa / single chain), it can freely pass through the glomerulus, and because its cross-linked structure is not degraded or reabsorbed by the renal tubules, its urinary concentration is significantly positively correlated with the degree of cartilage damage (r≈0.72). In OA animal models, every 1 μg increase in urinary CTX-II corresponds to approximately 0.3 mm² of collagen loss on the surface of articular cartilage; in a prospective human cohort, every 1 standard deviation increase in baseline urinary CTX-II increased the risk of knee OA structural progression by 64% over five years. Therefore, CTX-II is recognized by the Osteoarthritis Research Institute International (OARSI) as the most tissue-specific biomarker for cartilage degradation and is widely used for the early diagnosis, disease monitoring, and efficacy evaluation of diseases such as OA, rheumatoid arthritis (RA), and intervertebral disc degeneration.

[0007] Furthermore, CTX-II also exhibits therapeutic feedback: in multiple clinical intervention studies, oral salmon calcitonin, undenatured type II collagen, or exercise rehabilitation can all reduce urinary CTX-II by 15-25% within 12 weeks, in sync with improvements in WOMAC pain scores. More cutting-edgely, CTX-II is being developed as a targeted therapy carrier: by conjugating CTX-II antibodies to the surface of hyaluronic acid-gold nanoparticles, in-situ aggregation in degenerated cartilage can be achieved. This not only facilitates CT / fluorescence dual-mode imaging but also exerts a dual anti-inflammatory and anti-degradation effect by blocking the IL-17→MMP-13 signaling axis, reducing OARSI scores by 40%.

[0008] In summary, CTX-II is not only a structurally stable, conformationally rigid, and renal filtration-friendly biomarker, but also a multipotent molecular bridge connecting "cartilage destruction - system detection - precise intervention," holding an irreplaceable core position in the precision diagnosis and treatment system for OA.

[0009] Given the current state of osteoarthritis (OA) diagnosis, clinicians have been searching for a stable and reliable biomarker that can objectively diagnose OA and assess its treatment efficacy. Urinary CTX-II is transitioning from a research tool to a routine outpatient procedure, providing an economical, repeatable, and non-invasive "liquid biopsy" shortcut for early OA screening, precise follow-up, and personalized intervention. Its clinical value extends beyond diagnosis, serving as a non-invasive fulcrum for comprehensive OA management. Further research has revealed significantly elevated CTX-II expression levels in OA. It does not directly destroy collagen but rather activates the TLR2 / 4-NF-κB pathway as a damage-associated molecular pattern (DAMP), promoting the release of inflammatory mediators such as IL-6 and TNF-α from monocytes and macrophages, amplifying intra-articular inflammatory responses. Simultaneously, it can be taken up by dendritic cells and presented to CD4+. + T cells induce Th1 immune responses and participate in the expansion of cartilage autoantigen epitopes, thereby driving a vicious cycle of chronic inflammation and repair imbalance in osteoarthritis (OA). CTX-II, as a biomarker for OA, not only has diagnostic value but can also be used for prognostic assessment, helping to evaluate changes in the patient's condition and treatment efficacy.

[0010] In summary, CTX-II plays a crucial role in the development and progression of OA, and its research as a biomarker and therapeutic target provides new ideas and methods for the diagnosis and treatment of OA. However, current research on the specific detection of CTX-II is relatively limited. Developing new CTX-II specific detection technologies is of great significance and clinical application value for monitoring related diseases, evaluating treatment efficacy, and predicting long-term prognosis. Summary of the Invention

[0011] The purpose of this application is to provide a new antibody, kit, and application for detecting type II collagen C-terminal peptide.

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

[0013] The first aspect of this application discloses an antibody for detecting type II collagen C-terminal peptide. The specific antibody is prepared by immunizing animals with a specific antigenic epitope peptide of type II collagen C-terminal peptide conjugated with a carrier protein, wherein the amino acid sequence of the 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] Tyr-Arg-Glu-Lys-Gly-Pro-Asp-Pro-Leu

[0016] SEQ ID NO.2:

[0017] Glu-Lys-Gly-Pro-Asp-Pro-Leu-Lys-Tyr.

[0018] In one implementation of this application, the specific antibody is a polyclonal antibody prepared by immunizing animals with a specific antigenic epitope peptide of type II collagen C-terminal peptide coupled with a carrier protein.

[0019] It should be noted that the key to this application lies in the development of a novel specific antigenic epitope peptide for type II collagen C-terminal peptide, and based on this novel epitope peptide, a new antibody capable of specifically detecting type II collagen C-terminal peptide, namely the polyclonal antibody of this application, thus providing a new scheme and approach for the detection of type II collagen C-terminal peptide. 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.

[0020] The second aspect of this application discloses a kit for detecting type II collagen C-terminal peptide, the kit containing the antibody of this application that specifically detects type II collagen C-terminal peptide.

[0021] 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.

[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 conventionally used in this application. The key to this application lies in the development and preparation of a new antibody that specifically detects type II collagen C-terminal peptide. 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 antibody that specifically detects type II collagen C-terminal peptide.

[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 type II collagen C-terminal peptide standard.

[0028] 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.

[0029] In one implementation of this application, the chemiluminescence assay kit is prepared using a combination of chemiluminescence immunoassay and a double-antibody sandwich method.

[0030] In one implementation of this application, the kit further includes at least one of a magnetic separation reagent, a washing solution, and a chemiluminescent substrate.

[0031] 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.

[0032] The third aspect of this application discloses the use of the antibody that specifically detects type II collagen C-terminal peptide, or the kit of this application, in the preparation of products for diagnosing osteoarthritis.

[0033] It should be noted that the specific antibody or kit of this application can specifically detect type II collagen C-terminal peptide. According to existing research, CTX-II is recognized by OARSI as the most tissue-specific cartilage degradation biomarker and is widely used for the early diagnosis, disease monitoring and efficacy evaluation of diseases such as OA, rheumatoid arthritis (RA) and intervertebral disc degeneration. Therefore, the antibody or kit of this application that specifically detects CTX-II can be used to prepare products for diagnosing osteoarthritis.

[0034] The beneficial effects of this application are as follows:

[0035] The antibody for the specific detection of type II collagen C-terminal peptide in this application has the advantages of high sensitivity, good specificity, and good reproducibility. When used for the auxiliary diagnosis or efficacy evaluation of osteoarthritis, it has good diagnostic sensitivity, specificity, and reproducibility, which meets the needs of clinical application. It provides a more accurate and reliable objective indicator for the auxiliary diagnosis, disease monitoring, and prognostic assessment of osteoarthritis, and has important significance and clinical application value for the diagnosis and treatment of related diseases. Attached Figure Description

[0036] Figure 1 This is a comparative analysis of CTX-II levels in urine samples from healthy control groups and KOA patients in the embodiments of this application;

[0037] Figure 2 This is a comparative analysis of urinary CTX-II levels in KOA patients with different severity levels (0-4) and healthy control groups in the embodiments of this application. Detailed Implementation

[0038] With the in-depth research of CTX-II in related diseases, its use for early diagnosis, disease monitoring and efficacy evaluation of OA has been widely accepted; however, there are relatively few specific detection technologies for CTX-II.

[0039] Based on this research and understanding, this application develops a highly sensitive and specific CTX-II assay kit, and uses a specific polyclonal antibody against CTX-II to achieve accurate quantification of CTX-II in urine samples.

[0040] Specifically, the specific antibody for detecting type II collagen C-terminal peptide in this application is prepared by immunizing animals with a specific antigenic epitope peptide of type II collagen C-terminal peptide conjugated with a carrier protein. The amino acid sequence of the specific antigenic epitope peptide is at least one of the sequences shown in SEQ ID NO.1 and SEQ ID NO.2.

[0041] 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 osteoarthritis, the concentration of CTX-II in the subject sample measured by the kit is compared with the control reference range (i.e., the concentration level of healthy subjects). If the concentration of CTX-II in the subject sample is higher than the concentration in the control reference range, the subject has the risk of osteoarthritis, and the severity of the condition is positively correlated with the concentration level of CTX-II in the sample.

[0042] In chemiluminescence detection, it exhibits high sensitivity with a limit of detection of 15.6 ng / mL, a wide linear range of 3-6 orders of magnitude, and can directly measure samples with concentrations up to 1000 ng / mL without dilution. It also boasts high accuracy with a recovery rate of 97.86% in recovery tests, high precision with a CV of less than 10%, and short reaction time, providing results in 30 minutes, significantly improving detection efficiency. Furthermore, it is highly automated, requiring no manual operation and reducing human error.

[0043] In summary, the specific antibody and kit of this application have the following advantages and positive effects compared with the prior art:

[0044] 1. This application uses CTX-II as a biomarker to prepare a kit for auxiliary diagnosis or efficacy evaluation of osteoarthritis, which overcomes the shortcomings of the indicators used in the current clinical diagnosis of osteoarthritis, which are too subjective and lack objectivity. It provides a more accurate and reliable objective indicator for the auxiliary diagnosis, disease monitoring and prognosis assessment of osteoarthritis.

[0045] 2. The reagent kit prepared in this application has good diagnostic sensitivity, specificity and reproducibility, which meets the needs of clinical application.

[0046] 3. The CTX-II polyclonal antibody prepared in this application can bind to CTX-II in the sample with high specificity.

[0047] 4. The two CTX-II 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.

[0048] 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.

[0049] Unless otherwise specified, the technical means used in the embodiments are conventional means well known to those skilled in the art.

[0050] Example

[0051] I. Synthesis of CTX-II specific antigenic epitope peptides

[0052] In this case, two CTX-II specific antigenic epitope peptides were developed, namely the sequences shown in SEQ ID NO.1 and SEQ ID NO.2.

[0053] SEQ ID NO.1:

[0054] Tyr-Arg-Glu-Lys-Gly-Pro-Asp-Pro-Leu

[0055] SEQ ID NO.2:

[0056] Glu-Lys-Gly-Pro-Asp-Pro-Leu-Lys-Tyr.

[0057] The two CTX-II specific antigenic epitope peptides are synthesized as follows:

[0058] 1. Main raw materials

[0059] 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.).

[0060] 2. Main Instruments

[0061] 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.

[0062] 3. Synthesis methods and processes

[0063] 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:

[0064] (1) Activation of amino acids (HOBt / DCC method)

[0065]

[0066] Fmoc-protected amino acids

[0067]

[0068] (2) Connecting amino acids to resin (HO-Resin)

[0069]

[0070] (3) Removal of the Fmoc protecting group from amino acids

[0071]

[0072] (4) Activation of another amino acid (HOBt / DCC method)

[0073]

[0074] (5) Coupling

[0075]

[0076] (6) Repeat steps (3) to (5) until the synthesis is complete.

[0077] Peptide resins containing CTX-II antigenic epitope peptide (1) and CTX-II antigenic epitope peptide (2) were obtained. CTX-II antigenic epitope peptide (1) and CTX-II antigenic epitope peptide (2) are CTX-II specific antigenic epitope peptides with sequences shown in SEQ ID NO.1 and SEQ ID NO.2, respectively.

[0078] (7) Peptide cleavage resin

[0079] 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 CTX-II antigenic epitope peptide (1) and CTX-II antigenic epitope peptide (2).

[0080] 4. Purification of crude CTX-II antigenic epitope peptides (1) and (2)

[0081] Separation and purification were performed using high performance liquid chromatography:

[0082] Conditions: Column: C8 10×100mm, purchased from Waters Corporation, USA

[0083] Chromatograph: Waters 600, Waters Corporation, USA

[0084] Mobile phase: A: 0.1% TFA (trifluoroacetic acid) aqueous solution

[0085] B: 0.1% TFA (trifluoroacetic acid) in 60% acetonitrile

[0086] Detection wavelength: 214nm

[0087] Flow rate: 4 mL / min

[0088] Elution gradient: 20-60% B, 30 minutes

[0089] HPLC (High Performance Liquid Chromatography) Analysis

[0090] Column: C18 4.6×150mm, purchased from Waters Corporation, USA

[0091] Mobile phase: A: 0.1% TFA (trifluoroacetic acid) aqueous solution

[0092] B: 0.1% TFA (trifluoroacetic acid) in acetonitrile

[0093] Detection wavelength: 214nm

[0094] Flow rate: 1 mL / min

[0095] Elution gradient: 0-60% B, 30 minutes

[0096] Peptide analysis results showed that the purity of CTX-II antigenic epitope peptide (1) and CTX-II antigenic epitope peptide (2) prepared in this example was 98%, which met the requirements for subsequent use.

[0097] II. Preparation of CTX-II Specific Antibodies

[0098] 1. Preparation of antigens

[0099] CTX-II antigen (1) and CTX-II antigen (2) were prepared by linking the CTX-II specific antigen epitope peptide (1) or (2) to the carrier protein KLH (keyhole hemocyanin) using the BDB (Bis-diazotized benzidine dichloride) method.

[0100] Specifically, take 20.0 mg of CTX-II 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 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℃.

[0101] 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.

[0102] 2. Preparation of polyclonal antibodies by immunizing animals

[0103] 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 CTX-II 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.

[0104] 2.2. Antibody titer determination: The titer of CTX-II polyclonal antibody (1) or (2) was determined by indirect ELISA. Specifically, CTX-II 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.

[0105] The results showed that the titers of the CTX-II polyclonal antibody (1) and CTX-II polyclonal antibody (2) prepared in this example both reached 1:32000 or higher.

[0106] 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.

[0107] 3. Isolation and purification of antibodies

[0108] The CTX-II polyclonal antibody (1) and CTX-II polyclonal antibody (2) to be loaded were dialyzed with a balancing buffer (0.02M, PB, pH 8.0) until the pH was consistent. The column was packed and connected to the protein chromatography system, and washed with the balancing buffer until the pH was 8.0. The CTX-II polyclonal antibody (1) and CTX-II 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 detector showed the beginning of elution. The protein solution was then collected. Collection was stopped when the peak gradually decreased. The concentration of CTX-II polyclonal antibody (1) or CTX-II polyclonal antibody (2) was measured and calculated using an L5S UV-Vis spectrophotometer (Boda Precision). The results showed that the concentration of CTX-II polyclonal antibody (1) was 2.0 mg / mL and the concentration of CTX-II polyclonal antibody (2) was 1.0 mg / mL. The purified CTX-II polyclonal antibody (1) and CTX-II polyclonal antibody (2) were aliquoted and stored at -20℃.

[0109] Experiment 1: Preparation of the CTX-II chemiluminescence reagent kit

[0110] 1. Preparation of magnetic microparticle suspension (working solution of CTX-II polyclonal antibody (1) immunomagnetic beads)

[0111] (1) Washing of magnetic beads

[0112] 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.

[0113] (2) Activation of magnetic beads

[0114] 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.

[0115] (3) Coating CTX-II polyclonal antibody with activated magnetic beads (1)

[0116] Add 1 mL of 0.1 M MES (pH 6.0) buffer to the coating tube and vortex for 1 min. Add 10 μL of CTX-II polyclonal antibody (1) (2.0 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.

[0117] (4) Magnetic bead sealing

[0118] 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.

[0119] (5) Cleaning

[0120] 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.

[0121] (6) Preparation of magnetic microparticle suspension

[0122] The magnetic beads coated with CTX-II 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.

[0123] 2. Preparation of enzyme-labeled antibody (polyclonal antibody labeled with alkaline phosphatase CTX-II (2))

[0124] (1) Take 250 μL of 0.1M MES (pH 4.5) and soak the ultrafiltration centrifuge tube (30KD) for 2 min.

[0125] (2) Add 250 μL of 0.1 M MES (pH 4.5) to the centrifuge column and bring the volume to 500 μL.

[0126] (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.

[0127] (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.

[0128] 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.

[0129] (5) Take 5 μL of CTX-II polyclonal antibody (2) 1.0 mg / mL, mix well, centrifuge at 13000 rpm for 20 min and discard the waste liquid.

[0130] (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.

[0131] (7) Repeat the experimental steps (6) once.

[0132] (8) Add 0.1M PB (pH 9.0) to a final volume of 250μL and place on a shaker at 120rpm for 2h.

[0133] (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℃.

[0134] 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.

[0135] 3. Preparation of washing solution

[0136] It consists of 10 mM PBS (pH 7.2), 0.08% Tween-20 and 0.03% Proclin-300.

[0137] 4. Preparation of CTX-II calibrators and quality control samples

[0138] CTX-II recombinant protein (provided by Chengdu Cormorant Biotechnology, 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 (15.6–1000 ng / mL) and two quality control samples of high and low concentrations.

[0139] 5. Preparation of luminescent substrates

[0140] 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.

[0141] 6. Components of the reagent kit

[0142] The kit mainly consists of reagent compartments (reagent 1, reagent 2), CTX-II calibrator A (1.0mL×1 vial, concentration: 100 ng / mL), CTX-II calibrator B (1.0mL×1 vial, concentration: 800 ng / mL), quality control (1.0mL×1 vial, concentration: 300 ng / 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 magnetic microparticles coated with CTX-II polyclonal antibody (1), and reagent 2 contains alkaline phosphatase (AP) labeled CTX-II polyclonal antibody (2). The kit is available in two packaging specifications: 50 doses / box and 100 doses / box.

[0143] 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.

[0144] Experiment 2: Preparation of the CTX-II chemiluminescence reagent kit

[0145] 1. Preparation of magnetic microparticle suspension reagent (working solution of CTX-II monoclonal antibody immunomagnetic beads)

[0146] (1) Washing of magnetic beads

[0147] 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.

[0148] (2) Activation of magnetic beads

[0149] Add 800 μL of 0.1M MES (pH 6.0) buffer to the coated tube, then add 100 μL of NHS (10 mg / L) solution and vortex for 1 min. Add 100 μL of EDC (10 mg / L) 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.

[0150] (3) CTX-II monoclonal antibody was coated with activated magnetic beads.

[0151] 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 CTX-II monoclonal antibody (1 mg / mL) (provided by Chengdu Cormorant Biotechnology, catalog number CTX-II-Mab) 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.

[0152] (4) Magnetic bead sealing

[0153] 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.

[0154] (5) Cleaning

[0155] 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.

[0156] (6) Preparation of magnetic microparticle suspension reagent

[0157] The magnetic beads coated with CTX-II monoclonal antibody were diluted with magnetic bead diluent (0.05M TRIS (pH 7.4), 0.5% BSA, 0.9% NaCl) to obtain a final antibody concentration of 2 μg / mL.

[0158] 2. Preparation of enzyme-labeled antibody (polyclonal antibody labeled with alkaline phosphatase CTX-II (2))

[0159] Same as Experiment 1.

[0160] 3. Preparation of CTX-II calibrators / quality control samples, washing solution, and luminescent substrates

[0161] Same as Experiment 1.

[0162] 4. Components of the reagent kit

[0163] The kit mainly consists of reagent compartments (reagent 1, reagent 2), CTX-II calibrators A and B (1.0mL×1 vial), quality control (1.0mL×1 vial), substrate solution (12.0mL / 25.0mL), concentrated washing solution (25×) (60.0mL×1 vial / 60.0mL×2 vials) and calibration / quality control information cards. Reagent 1 in the reagent compartment contains magnetic microparticles coated with CTX-II monoclonal antibody, and Reagent 2 contains alkaline phosphatase (AP) labeled CTX-II polyclonal antibody (2). The kit is available in two packaging specifications: 50 doses / box and 100 doses / box.

[0164] 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.

[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] CTX-II specific polyclonal antibody (1) was dissolved in coating buffer to prepare a pre-coating solution. 100 μL of 0.1 μg was added to each well of an enzyme-labeled plate (purchased from Shenzhen Jincanhua Company). The plate was placed at 4°C for 18-24 hours. The plate was then removed, the coating solution was discarded, and the plate was washed. 100 μL of blocking solution was added to each well and the plate was blocked at 4°C for 16 hours. The blocking solution was discarded, the plate was dried, and the plate was sealed in an aluminum foil bag and stored at 4°C.

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

[0186] The conjugate antibody (CTX-II 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.

[0187] 4. Preparation of CTX-II calibrators and quality control samples

[0188] The recombinant CTX-II (provided by Chengdu Cormorant Biotechnology, concentration 2 mg / mL) was serially diluted with sample diluent to prepare CTX-II calibrators 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 CTX-II calibrators, CTX-II quality control samples (high and low concentrations), binding antibody (10 mL), enzyme marker (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 CTX-II Fluorescence Chromatography Assay Kit

[0192] 1. Wrapped with padding

[0193] 1.1 Fluorescent microspheres labeled with CTX-II 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 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.

[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. Labeling CTX-II 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 CTX-II 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 CTX-II 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] CTX-II 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. CTX-II 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 CTX-II 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 CTX-II 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 CTX-II 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 CTX-II polyclonal antibody (1).

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

[0215] 1. Urine sample: The sample does not involve preservatives and should not be left at room temperature for more than 1 hour. It can be refrigerated at 2-8℃ for 7 days. Refrigerated samples need to be brought to room temperature before testing. The sample should not be frozen.

[0216] 2. The CTX-II concentration was determined using a chemiluminescent immunoassay (CLIA) kit. 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 sampling 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 to perform the test (total test 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, 50% means 150 μL of calibrator is mixed with 150 μL of diluent, and so on.

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

[0235] Sample dilution Measurement 1 Measurement 2 mean 100% 989 997 993 50% 513 502 507.5 25% 246 251 248.5 10% 96 98 97 5% 56 49 52.5 2.5% 20.5 23.6 22.05

[0236] The test results show that the kit prepared in Experiment 1 has a wide linear range within the range of 15.6 to 1000 ng / mL, with the linear equation formula: y = 996.55x + 0.3667 and a linear correlation coefficient of 0.999, indicating 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 15.6 ng / 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 100 ng / mL and 900 ng / 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 100 ng / mL Test concentration 900 ng / mL 1 102.01 901.02 2 96.32 896.32 3 95.65 890.13 4 89.25 879.39 5 96.14 905.96 6 91.35 900.13 7 89.73 887.36 8 87.95 879.58 9 88.74 883.21 10 99.36 910.36 mean 93.65 893.346 Standard deviation SD 4.65 10.47 CV% 4.97% 1.17%

[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 97.86%, 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 1000 ng / mL without dilution; it has high sensitivity with a limit of detection of 15.6 ng / mL; it has high accuracy with a recovery rate of 97.86%; it has good precision with CV% of 4.97% for high concentration and 1.17% for low concentration; 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 CTX-II using the chemiluminescence reagent kit from Experiment 2 and validation of kit performance.

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

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

[0258] 3. Reagent kit performance verification

[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: ng / mL)

[0263] Sample dilution Measurement 1 Measurement 2 mean 100% 989.35 990.14 989.75 50% 450.21 420.98 435.60 25% 200.96 220.14 210.55 10% 101.32 80.69 91.01 5% 50.36 48.67 49.52 2.50% 22.32 20.58 21.45

[0264] The test results show that the kit prepared in Experiment 2 has a wide linear range in the range of 20-1000 ng / mL, with the linear equation formula: y=982.29x+15.507 and a linear correlation coefficient of 0.9956, 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 20 ng / mL, indicating that the kit has high sensitivity.

[0268] 3.3 Precision

[0269] The validation method was the same as in application test 1. The two concentrations used for precision validation were 150 ng / mL and 800 ng / mL.

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

[0271] Table 4 Precision Test Results

[0272] Serial Number Test concentration 150 ng / mL Test concentration 800 ng / mL 1 160.23 804.32 2 145.32 789.21 3 140.36 779.36 4 143.69 780.36 5 140.36 791.25 6 132.56 805.36 7 150.87 789.19 8 139.21 795.13 9 140.01 796.17 10 141.35 786.32 mean 143.396 791.667 Standard deviation SD 7.16 8.37 CV% 4.99% 1.06%

[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 95.41%, indicating that the kit has high accuracy.

[0277] Verification conclusion:

[0278] Experiment 2 validation results show that: the kit has a wide linear range and can directly measure samples with concentrations up to 1000 ng / mL without dilution; it has high sensitivity with a limit of detection of 20 ng / mL; it has high accuracy with a recovery rate of 95.41%; it has good precision with CV% of 4.99% and 1.06% for high and low concentrations, respectively; and the entire reaction time is short, with results available in 30 minutes, which greatly improves detection efficiency.

[0279] Application Experiment 3: Determination of CTX-II 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 CTX-II concentration was determined using an enzyme-linked immunosorbent assay (ELISA) kit. The specific steps are as follows:

[0282] (1) Preparation of washing solution: 25 times concentrated washing solution is diluted with deionized water at a ratio of 1:25;

[0283] (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.

[0284] (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 pat to mix, seal the microplate with sealing film, and incubate at 37°C for 30 minutes.

[0285] (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;

[0286] (5) Add two drops or 100 μL of enzyme-labeled material to each well, seal the plate with sealing film, and incubate at 37°C for 30 minutes;

[0287] (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;

[0288] (7) Add 50 μL of colorimetric reagent A and B solution to each well, mix thoroughly, and incubate at 37°C for 15 minutes;

[0289] (8) Add 1 drop (50 μL) of stop solution to each well as soon as possible and gently tap to mix;

[0290] (9) Measure the OD value of each well using an ELISA reader (with dual wavelengths of 450 / 630 nm);

[0291] (10) Plot the calibration curve and calculate the CTX-II concentration in the sample based on the calibration curve.

[0292] 3. Reagent kit performance verification

[0293] 3.1 Linearity Range Verification

[0294] The verification method is the same as that used in application experiment 1.

[0295] The test results are shown in Table 5 below.

[0296] Table 5. Linearity range test results and calculations (unit: ng / mL)

[0297] Sample dilution Measurement 1 Measurement 2 mean 100% 95.36 101.23 98.30 50% 49.33 45.1 47.22 25% 22.36 25.31 23.84 10% 9.65 8.21 8.93 5% 4.27 4.58 4.43 2.50% 2.39 2.16 2.28

[0298] The test results show that the kit prepared in Experiment 3 has a wide linear range in the range of 2-100 ng / mL, with the linear equation formula: y=98.42x+0.75 and a linear correlation coefficient of 0.9997, indicating that the kit has a wide linear range.

[0299] 3.2 Minimum Detection Limit

[0300] The verification method is the same as that used in application experiment 1.

[0301] The test results showed that the limit of detection was 2 ng / mL, indicating that the kit has high sensitivity.

[0302] 3.2 Precision

[0303] The validation method was the same as in application test 1. The two concentrations used for precision validation were 8.5 ng / mL and 90 ng / mL.

[0304] The test results are shown in Table 6 below.

[0305] Table 6 Precision Test Results

[0306] Serial Number Test concentration: 8.5 ng / mL Test concentration 90 ng / mL 1 8.23 90.21 2 8.54 90.14 3 8.36 89.65 4 8.57 88.11 5 7.52 90.36 6 7.23 87.25 7 7.39 84.36 8 7.89 86.41 9 8.32 89.12 10 8.01 84.12 mean 8.006 87.973 Standard deviation SD 0.46 2.25 CV% 5.74% 2.55%

[0307] 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.

[0308] 3.4 Accuracy

[0309] The verification method is the same as that used in application experiment 1.

[0310] The recovery rate was calculated to be 96.81%, indicating that the kit has high accuracy.

[0311] Verification conclusion:

[0312] The validation results of the kit prepared in Experiment 3 showed that the kit had good linearity in the range of 2-100 ng / mL, reaching 0.9997; high sensitivity, with a limit of detection of 2 ng / mL; recovery rate of 96.81%; 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.

[0313] Application Experiment 4: Validation of the fluorescence immunochromatographic kit used in Experiment 4 for CTX-II assay and kit performance.

[0314] 1. Sample collection: Same as application experiment 1.

[0315] 2. The concentration of CTX-II was determined using a fluorescence immunochromatographic assay kit. The specific steps are as follows:

[0316] (1) Before testing, bring the reagents to room temperature (20±5℃), and conduct the test at room temperature;

[0317] (2) Turn on the fluorescence analyzer and adjust the instrument to the ready-to-detect state;

[0318] (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;

[0319] (4) After the reaction is complete, insert the test card into the fluorescence analyzer for scanning test, and calculate the CTX-II concentration of each sample according to the built-in calibration curve of the analyzer.

[0320] 3. Reagent kit performance verification

[0321] 3.1 Linearity Range Verification

[0322] The testing method is the same as in application experiment 1.

[0323] The test results are shown in Table 7 below.

[0324] Table 7. Linearity Range Test Results and Calculations (Unit: ng / mL)

[0325] Sample dilution Measurement 1 Measurement 2 mean 100% 96.32 98.21 97.27 50% 51.24 50.14 50.69 25% 34.12 33.54 33.83 10% 8.45 8.21 8.33 5% 4.36 4.69 4.53 2.50% 2.32 2.69 2.51

[0326] The test results show that, within the range of 2–100 ng / mL, the linear equation of the test kit in Experiment 4 is: y = 97.368x + 1.6185, and the linear correlation coefficient can reach 0.9882, indicating that the kit has a wide linear range.

[0327] 3.2 Minimum Detection Limit

[0328] The testing method is the same as in application experiment 1.

[0329] The test results showed that the limit of detection was 2 ng / mL.

[0330] 3.2 Precision

[0331] The test method was the same as in application test 1. The two concentrations used for precision verification were 5 ng / mL and 90 ng / mL.

[0332] The test results are shown in Table 8 below.

[0333] Table 8 Precision Test Results

[0334] Serial Number Test concentration 5ng / mL Test concentration 90 ng / mL 1 4.93 89.63 2 4.21 85.12 3 4.36 89.32 4 5.02 86.14 5 4.69 81.23 6 4.52 86.36 7 5.01 87.65 8 5.36 88.11 9 5.01 83.14 10 4.36 90.27 mean 4.747 86.697 Standard deviation SD 0.36 2.77 CV% 7.49% 3.19%

[0335] 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.

[0336] 3.4 Accuracy

[0337] The verification method is the same as that used in application experiment 1.

[0338] The recovery rate was calculated to be 97.36%, which meets the recovery rate requirement (90%–110%).

[0339] Verification conclusion:

[0340] The validation results of the kit prepared in Experiment 4 showed that the kit had good linearity in the range of 2-100 ng / mL, reaching 0.9882, with a sensitivity of 2 ng / mL, a recovery rate of 97.36%, 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.

[0341] Test Comparison Example 1:

[0342] A chemiluminescence kit (comparative kit) was prepared using the CTX-II paired antibody of PSA686Hu11 (purchased from Bioscient Biotech) according to the method in Experiment 1, and its analytical performance was compared with that of the kit in Experiment 1.

[0343] 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.

[0344] Table 9 Performance comparison results between the comparison kit and the kit in Experiment 1

[0345]

[0346] In the comparison of various analytical performances, the kit prepared in Experiment 1 has a wider linear detection range, higher sensitivity, and better other indicators.

[0347] Clinical Trial 1: CTX-II assay kit for the auxiliary diagnosis and disease monitoring of knee osteoarthritis (KOA)

[0348] 1. Research Subjects

[0349] Patients with knee osteoarthritis: 70 patients with knee osteoarthritis (KOA) were included in the study group, including 36 males and 34 females, with an average age of (67±7.9) years, years of education of (13.7±5.2), BMI of (23.52±3.1), and a disease duration of 2-6 years. The Kellgren-Lawrence grading system (KL grading) was used to classify the severity of knee osteoarthritis: Grade 0-1 was mild, with suspected joint space narrowing and lip-like osteophyte formation; Grade 2-3 was moderate, with suspected joint space narrowing and significant osteophyte formation; Grade 4 was severe, with significant joint space narrowing, large osteophytes, severe osteosclerosis, and deformity.

[0350] Healthy control group: 62 healthy individuals served as the control group, including 35 males and 27 females, with an average age of (67±6.9) years, years of education of (12.8±4.6), and BMI of (23.62±3.1).

[0351] There were no statistically significant differences in general characteristics between the two groups, and all participants gave informed consent to this study.

[0352] 2. Experimental Methods

[0353] The kits prepared in Experiment 1 were used to test the CTX-II levels in urine samples from enrolled healthy controls and KOA patients.

[0354] The kits prepared in Experiment 1 were used to test the urinary CTX-II levels of enrolled KOA patients with different severity levels (0-4) and compared them with those of healthy controls.

[0355] 3. Results

[0356] 3.1 Comparative analysis of CTX-II levels in urine samples from healthy control group and KOA patients

[0357] like Figure 1 As shown, a comparative analysis of urinary CTX-II levels in 70 KOA patients and 62 healthy controls revealed that, under CI (95%) conditions, the urinary CTX-II level in KOA patients (255.73±7.69 ng / mL) was significantly higher than that in the control group (138.91±2.96 ng / mL), with a statistically significant difference (p<0.001). This indicates that urinary CTX-II levels have good value in the auxiliary diagnosis of KOA patients.

[0358] 3.2 Comparative analysis of urinary CTX-II levels in patients with different degrees of KOA (0-4) and healthy controls.

[0359] like Figure 2 As shown, a comparative analysis of urinary CTX-II levels in 24 patients with mild knee osteoarthritis (grades 0-1), 20 patients with moderate knee osteoarthritis (grades 2-3), 16 patients with severe knee osteoarthritis (grade 4), and 62 healthy controls revealed the following: Under CI (95%) conditions, the urinary CTX-II levels in patients with grade 0-4 KOA were 189.29±4.82 ng / mL, 259.55±8.30 ng / mL, and 292.78±8.82 ng / mL, respectively, while the urinary CTX-II level in the control group was 138.91±2.96 ng / mL. This indicates that CTX-II levels vary among patients with different degrees of KOA severity, with higher severity patients exhibiting higher CTX-II levels. Furthermore, all CTX-II levels were higher than those in the healthy control group. This suggests that urinary CTX-II levels are of significant importance in the auxiliary diagnosis of KOA patients with different degrees of severity.

[0360] 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 that detects a C-terminal peptide of collagen type II, characterized in that: The specific antibody is prepared by immunizing animals after coupling a specific antigen epitope peptide of the C-terminal peptide of collagen type II with a carrier protein, and the amino acid sequence of the specific 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. SEQ ID NO. 1: Tyr-Arg-Glu-Lys-Gly-Pro-Asp-Pro-Leu SEQ ID NO. 2: Glu-Lys-Gly-Pro-Asp-Pro-Leu-Lys-Tyr.

2. The antibody of claim 1, wherein: The specific antibody is a polyclonal antibody prepared by immunizing animals after coupling the specific antigen epitope peptide with a carrier protein.

3. A kit for detecting a C-terminal peptide of collagen type II, characterized in that: The antibody of claim 1 or 2.

4. The kit of claim 3, wherein: The kit further comprises a solid phase carrier and the antibody, wherein the antibody is directly or indirectly connected to the solid phase carrier. Optionally, the solid phase carrier is at least one of a microwell reaction plate, a magnetic microsphere and a nitrocellulose membrane.

5. The kit of claim 4, wherein: The kit further comprises a label for labeling the antibody. Optionally, the label is at least one of horseradish peroxidase (HRP), alkaline phosphatase (AP), a luminescent substance, a fluorescent substance, a dye and colloidal gold.

6. The kit of claim 4, wherein: The kit further comprises a standard sample of the C-terminal peptide of collagen type II.

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

8. The kit of claim 7, wherein: The chemiluminescence assay kit is prepared by chemiluminescence immunoassay combined with double antibody sandwich method.

9. The kit of claim 7, wherein: The kit further comprises a magnetic microsphere suspension, a washing solution and a chemiluminescence substrate. Optionally, the chemiluminescence substrate comprises at least one of luminol, isoluminol and derivatives thereof, (adamantane)-1,2-dioxyethane and derivatives thereof.

10. 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 diagnosing osteoarthritis.

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