Type I collagen cross-linked N-terminal peptide monitoring kit for monitoring tumor bone metastasis

Abstract

The invention discloses an I-type collagen cross-linked N-terminal peptide monitoring kit for monitoring tumor bone metastasis, and relates to the field of kits, the I-type collagen cross-linked N-terminal peptide monitoring kit comprises: a solid phase carrier coated with a synthetic NTx peptide; an enzyme labeled anti-NTx monoclonal antibody conjugate; a chemiluminescent substrate solution; the invention relates to a calibrator for quantitatively determining the concentration of I-type collagen cross-linked N-terminal peptide in a urine sample. According to the invention, the multi-gradient calibrator adaptive to the morning urine characteristics of the tumor patient is designed, and the integrated creatinine detection module is integrated to realize synchronous correction, so that the problem of insufficient standardization caused by mismatching of a calibration system and external creatinine detection is effectively solved; meanwhile, the anti-interference capability and the detection stability of the kit in the presence of common endogenous interfering substances of tumor patients such as hemoglobin, bilirubin and chyle are remarkably improved, so that the accuracy of bone resorption degree evaluation and treatment response dynamic monitoring of the bone metastasis patients is comprehensively enhanced.

Description

Technical Field

[0001] This invention relates to the field of reagent kits, specifically a type I collagen cross-linked N-terminal peptide monitoring kit for monitoring tumor bone metastasis. Background Technology

[0002] Bone metastasis is a common complication of advanced prostate cancer and other malignant tumors, affecting approximately 80-90% of patients with metastatic prostate cancer. Increased bone resorption activity leads to abnormally elevated levels of bone metabolism markers such as NTx. Therefore, developing highly sensitive NTx detection tools is of significant clinical value for assessing the extent of bone metastasis and monitoring the effectiveness of anti-bone resorption therapy.

[0003] Existing technologies include NTx detection kits using chemiluminescence immunoassay, such as the VITROS NTx kit based on the principle of competitive immunoassay. These kits utilize streptavidin-coated reaction cups containing synthetic NTx peptides to competitively bind horseradish peroxidase-labeled monoclonal antibodies to NTx in the sample, achieving quantitative detection through the binding of the chemiluminescent substrate. These kits are primarily used for osteoporosis diagnosis, with sample concentrations ranging from 15 to 3000 nM bone collagen equivalents (BCE), and support fully automated immunoassay analyzer operation. Clinical studies have shown a significant positive correlation between urinary NTx levels and the maximum standardized uptake value (SUVmax) of bone metastases on PET imaging, confirming that NTx can serve as an effective biomarker for bone resorption activity.

[0004] However, existing reagent kits are mainly designed for metabolic bone diseases such as osteoporosis and have not been optimized for the specific detection needs of tumor bone metastases. In particular, they lack a calibration system adapted to the characteristics of urine samples from cancer patients. The creatinine correction step in the detection process relies on external components and does not integrate an integrated creatinine detection module, resulting in insufficient standardization of results. In addition, cancer patients often have endogenous interferences such as hemolysis, lipemia, and jaundice. Existing reagent kits have not systematically verified their anti-interference ability in complex sample matrices, especially the stability of creatinine detection in the presence of interfering substances such as hemoglobin, bilirubin, and chyle, which affects their reliability in dynamic tumor monitoring. Summary of the Invention

[0005] Based on this, the purpose of this invention is to provide a type I collagen cross-linked N-terminal peptide monitoring kit for monitoring tumor bone metastasis, in order to solve the technical problems in the prior art caused by quantitative deviation due to mismatch between the calibration system and the characteristics of urine samples from tumor patients, insufficient standardization caused by creatinine correction relying on external components, and the influence of common endogenous interfering substances in tumor samples on the stability of creatinine detection.

[0006] To achieve the above objectives, the present invention provides the following technical solution: a type I collagen cross-linked N-terminal peptide monitoring kit for monitoring tumor bone metastasis, comprising: a solid-phase carrier coated with synthetic NTx peptide; an enzyme-labeled anti-NTx monoclonal antibody conjugate; a chemiluminescent substrate solution; and a calibrator for quantitatively determining the concentration of type I collagen cross-linked N-terminal peptide in urine samples.

[0007] The present invention is further configured such that the solid-phase support is a streptavidin-coated reaction cup; and the enzyme-labeled anti-NTx monoclonal antibody conjugate is an enzyme-labeled anti-NTx monoclonal antibody.

[0008] The present invention is further configured such that the chemiluminescent substrate solution contains a luminol derivative, a peroxide, and an electron transfer enhancer.

[0009] The present invention is further configured to include: a sample diluent, a washing solution, and a creatinine detection component for creatinine correction.

[0010] The present invention is further configured such that the calibrator comprises 0 nM BCE and NTx standards at two or more non-zero concentration points, the non-zero concentration points including 250 nM BCE and 850 nM BCE.

[0011] The present invention is further configured such that the reagent kit is compatible with a fully automated immunoassay analyzer.

[0012] The present invention is further configured such that the detection method of the kit is a competitive immunoassay, comprising the following steps: Step 1: urine sample and enzyme-labeled anti-NTx antibody competitively bind on a solid-phase carrier; Step 2: washing away unbound substances; Step 3: adding chemiluminescent substrate to trigger luminescence reaction; Step 4: calculating NTx concentration by means of light signal intensity.

[0013] The present invention is further configured such that the detection results are corrected for urinary creatinine concentration and reported in nM BCE / mM creatinine units.

[0014] The present invention is further configured such that the urine sample is the second morning urine.

[0015] In summary, the present invention has the following main beneficial effects:

[0016] This invention designs a multi-gradient calibrator adapted to the characteristics of morning urine in cancer patients and integrates an integrated creatinine detection module to achieve synchronous calibration, effectively solving the problem of insufficient standardization caused by mismatched calibration systems and external creatinine detection. At the same time, it significantly improves the anti-interference ability and detection stability of the reagent kit in the presence of common endogenous interfering substances in cancer patients such as hemoglobin, bilirubin, and chyle, thereby comprehensively enhancing the accuracy of bone resorption assessment and dynamic monitoring of treatment response in patients with bone metastases. Attached Figure Description

[0017] Figure 1 This is a schematic diagram of the detection process of the present invention. Detailed Implementation

[0018] The technical solutions of the embodiments of the present invention will be clearly and completely described below with reference to the accompanying drawings. The embodiments described below with reference to the accompanying drawings are exemplary and are only used to explain the present invention, and should not be construed as limiting the present invention.

[0019] The embodiments of the present invention will now be described.

[0020] Example 1

[0021] This embodiment provides a type I collagen cross-linked N-terminal peptide (NTx) monitoring kit for monitoring tumor bone metastasis, comprising the following components:

[0022] Solid support: 100-well polystyrene reaction cups coated with streptavidin, each well was coated to synthesize 18 ng of NTx peptide;

[0023] Enzyme-labeled antibody: Horseradish peroxidase (HRP)-labeled mouse anti-human NTx monoclonal antibody conjugate, which can bind ≥740 pmol BCE NTx per milliliter;

[0024] Chemiluminescent substrate: Buffer solution containing luminol derivative, trichlorotetrahydroxyacetanilide and peroxyate;

[0025] Calibrators: NTx standards in three concentration gradients (0, 250, 850 nM BCE), prepared with specific buffer matrix;

[0026] Creatinine detection module: Dry chemical enzymatic creatinine detection reagent, per cm 2 The dried tablets contain 0.2 U of creatinine aminohydrolase, 3.6 U of creatinine amidohydrolase, 0.55 U of amino acid oxidase, 1.6 U of horseradish peroxidase, and 32 μg of 2-(3,5-dimethoxy-4-hydroxyphenyl)-4,5-p-(4-dimethylaminophenyl)imidazolium (colorless dye).

[0027] Auxiliary reagents: Sample diluent (treated water containing processed human serum, preservatives, and inorganic salts), washing solution (containing nonionic surfactants Bronidox-K and Triton-X 100).

[0028] Testing steps:

[0029] Take 25 μL of the second morning urine sample (supernatant after centrifugation) and mix it with 135 μL of enzyme-labeled antibody. Add the mixture to a reaction vessel and incubate for 30 min (37℃).

[0030] After washing three times, add 200 μL of chemiluminescent substrate;

[0031] The immunoassay analyzer measures the signal at fixed time points after the addition of the chemiluminescent substrate and calculates the result based on the stored calibration curve.

[0032] Simultaneous detection of urinary creatinine: Add 6 μL of urine to the creatinine dry slide, and perform colorimetric analysis at 670 nm;

[0033] Result calculation: NTx concentration (nM BCE) = calibration curve fitting value × creatinine correction factor.

[0034] Example 2:

[0035] To address the high solute content in the morning urine of cancer patients, a three-gradient calibrator (0, 250, 850 nM BCE) was designed for comparison with a conventional two-gradient calibrator (0, 850 nM BCE). The test sample consisted of morning urine from 50 patients with bone metastases, with standard NTx added to a final concentration of 500 nM BCE.

[0036] Table 1: Comparison of Recovery Rates of Gradient Calibration Systems

[0037] Calibrator gradient number Average recovery rate (%) Tumor sample fitting R² 2 gradients 82.6±7.3 0.891 3 gradients 98.2±2.1 0.997

[0038] Conclusion: For tumor patient samples, the three-gradient calibration system with 0, 250, and 850 nM BCE significantly improves the accuracy and goodness of fit compared to the two-gradient system with 0 and 850 nM BCE.

[0039] Example 3

[0040] The anti-interference performance of the integrated creatinine detection module of this invention was verified. The results showed that the creatinine detection kit of this invention exhibited excellent stability under interference from hemoglobin, bilirubin, and chyle.

[0041] Table 2: Recovery rate of creatinine detection under hemoglobin (Hb) interference (n=5)

[0042] Hb (g / L) COBA C501 system recovery rate (%) Mike-7180 ​​system recovery rate (%) Recovery rate (%) of the reagent kit of this invention 0 (Control) 100.0 100.0 100.0 0.92 93.1±6.9 94.5±5.5 98.5±2.1 4.60 115.0±15.0 92.8±16.1 99.2±1.8

[0043] Table 3: Recovery rate of creatinine detection under bilirubin interference (n=5)

[0044] Bilirubin (μmol / L) COBA C501 system recovery rate (%) Mike-7180 ​​system recovery rate (%) Recovery rate (%) of the reagent kit of this invention 0 (Control) 100.0 100.0 100.0 309.0 94.1±5.9 95.9±4.1 98.8±1.5

[0045] Table 4: Recovery rate of creatinine detection under chylous interference (n=5)

[0046] Chyre (FTU) COBA C501 system recovery rate (%) Mike-7180 ​​system recovery rate (%) Recovery rate (%) of the reagent kit of this invention 0 (Control) 100.0 100.0 100.0 1534 67.0±33.0 92.0±8.0 97.6±2.3

[0047] This invention's kit, employing optimized tri-gradient calibrators (0, 250, 850 nM BCE) and an integrated creatinine correction module, constitutes a specific detection scheme for urine samples from patients with bone metastases. In anti-interference validation, the kit maintained stable detection performance under interference from hemoglobin (0.92-4.60 g / L), bilirubin (309.0 μmol / L), and chyle (1534 FTU), with recoveries maintained between 97.6% and 99.2%, significantly superior to the control system, demonstrating its ability to meet the accurate detection needs of complex urine samples from patients with bone metastases.

[0048] This invention designs a three-gradient calibrator (0, 250, 850 nM BCE) to better match the concentration distribution characteristics of NTx in the morning urine of tumor patients. It employs a high-affinity binding system of streptavidin solid-phase carrier and horseradish peroxidase-labeled antibody to ensure detection sensitivity, and integrates a dry chemistry creatinine detection module to achieve batch-wise calibration. Its core mechanism lies in effectively suppressing spectral absorption interference from hemoglobin, chemical oxidation interference from bilirubin, and light scattering interference from chylomicrons by optimizing the pH environment, substrate components, and detection wavelength of the reaction system. This maintains the stability of the enzymatic reaction during competitive immunoassay, ensuring accurate resolution of NTx concentration and simultaneous creatinine correction in complex tumor sample matrices. Ultimately, this forms a comprehensive anti-interference detection solution for highly interfering samples from bone metastasis patients.

[0049] Although embodiments of the present invention have been shown and described, these specific embodiments are merely explanations of the invention and are not intended to limit it. The specific features, structures, materials, or characteristics described may be combined in any suitable manner in one or more embodiments or examples. After reading this specification, those skilled in the art may make modifications, substitutions, and variations to the embodiments as needed without departing from the principles and spirit of the invention, but such modifications, substitutions, and variations are protected by patent law as long as they are within the scope of the claims of the present invention.

Claims

1. A type I collagen cross-linked N-terminal peptide monitoring kit for monitoring tumor bone metastasis, characterized in that: include: A solid-phase support coated with synthetic NTx peptides; Enzyme-labeled anti-NTx monoclonal antibody conjugate; Chemiluminescent substrate solution; A calibrator used for the quantitative determination of the concentration of type I collagen cross-linked N-terminal peptide in urine samples.

2. The type I collagen cross-linked N-terminal peptide monitoring kit for monitoring tumor bone metastasis according to claim 1, characterized in that: The solid support is a streptavidin-coated reaction vessel; The enzyme-labeled anti-NTx monoclonal antibody conjugate is an enzyme-labeled anti-NTx monoclonal antibody.

3. The type I collagen cross-linked N-terminal peptide monitoring kit for monitoring tumor bone metastasis according to claim 1, characterized in that: The chemiluminescent substrate solution contains luminol derivatives, peroxides, and electron transfer enhancers.

4. The type I collagen cross-linked N-terminal peptide monitoring kit for monitoring tumor bone metastasis according to claim 1, characterized in that: Also includes: Sample diluent, washing solution, and creatinine detection components used for creatinine correction.

5. The type I collagen cross-linked N-terminal peptide monitoring kit for monitoring tumor bone metastasis according to claim 1, characterized in that: The calibrators include 0 nM BCE and NTx standards at two or more non-zero concentration points, including 250 nM BCE and 850 nM BCE.

6. The type I collagen cross-linked N-terminal peptide monitoring kit for monitoring tumor bone metastasis according to claim 1, characterized in that: The kit is compatible with fully automated immunoassay analyzers.

7. The type I collagen cross-linked N-terminal peptide monitoring kit for monitoring tumor bone metastasis according to claim 1, characterized in that: The detection method of the kit is a competitive immunoassay, which includes the following steps: Step 1: Urine samples compete for binding with enzyme-labeled anti-NTx antibodies on a solid-phase support; Step 2: Wash away unbound substances; Step 3: Add a chemiluminescent substrate to trigger the luminescence reaction; Step 4: Calculate the NTx concentration by using the light signal intensity.

8. The type I collagen cross-linked N-terminal peptide monitoring kit for monitoring tumor bone metastasis according to claim 7, characterized in that: Test results need to be corrected for urine creatinine concentration and reported in nM BCE / mM creatinine units.

9. The type I collagen cross-linked N-terminal peptide monitoring kit for monitoring tumor bone metastasis according to claim 1, characterized in that: The urine sample was the second morning urine.

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