Use of a polypeptide as a marker in the diagnosis of primary osteoporosis

Abstract

This invention discloses the application of a polypeptide as a biomarker in the diagnosis of primary osteoporosis, wherein the polypeptide has the amino acid sequence shown in SEQ ID NO.3. This invention uses a combination of two peptides for the diagnosis of primary osteoporosis, achieving high diagnostic efficacy. ROC analysis shows an AUC value of 0.945 for diagnostic significance, with a sensitivity of 92.7% and a specificity of 85.3%. Furthermore, the polypeptide with the aforementioned amino acid sequence shown in SEQ ID NO.3, obtained by linking the two peptides using a flexible GGS linker, also exhibits the same diagnostic efficacy and can be used to develop diagnostic kits for primary osteoporosis.

Description

Technical Field

[0001] This invention relates to the field of biomedical technology, specifically to the application of a polypeptide as a biomarker in the diagnosis of primary osteoporosis. Background Technology

[0002] Osteoporosis is mainly divided into primary osteoporosis and secondary osteoporosis. Primary osteoporosis is a systemic bone disease characterized by low bone mass, damage to bone microstructure leading to increased bone fragility, and susceptibility to fractures. Primary osteoporosis is further divided into postmenopausal osteoporosis (Type I), senile osteoporosis (Type II), and idiopathic osteoporosis. Typical symptoms include lower back or generalized bone pain, decreased height, kyphosis, and fragility fractures. Spinal compression fractures may lead to chest deformities and respiratory dysfunction.

[0003] Existing research suggests that serum biomarkers have the potential to serve as diagnostic indicators for osteoporosis. For example, Kerschan-Schindl (characterized by 19 emerging miRNA bone biomarkers) can be detected in human serum samples, forming an independent set of bone and muscle biomarkers, which, in combination, could serve as diagnostic biomarkers for osteoporosis in postmenopausal women. However, the diagnostic efficacy of this study is limited. Ladang et al. explored the prognostic value of OsteomiRs in fracture risk assessment using a multivariate model, obtaining only a positive predictive value of 68% and a sensitivity of 76%.

[0004] Therefore, developing new diagnostic biomarkers to increase the sensitivity and specificity of osteoporosis diagnosis is of great significance. Summary of the Invention

[0005] In view of the shortcomings of the prior art, the purpose of this invention is to provide an application of polypeptide as a biomarker in the diagnosis of primary osteoporosis.

[0006] The objective of this invention is achieved through the following technical solution: In a first aspect, the present invention provides a polypeptide having an amino acid sequence as shown in SEQ ID NO.3.

[0007] Specifically, the amino acid sequence of the polypeptide is: CSPSSAYPRHGPDGGSCDLYWLRKGGSDLPTLRKGGSDLPFLLK (SEQ ID NO.3).

[0008] Secondly, the present invention provides a polynucleotide encoding a nucleotide sequence of a polypeptide as described above.

[0009] Thirdly, the present invention provides the application of a polypeptide as a biomarker in the preparation of products for diagnosing primary osteoporosis, said polypeptide having an amino acid sequence as shown in SEQ ID NO.3.

[0010] As a preferred embodiment, the primary osteoporosis is postmenopausal primary osteoporosis.

[0011] As a preferred embodiment, the primary osteoporosis is diabetic primary osteoporosis.

[0012] As a preferred option, the product is a reagent kit.

[0013] As a preferred embodiment, the kit includes a reagent for detecting the antibody level of a polypeptide with an amino acid sequence as shown in SEQ ID NO.3 in the sample to be tested, which serves as an antigen.

[0014] As a preferred option, the sample to be tested is any one of whole blood, plasma, or serum.

[0015] As a preferred embodiment, when the antibody level of the polypeptide with the amino acid sequence shown in SEQ ID NO.3 in the sample to be tested is higher than 0.458, it can be diagnosed as primary osteoporosis.

[0016] As a preferred embodiment, the reagents include standards, antigen coating solution, blocking solution, sample diluent, stop solution, enzyme-labeled reagent, colorimetric reagent, and washing solution.

[0017] As a preferred option, the standard is an IgG antibody; The antigen coating solution is TBST; The sealing solution is TBST containing 3% BSA; The sample diluents include protein diluents and serum diluents; the protein diluent is PBS containing 5% glycerol; the serum diluent is TBST containing 10% BSA. The terminating solution is a 1M H2SO4 solution; The enzyme-labeled reagent contains PBST with enzyme-labeled antibodies; The colorimetric reagent is TMB; The washing solution is PBST.

[0018] Fourthly, the present invention provides an application of a polypeptide combination as a biomarker in the preparation of products for diagnosing primary osteoporosis, wherein the polypeptide combination comprises peptide 1 and peptide 2. Peptide 1 has the amino acid sequence shown in SEQ ID NO.1, and peptide 2 has the amino acid sequence shown in SEQ ID NO.2.

[0019] Specifically, the amino acid sequence of peptide 1 is: CSPSSAYPRHGPD (SEQ ID NO.1).

[0020] The amino acid sequence of peptide 1 is: CDLYWLRKGGSDLPTLRKGGSDLPFLLK (SEQ ID NO.2).

[0021] As a preferred embodiment, the primary osteoporosis is postmenopausal primary osteoporosis.

[0022] As a preferred embodiment, the primary osteoporosis is diabetic primary osteoporosis.

[0023] As a preferred option, the product is a reagent kit.

[0024] As a preferred embodiment, the product includes a reagent for detecting antibody levels in a sample containing a combination of peptides as an antigen.

[0025] As a preferred option, the sample to be tested is any one of whole blood, plasma, or serum.

[0026] As a preferred embodiment, when the antibody level of the polypeptide with the amino acid sequence as shown in SEQ ID NO.1 as an antigen in the sample to be tested is higher than 0.723, or the antibody level of the polypeptide with the amino acid sequence as shown in SEQ ID NO.2 as an antigen is higher than 0.893, it can be used to jointly diagnose primary osteoporosis.

[0027] As a preferred embodiment, the reagents include standards, antigen coating solution, blocking solution, sample diluent, stop solution, enzyme-labeled reagent, colorimetric reagent, and washing solution.

[0028] As a preferred option, the standard is an IgG antibody; The antigen coating solution is TBST; The sealing solution is TBST containing 3% BSA; The sample diluents include protein diluents and serum diluents; the protein diluent is PBS containing 5% glycerol; the serum diluent is TBST containing 10% BSA. The terminating solution is a 1M H2SO4 solution; The enzyme-labeled reagent contains PBST with enzyme-labeled antibodies; The colorimetric reagent is TMB; The washing solution is PBST.

[0029] Compared with the prior art, the present invention has the following beneficial effects: This invention utilizes a combination of two peptides for the diagnosis of primary osteoporosis, exhibiting high diagnostic efficacy. ROC analysis showed a diagnostic AUC value of 0.945, with a sensitivity of 92.7% and a specificity of 85.3%. Furthermore, the peptide obtained by linking the two peptides using a flexible GGS linker also possesses the same diagnostic efficacy and can be used to develop diagnostic kits for primary osteoporosis. Attached Figure Description

[0030] Other features, objects, and advantages of the present invention will become more apparent from the following detailed description of non-limiting embodiments with reference to the accompanying drawings: Figure 1 ROC analysis results for peptide 1 as a single diagnostic diagnose of primary osteoporosis; Figure 2 ROC analysis results for the combined diagnosis of primary osteoporosis using peptide combination (peptide 1 + peptide 2); Figure 3 ROC analysis results for the combined diagnosis of primary osteoporosis in different genders using peptide combinations (peptide 1 + peptide 2); Figure 4 ROC analysis results for the combined diagnosis of primary osteoporosis in different age-stratified populations using peptide combinations (peptide 1 + peptide 2); Figure 5 ROC analysis results for the combined diagnosis of primary osteoporosis (OP) and diabetic osteoporosis (DM-OP) using peptide combinations (peptide 1 + peptide 2). Detailed Implementation

[0031] The present invention will now be described in detail with reference to specific embodiments. These embodiments will help those skilled in the art to further understand the present invention, but do not limit the invention in any way. It should be noted that those skilled in the art can make various modifications and improvements without departing from the concept of the present invention. These all fall within the scope of protection of the present invention.

[0032] In the following examples, peptide 1 was obtained using phage immunoprecipitation sequencing (PhIP-seq) technology. Specifically, serum samples (from the First Affiliated Hospital of Fujian Medical University) from 25 osteoporosis (OP) patients and 30 non-OP controls (with no significant differences in age or sex between groups) were screened using an M13 phage display random peptide library. Each serum sample was incubated with the phage library, and IgG-bound phages were enriched with Protein G magnetic beads. After recovery, high-throughput sequencing was used to identify peptide 1, which was specifically recognized by the sample antibodies. Subsequently, the diagnostic potential of peptide 1 was validated in the screening cohort (the aforementioned 25 OP patients and 30 non-OP controls) using ELISA. ROC analysis showed that the AUC value of peptide 1 was 0.75, indicating its potential for diagnosing primary osteoporosis.

[0033] In the following examples, peptide 2 is an engineered multi-motif construct containing four tandem GIEXLLX motifs, which is a polypeptide obtained by linking them together via a flexible GGS linker.

[0034] In the following examples, peptide 3 is a polypeptide obtained by integrating peptide 1 and peptide 2 through a flexible connector GGS.

[0035] Unless otherwise specified, the experimental methods used in the following examples are conventional methods. Unless otherwise specified, the materials and reagents used in the following examples are commercially available.

[0036] Example 1 verifies the diagnostic efficacy of the peptide in primary osteoporosis. 1. Verification Queue The validation cohort used in this embodiment included 150 patients with osteoporosis (OP) and 150 age- and sex-matched non-OP controls. History of fractures and tumors was recorded using a standardized questionnaire. Participant demographic characteristics are shown in Table 1. All participants signed written informed consent forms, and the study was approved by the Ethics Committee of the First Affiliated Hospital of Fujian Medical University. All serum samples were collected according to a standardized protocol at the Fujian Provincial Key Laboratory of Glycolipid and Bone Metabolism. Inclusion criteria: Age > 50 years, all female participants were postmenopausal. Exclusion criteria: Secondary OP cases, including advanced cancer cachexia, long-term use of glucocorticoids, Cushing's syndrome, hyperparathyroidism, hematologic malignancies, and bone metastases. Diagnostic criteria for OP and fragility fractures: Following the "Chinese Guidelines for the Diagnosis and Treatment of Primary Osteoporosis (2022)".

[0037] Table 1. 300 participants included in the validation cohort. 2. Detection was performed using the ELISA assay method. 2.1 Synthesis of peptides: The amino acid sequences of peptide 1 are: CSPSSAYPRHGPD (SEQ ID NO.1), peptide 2 are: CDLYWLRKGGSDLPTLRKGGSDLPFLLK (SEQ ID NO.2), and peptide 3 are: CSPSSAYPRHGPDGGSCDLYWLRKGGSDLPTLRKGGSDLPFLLK (SEQ ID NO.3). All peptides were synthesized by Sangon Biotech. In peptides 1 and 2, an additional cysteine ​​residue (C in SEQ ID NO.1 and 2) was added to the N-terminus. BSA was activated with SMCC (ThermoFisher, 22322) and then coupled to the synthesized peptides.

[0038] 2.2 ELISA assay: 100 μL of 5 μg / mL BSA-conjugated peptide was added to and immobilized in each well of a 96-well ELISA plate (Corning, 42592) and incubated overnight at 4°C. The plate was washed twice with PBS containing 0.1% Tween-20 (PBST), and then blocked with PBST containing 3% BSA at room temperature. After washing twice with PBST, serum samples (diluted 1:100 with PBS) were added to the wells, and the plate was incubated at 37°C for 2 hours. After incubation, the plate was washed six times with PBST, and excess buffer was removed after the last wash. Then, 1:1000 diluted anti-human IgG secondary antibody (Sangon Biotech, D110149) was added, and the plate was incubated at 37°C for 1 hour. Wash six times with PBST. After the final wash, remove excess buffer and add 100 μL of tetramethylbenzidine substrate (Sigma-Aldrich, T4444). Incubate at 37°C for 30 minutes. Then, add 100 μL of 1 M H2SO4 to terminate the reaction and measure the absorbance at 450 nm.

[0039] 2.3 Statistical Analysis All statistical analyses were performed using SPSS Statistics 17.0 and R. Independent samples t-tests were used to compare clinical data from populations, and the Mann-Whitney U test was used to compare the means of unconverted ELISA data.

[0040] 3. Results When peptide 1 was used alone to verify its diagnostic efficacy, peptide 1 showed a significant difference in response between OP patients and non-OP controls (p<0.001, ANOVA with Tukey's post-hoc test). Figure 1 The diagnostic threshold was 1.94, and ROC analysis showed that the AUC value of diagnostic significance was 0.747 (95% CI: 0.691–0.803).

[0041] When peptide 2 was used alone to verify its diagnostic efficacy, it showed a significant difference in response between OP patients and non-OP controls (p<0.001, ANOVA with Tukey's post-hoc test), with a diagnostic threshold of 0.385. ROC analysis showed that the AUC value of diagnostic significance was 0.856 (95% CI: 0.812–0.899).

[0042] When using a combination of peptide 1 and peptide 2 for combined diagnosis, a significant difference was observed between OP patients and non-OP controls (p<0.001, ANOVA with Tukey's post-hoc test). Figure 2 ROC analysis showed that the AUC value with diagnostic significance was 0.945 (95% CI: 0.919–0.971), with a sensitivity of 92.7% and a specificity of 85.3%.

[0043] The diagnostic efficacy of using peptide 3 is consistent with that of using a combination of peptide 1 and peptide 2 for combined diagnosis.

[0044] Therefore, it is evident that the combination of peptide 1 and peptide 2 or the use of peptide 3 has very good diagnostic efficacy for primary osteoporosis.

[0045] 4. Clinical applicability analysis of peptide-based diagnostic groups To assess clinical efficacy in heterogeneous populations, the diagnostic performance of the peptide 1 + peptide 2 combination was systematically evaluated in stratified subgroups, considering the effects of sex, age, and diabetes mellitus (DM) on diagnostic efficacy. The validation cohort was divided into a male validation cohort and a female validation cohort (the female validation cohort consisted entirely of postmenopausal osteoporosis patients). The peptide 1 + peptide 2 combination was used to assess its diagnostic efficacy for osteoporosis in different sexes. ROC analysis showed the following diagnostic significance values: AUC value for men was 0.887 (95% CI: 0.815–0.959); AUC value for women was 0.968 (95% CI: 0.937–0.999). Figure 3 The results show that the combination of peptide 1 and peptide 2 exhibits gender-independent diagnostic accuracy.

[0046] The validation cohorts were divided into different age groups, and the efficacy of peptide 1 + peptide 2 combinations in diagnosing osteoporosis in different age stratifications was evaluated. ROC analysis showed the following diagnostic significance values: 50–60 years old, AUC value 0.869; 60–70 years old, AUC value 0.978; >70 years old, AUC value 0.989. Figure 4 Age stratification analysis showed increased efficiency in the elderly population.

[0047] Given the interaction between the pathogenesis of diabetes mellitus (DM) and osteoporosis (OP), the diagnostic specificity of DM-OP comorbidities was specifically evaluated. 150 OP / DM-OP patients in the validation cohort were divided into a normal OP cohort and a DM-OP cohort, and the diagnostic efficacy was validated using a combination of peptide 1 and peptide 2. ROC analysis showed that the combination of peptide 1 and peptide 2 maintained high accuracy in normal OP (AUC = 0.915). Figure 5 Non-diabetes in DM-OP patients achieved near-perfect discrimination (AUC=0.998). Figure 5 Diabetes (in the middle).

[0048] This invention has many specific applications, and the above description is only a preferred embodiment. It should be noted that the above embodiments are for illustrative purposes only and are not intended to limit the scope of protection of this invention. For those skilled in the art, several improvements can be made without departing from the principle of this invention, and these improvements should also be considered within the scope of protection of this invention.

Claims

1. A polypeptide, characterized in that, The polypeptide has the amino acid sequence shown in SEQ ID NO.

3.

2. A polynucleotide, characterized in that, The nucleotide sequence encoding the polypeptide as described in claim 1.

3. The application of a polypeptide as a biomarker in the preparation of products for diagnosing primary osteoporosis, characterized in that, The polypeptide has the amino acid sequence shown in SEQ ID NO.

3.

4. The application of a polypeptide combination as a biomarker in the preparation of products for diagnosing primary osteoporosis, characterized in that, The polypeptide combination includes peptide 1 and peptide 2; Peptide 1 has the amino acid sequence shown in SEQ ID NO.1, and peptide 2 has the amino acid sequence shown in SEQ ID NO.

2.

5. The application according to claim 3 or 4, characterized in that, The product in question is a reagent kit.

6. The application according to claim 5, characterized in that, The product includes a reagent for detecting antibody levels in a sample as an antigen, specifically a polypeptide or combination of polypeptides.

7. The application according to claim 5, characterized in that, The sample to be tested can be any one of whole blood, plasma, or serum.

8. The application according to claim 6 or 7, characterized in that, When the antibody level of the polypeptide with the amino acid sequence shown in SEQ ID NO. 3 in the sample to be tested is higher than 0.458, it can be diagnosed as primary osteoporosis; or When the antibody level of the polypeptide with the amino acid sequence as shown in SEQ ID NO.1 as an antigen in the sample to be tested is higher than 0.723, or the antibody level of the polypeptide with the amino acid sequence as shown in SEQ ID NO.2 as an antigen is higher than 0.893, a combined diagnosis of primary osteoporosis can be made.

9. The application according to claim 6, characterized in that, The reagents include standards, antigen coating solution, blocking solution, sample diluent, stop solution, enzyme-labeled reagent, colorimetric reagent, and washing solution.

10. The application according to claim 9, characterized in that, The standard is an IgG antibody; The antigen coating solution is TBST; The sealing solution is TBST containing 3% BSA; The sample diluents include protein diluents and serum diluents; the protein diluent is PBS containing 5% glycerol; the serum diluent is TBST containing 10% BSA. The terminating solution is a 1M H2SO4 solution; The enzyme-labeled reagent contains PBST with enzyme-labeled antibodies; The colorimetric reagent is TMB; The washing solution is PBST.

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