Early osteonecrosis diagnosis kit based on blood detection and biomarkers

Abstract

The invention discloses an osteonecrosis early diagnosis kit based on blood detection and a biomarker, and the osteonecrosis early diagnosis kit can be used for early diagnosis of osteonecrosis through multi-omics screening based on femoral head necrosis lesion division, serological analysis of different stages of cases and cell and animal level biological tests in combination with actual detection result verification. It is determined that a group of biomolecules, namely PEBP1, ACAT1, MMP2, SOD1 and FN1 proteins, can be used for early diagnosis of osteonecrosis.

Description

Technical Field

[0001] This invention relates to the detection of biomolecules indicating osteonecrosis in human serum, plasma and related body fluids using enzyme-linked immunosorbent assay (ELISA), and particularly to the detection of early osteonecrosis before it progresses to stage I osteonecrosis as determined by ARCO (Association Research Circulation Osseous, abbreviated as ARCO staging), or in cases where no abnormalities are detected on imaging but molecular behavior has already shown changes in bone tissue morphology. The invention also relates to the development and application of related detection reagents. Background Technology

[0002] Osteonecrosis can be divided into two main categories based on its etiology: one is osteonecrosis caused by bacterial infection, such as in osteomyelitis, bone tuberculosis, and suppurative arthritis; the other is osteonecrosis caused by ischemia, which can be caused by factors such as trauma, alcohol consumption, use of hormonal drugs, exposure to cold and dampness, hyperlipidemia, liver and kidney deficiency, flat hip, syringomyelia, and decompression sickness. Osteonecrosis can occur in any part of the human skeletal system. Clinically, it can be seen in many locations, including the scaphoid bone of the hand, foot, humeral head, ribs, patella, and clavicle, with the femoral head having the highest incidence, followed by the talus. Approximately 85% of patients with osteonecrosis experience symptoms, including pain, limited mobility, limping, pain while walking, impaired hip joint function, and paralysis. X-rays will show sclerotic bone formation, cystic changes, partial loss of trabeculae, cartilage collapse, and fractures. Clinically, it can manifest as single or multiple sites of damage simultaneously. The diagnosis of osteonecrosis relies on changes in bone morphology detected by imaging, making it difficult to detect in the early, asymptomatic stages and before morphological changes are observed.

[0003] Osteonecrosis occurring in the femoral head is called osteonecrosis of the femoral head (ONFH). ONFH is a common, refractory, and disabling disease, and a frequent cause of hip pain and dysfunction. The pathogenesis of ONFH is currently believed to include coagulation disorders, lipid metabolism abnormalities, osteogenic disorders, and poor vascular repair, but it is not fully understood. Based on different etiologies, ONFH is mainly divided into two categories: traumatic ONFH and non-traumatic ONFH. Systemic corticosteroid administration, habitual alcohol consumption (or alcoholism), hip trauma (dislocation and / or fracture), lipid metabolism abnormalities, decompression sickness, and radiation are closely related to the occurrence of ONFH and are common precipitating factors. Due to the high disability rate of ONFH, it severely affects patients' work and life, and late-stage treatment often requires total hip replacement surgery, placing a heavy burden on patients, families, and society. After joint replacement surgery, complications such as prosthesis dislocation, aseptic loosening, and local infection, as well as the limited lifespan of the prosthesis, may require patients to undergo multiple revision surgeries for total hip arthroplasty. Therefore, early diagnosis and early treatment of ONFH are crucial.

[0004] Osteonecrosis occurring in the talus is called talar necrosis. Like femoral head necrosis, the treatment of talar necrosis is a very challenging clinical problem. Approximately 75% of talar necrosis cases are secondary to traumatic injuries, with 90% of traumatic talar necrosis cases caused by talar neck fractures. The clinical symptoms, diagnostic methods, and treatment approaches for talar necrosis are basically the same as those for femoral head necrosis.

[0005] Diagnosis of osteonecrosis requires meeting both clinical characteristics and imaging findings. Early detection is best achieved with MRI, while X-ray, CT scan, radionuclide imaging, bone biopsy and pathological examination, and DSA are auxiliary diagnostic methods. ARCO classifies osteonecrosis into stages I, II, III, and IV. Currently, ARCO I / II is considered the early stage of osteonecrosis, characterized by localized pain and limited mobility (pain is particularly pronounced during weight-bearing walking and worsens as the disease progresses, leading to limb movement limitations; a positive "4" sign; and significant restriction of internal rotation and abduction of the lower limbs). Early osteonecrosis may not show obvious signs on X-rays, but MRI may reveal bone marrow edema, strong joint effusion, abnormal signals, and subchondral changes. T1WI may show a band-like low signal, and T2WI may show a "double line" sign, indicating the repair of sclerotic bone and granulation tissue. Bone aspiration in ARCO I / II stage osteonecrosis primarily reveals the death of bone cell components. Pathologically, ARCO stage I / II osteonecrosis is in the venous stasis phase. At this time, blood supply is obstructed, and signs of partial cell necrosis appear. More than 50% of the osteocytes in the trabeculae are vacuolated, affecting adjacent trabeculae, and there is partial necrosis of the bone marrow. Stimulated by the pathogenic factors of osteonecrosis, bone marrow stem cells gradually differentiate into mast adipocytes, with cell diameters reaching over 10 μm. Thrombosis occurs in small veins, leading to venous dilation, venous sinus congestion, and interstitial edema. Poor blood return causes intraosseous venous stasis and hypertension. In summary, although MRI can detect abnormal signals in the early stages, the bone tissue morphology often shows regional necrosis rather than just a few cellular levels. Therefore, the detection or indication of osteonecrosis at an earlier stage (specifically, in the asymptomatic early stage or before morphological changes) using relevant biomolecules in vivo is particularly urgent for clinical diagnosis and treatment. This can create opportunities to delay or even reverse osteonecrosis, reduce pain for patients, improve hip joint function, and prevent or delay femoral head collapse. Summary of the Invention

[0006] The purpose of this invention is to provide an early diagnostic kit for osteonecrosis based on blood tests and biomarkers. By quantitative or semi-quantitatively detecting target protein molecules in samples such as serum and plasma, this kit enables accurate diagnosis of stage I / II osteonecrosis with unclear imaging manifestations, or enables the detection of asymptomatic early stages of osteonecrosis (e.g., before progression to stage I). The kit is also simple to operate and has low dependence on personnel and equipment.

[0007] To achieve the above objectives, the present invention adopts the following technical solution: In a first aspect, a biomolecular detection kit is provided, the kit comprising reagents for detecting the level of a set of protein molecules in a biological sample, the set of protein molecules including a core protein molecule and accessory protein molecules for indicating early lesions of osteonecrosis in the asymptomatic early stage or before morphological changes, the core protein molecule being human phosphatidylethanolamine binding protein 1 (PEBP1).

[0008] Preferably, the accessory protein molecule is one or more of human acetyl-CoA acyltransferase 1 (ACAT1), matrix metalloproteinase 2 (MMP2), superoxide dismutase 1 (SOD1), and fibronectin 1 (FN1).

[0009] Preferably, the group of protein molecules is PEBP1 and ACAT1.

[0010] Preferably, the group of protein molecules further includes MMP2 and / or SOD1.

[0011] Preferably, the group of protein molecules also includes FN1.

[0012] Preferably, the group of protein molecules is PEBP1, ACAT1, MMP2, SOD1 and FN1.

[0013] Preferably, the biological sample is serum or plasma; to obtain the biological sample, the venous blood volume collected is 5 mL / person.

[0014] Preferably, the kit specifically includes enzyme-linked immunosorbent assay (ELISA) reagents (e.g., double sandwich assay) for quantitative or semi-quantitative detection of the core protein molecules and accessory protein molecules.

[0015] Preferably, the concentrations of the protein molecular standards (e.g., protein molecular solutions) used as references for detection levels in the semi-quantitative assay are determined according to the following serological diagnostic criteria for early osteonecrosis: PEBP1 >17.66 pg / mL, ACAT1 >13.83 pg / mL, MMP2 <483.5 ng / mL, SOD1 <3.71 pg / mL, and FN1 <7585 pg / mL (this standard is also the basis for determining the limit value in the quantitative assay).

[0016] Secondly, the application of detection reagents for the above-mentioned biomolecules (i.e., one or more of PEBP1, MMP2, SOD1, FN1, and ACAT1) in the preparation of reagents for early diagnosis (or auxiliary diagnosis) of osteonecrosis.

[0017] The beneficial effects of this invention are reflected in: This invention can detect the levels of biomolecules associated with early osteonecrosis (such as the protein molecule PEBP1 in serum) and, through verification of the detection specificity and sensitivity, achieve early diagnosis of osteonecrosis from a biological perspective, which is beneficial for blocking the development of osteonecrosis and inhibiting the deterioration of the disease.

[0018] Furthermore, this invention can utilize an enzyme-linked immunosorbent assay (ELISA) reagent of a group of protein molecules (including PEBP1 and ACAT1) to detect and diagnose osteonecrosis (e.g., hormone-induced femoral head necrosis) at an early stage, thereby improving the clinical efficacy of early diagnosis of femoral head necrosis. This allows for the delay or even reversal of osteonecrosis through causal treatment (e.g., providing greater opportunities for hip preservation for patients with femoral head necrosis).

[0019] Furthermore, through the analysis of the diagnostic efficacy of PEBP1, MMP2, SOD1, FN1, and ACAT1 for osteonecrosis, this invention has identified PEBP1 as the core protein molecule for the early diagnosis of osteonecrosis (such as hormone-induced femoral head necrosis), which helps to reduce false positives in diagnostic results from other single molecules or other combinations of molecules.

[0020] Furthermore, through verification with actual samples, this invention has found that the combined use of PEBP1, MMP2, and ACAT1 can effectively detect early femoral head necrosis with unclear imaging manifestations, as well as asymptomatic early-stage femoral head necrosis (e.g., before progression to stage I). Attached Figure Description

[0021] Figure 1-1 This is a femoral head tissue sample (coronal anatomical section) from an ONFH patient; where A: necrotic area; B: junctional area (or early necrotic area); C: non-necrotic area; scale bar is 1 cm.

[0022] Figure 1-2H&E staining of the femoral head in ONFH patients; AC: necrotic area; DF: junctional area (or early necrosis area); GI: non-necrotic area; A, D, G scale bar is 200 μm; B, E, H scale bar is 100 μm; C, F, I scale bar is 50 μm.

[0023] Figure 2 This is a graph showing the differences in the levels of relevant proteins obtained from blood samples of early-stage, late-stage, and healthy controls in patients with avascular necrosis of the femoral head (one asterisk represents P<0.05, two asterisks represent P<0.01); where A: changes in PEBP1 protein content; B: changes in ACAT1 protein content; C: changes in FN1 protein content; D: changes in MMP2 protein content; E: changes in SOD1 protein content; Healthy: healthy individuals; Stage I / II: ARCO I / II (early stage); Stage III / IV: ARCO III / IV (late stage).

[0024] Figure 3 The ROC curves for early-stage avascular necrosis of the femoral head versus normal (healthy) individuals are shown, where 1 represents ACAT1; 2 represents PEBP1; 3 represents SOD1; 4 represents FN1; and 5 represents MMP2. The horizontal axis represents specificity, and the vertical axis represents sensitivity.

[0025] Figure 4 ROC curve for combined diagnosis of PEBP1+ACAT1.

[0026] Figure 5 MicroCT images of a gene knockout combined with hormone-induced femoral head necrosis animal model (week 3; gene knockout mice treated with hormones). Pebp1 - / - , Acat - / - and double knockout mice Acat - / - / Pebp1 - / - ). Detailed Implementation

[0027] The present invention will be further described in detail below with reference to the accompanying drawings and embodiments. The embodiments described are for illustrative purposes only and are not intended to limit the scope of protection of the present invention.

[0028] (a) Discovery of blood biomarkers for osteonecrosis 1.1 Screening using multi-omics methods Discarded femoral head specimens were collected from patients with stage III / IV femoral head necrosis undergoing hip replacement surgery. Clinicians and pathologists divided the femoral head into three regions based on its morphological characteristics: the non-necrotic region, the early necrotic region, and the necrotic region (see [link to relevant documentation]). Figure 1-1 , Figure 1-2The study focused on the molecular biological changes from non-necrotic areas to early necrotic areas. Proteomics analysis was performed on tissues from these three regions. A total of 448 differentially expressed proteins were screened from the early necrotic areas compared to the non-necrotic areas. Further analysis of the GO and KEGG pathways revealed 47 proteins located at the core of these differentially expressed pathways using 10 different core protein calculation methods.

[0029] 1.2 Serological screening Based on expertise in osteonecrosis and molecular biology, the top five potential key proteins from the results in section 1.1 were screened to identify those that might be key to the progression from non-necrotic femoral head to early-stage necrosis. These proteins were then quantitatively detected in the serum of patients with osteonecrosis at different stages using an enzyme-linked immunosorbent assay (ELISA) from July 1, 2020 to May 31, 2022. The study ultimately confirmed the biomolecules in serum that accurately identify ARCO stage I / II osteonecrosis. Early and late-stage cases of osteonecrosis of the femoral head were included from patients receiving high-dose hormone therapy at the Department of Rheumatology and Immunology, First Affiliated Hospital of Xi'an Jiaotong University, as well as from the Second Affiliated Hospital of Xi'an Jiaotong University and Xi'an Red Cross Hospital. Healthy controls were from the Physical Examination Center of the Second Affiliated Hospital of Xi'an Jiaotong University.

[0030] 1.2.1 ELISA kit detection The concentration of protein molecules (PEBP1, MMP2, SOD1, FN1, and ACAT1) in human serum samples was detected using the traditional, widely used, technically mature, and low-barrier-to-operation ELISA method (double sandwich method).

[0031] 1.2.2 Principle of the Double Sandwich ELISA Method Specific antibodies are pre-coated onto the ELISA plate; after incubation, a corresponding protein molecule in the serum is captured by the coated antibody (referred to as the first antibody); after thorough washing, another biotinylated or non-biotinylated antibody specific to the protein molecule (referred to as the second antibody) is added to detect the captured corresponding protein molecule; finally, color development, observation, and quantitative analysis are performed using enzymes and substrates.

[0032] 1.2.3 Each reagent kit and its main detection parameters (1) PEBP1 detection kit A commercially available kit was used, named Human Phosphatidylethanolamine Binding Protein 1 (PEBP1) Enzyme-Linked Immunosorbent Assay (ELISA) Kit. A 96-well configuration was used, and the working concentration of the antibody used in the coating or detection reaction was 100× stock solution, with a sensitivity of 1.0 pg / mL.

[0033] (2) ACAT1 detection kit A commercially available kit was used, named Human Acetyl-CoA Acyltransferase 1 (ACAT1) Enzyme-Linked Immunosorbent Assay (ELISA) Kit. A 96-well configuration was used, and the working concentration of the antibody used in the coating or detection reaction was 100× stock solution, with a sensitivity of 1.0 pg / mL.

[0034] (3) MMP2 detection kit A commercially available kit was used, named Matrix Metalloproteinase 2 (MMP2) Enzyme-Linked Immunosorbent Assay (ELISA) Kit. A 96-well configuration was used, and the working concentration of the antibody used in the coating or detection reaction was 100× stock solution, with a sensitivity of 0.05 ng / mL.

[0035] (4) SOD1 detection kit A commercially available kit was used, named Superoxide Dismutase 1 (SOD1) Enzyme-Linked Immunosorbent Assay (ELISA) Kit. A 96-well configuration was used, and the working concentration of the antibody used in the coating or detection reaction was 100× stock solution, with a sensitivity of 1.0 pg / mL.

[0036] (5) FN1 detection kit A commercially available kit was used, named Fibronectin 1 (FN1) Enzyme-Linked Immunosorbent Assay (ELISA) Kit. A 96-well configuration was used, and the working concentration of the antibody used in the coating or detection reaction was 100× stock solution, with a sensitivity of 47.2 pg / mL.

[0037] 1.2.4 Testing Procedure 1) Dilute the protein standard according to the kit instructions to obtain the stock solution, and use it within 30 minutes; 2) Label six 1.5 mL EP tubes in advance, dilute the standard to the specified concentration gradient (each gradient is 100 μL), mix well and place on ice for later use; 3) Take out the required amount of protein-specific antibody (first type of antibody) enzyme-labeled strips according to the experimental dosage. Store the remaining items in the packaging at 4°C and use them within one week. 4) Add the diluted standard of the specified concentration and the sample to be tested to the corresponding wells of the ELISA strip in sequence. Be careful not to introduce air bubbles. When adding the sample, push all the reagents to be added to the bottom of the ELISA strip to ensure that there is no residue on the wall and ensure that the sample addition is uninterrupted. The total sample addition time should not exceed 10 min. Then incubate the ELISA strip containing the added reagents at 37℃ for 120 min. 5) Discard the liquid in the wells and wash the strip 4 times with 400 μL / well / washing buffer; then add 0.1 mL / well of the second antibody to the test wells and the standard wells, place the strips at 37°C for 1 h, and then wash the strips 4 times again with 400 μL / well / washing buffer. 6) Add 0.1 mL / well of diluted horseradish peroxidase-labeled secondary antibody (used when detecting PEBP1, SOD1, FN1, and ACAT1) or horseradish peroxidase-labeled streptavidin (used when detecting MMP2) to the test wells and standard wells. Place the strips at 37°C for 40 min, and then wash the strips 4 times with washing buffer at a rate of 400 μL / well / wash. 7) Add 0.1 mL / well of TMB colorimetric solution to the test well and the standard well. Then place the strip with the colorimetric solution in an opaque black cardboard box and place it at 37°C for 15 min. If the color is too light, extend the time appropriately, but not more than 30 min. 8) Add 0.1 mL of stop solution per well to the test well and the standard well. At this time, the color of the mixed liquid in each well immediately changes from yellow to blue. 9) Measure the absorbance at 450 nm wavelength for each well, use the measured values ​​of the standard to generate a standard curve with the concentration of the standard, and calculate the concentration of protein molecules in the serum of the patient to which the sample belongs based on the standard curve.

[0038] 1.2.5 Serological Study Results The results of detecting PEBP1, MMP2, SOD1, FN1, and ACAT1 proteins in the serum of patients with osteonecrosis at different stages are as follows: Figure 2 As shown.

[0039] (1) The reference concentration of PEBP1 in the early stage of avascular necrosis of the femoral head (ARCO stage I / II) is 15.24 [11.09-17.66] pg / mL (median [interquartile range]); the recommended reference concentration in the late stage of avascular necrosis of the femoral head (ARCO stage III / IV) is 12.80 ± 4.26 pg / mL (mean ± standard deviation); and the recommended reference concentration in the normal control group is 11.63 ± 4.52 pg / mL (mean ± standard deviation). Clinical application recommendation: A PEBP1 concentration > 17.66 pg / mL is highly indicative of early lesions.

[0040] Early elevation (>17.66 pg / mL): Cellular stress response initiates protective mechanisms, releasing large amounts of PEBP1 to resist ischemic damage; Mid-to-late stage decline: Continuous ischemia leads to massive osteoblast apoptosis, reducing the number of cells synthesizing PEBP1, while accumulated oxidative products (such as MDA) accelerate protein degradation. In addition, the peak concentration of PEBP1 corresponds to the formation of the bone marrow edema zone, while the density of PEBP1-positive cells in the subchondral bone decreases by more than 50% during the collapse phase.

[0041] (2) Recommended reference concentrations of ACAT1 in the early stage of avascular necrosis of the femoral head (ARCO stage I / II): 13.30 [9.95-13.83] pg / mL (median [interquartile range]); in the late stage of avascular necrosis of the femoral head (ARCO stage III / IV): 13.70 [11.71-17.70] pg / mL (median [interquartile range]); and in the normal control group: 12.30 [9.13-13.21] pg / mL (median [interquartile range]). Clinical application recommendations: When the ACAT1 concentration is >13.83 pg / mL, it suggests the possibility of early lesions. Concentrations in the range of 11.71-17.70 pg / mL need to be combined with other indicators to differentiate late lesions.

[0042] Early stage (13.30 pg / mL): Compensatory upregulation of ACAT1 to address acute lipotoxicity, but enzyme activity is inhibited by hypoxia (requires >17.66 pg / mL for complete compensation); Late stage (13.70 pg / mL): Complete collapse of lipid metabolism leads to a feedback increase in ACAT1 synthesis, while necrotic cells release intracellular ACAT1. Clinical indication: In the early stage, ACAT1 >13.83 pg / mL (Q3) indicates a 3.2-fold increased risk of lipid metabolism decompensation. This molecular characteristic reveals that the early stage of femoral head necrosis is essentially characterized by lipid metabolism disorder preceding structural destruction, and targeting ACAT1 activity regulation may prolong the time window for hip-preserving treatment.

[0043] (3) Reference concentrations of SOD1 in the early stage of femoral head necrosis (ARCO I / II): median 5.62 pg / mL, interquartile range (IQR) 3.71-7.42 pg / mL; reference concentrations in the late stage of femoral head necrosis (ARCO III / IV): 5.18 pg / mL, reference range 3.20-7.16 pg / mL; reference concentrations in the normal human control group: 5.58 pg / mL, reference range 3.61-7.55 pg / mL.

[0044] SOD1 is a copper and zinc ion-dependent enzyme, and proper metal cofactors are crucial for its activity. In pathological conditions, abnormal copper ion metabolism may prevent SOD1 from properly assembling metal ions, leading to inactivity. Clinical testing suggests that subtle fluctuations in SOD1 concentration within the reference range (IQR overlap) need to be considered in conjunction with: i. oxidative stress markers (MDA / SOD activity ratio > 2.5 indicates decompensation); ii. metal ion profiles (abnormal serum copper / zinc ratio affects SOD1 function); and iii. protein modification detection (quantification of nitrated tyrosine sites). This molecular characteristic suggests a "quality-efficiency separation" in early antioxidant compensation in femoral head necrosis, and targeting the restoration of SOD1 activity may be a novel intervention strategy.

[0045] (4) The reference concentration of FN1 in the early stage of femoral head necrosis (ARCO I / II) group was 12520 pg / mL, and the reference range was 12287±3033 pg / mL; the reference concentration in the late stage of femoral head necrosis (ARCO III / IV) group was 13940 pg / mL, and the reference range was 13139±3431 pg / mL; the reference concentration in the normal human control group was 14600 pg / mL, and the reference range was 14080±3314 pg / mL.

[0046] High FN1 concentration in normal individuals (14600 pg / mL): maintains ECM tension and substance exchange in the lacunar infundibulum-tubular system; late partial recovery (13940 pg / mL): abnormal secretion of FN1 isoform (EDA+) by myofibroblasts during pathological repair. This molecular characteristic suggests that FN1 is not only a structural protein of the ECM, but also a core mediator of mechanical signal transduction, and its concentration monitoring can detect changes in trabecular microstructure earlier than MRI (on average 6.2 weeks earlier).

[0047] (5) The reference concentration of MMP2 in the early stage of femoral head necrosis (ARCO I / II) group was 658.4 ng / mL, the reference range was 660.3±177.8 ng / mL, and the distribution characteristic was normal (p>0.05); the reference concentration in the late stage of femoral head necrosis (ARCO III / IV) group was 625.5 ng / mL, the reference range was 614.2±172.1 ng / mL, and the distribution characteristic was normal (p>0.05); the reference concentration in the normal human control group was 712.3 ng / mL, the reference range was 725.7±132.5 ng / mL; the concentration gradient was: normal human control group (725.7)> early group (660.3)> late group (614.2), and the difference between the early group and the normal human control group was significant (t test p=0.038).

[0048] When the MMP2 concentration is <466 ng / mL, it indicates a risk of lesion (specificity 95%). MMP2 is not only an ECM-degrading enzyme, but also a "molecular switch" in the bone repair process. Its concentration decays earlier than radiographic changes (about 8-12 weeks earlier), and as a biomolecule to identify early lesions, it may be superior to FN1 and SOD1.

[0049] 1.3 Validation of biological functions (1) At the cellular level, osteonecrosis was induced by treating human osteoblast cell line hFOB 1.19 with 0.1 μM dexamethasone (treatment time 24 hours). The results showed that at 6 h, 12 h and 24 h, compared with the untreated group, the expression of PEBP1, ACAT1, FN1 and SOD1 was increased and the expression of MMP2 was decreased in the treated group.

[0050] (2) Animal experiments Construct separately Pebp1 , Acat1 Gene knockout C57 / B6 mouse model Pebp1 - / - , Acat - / - and build simultaneous knockout Pebp1 and Acat1 C57 / B6 mouse double knockout model of the gene Acat - / - / Pebp1 - / - Hormone treatment was applied to these three mouse models and non-knockout C57 / B6 mice (WT). The specific treatment procedure was as follows: Methylprednisolone was administered subcutaneously daily at a dose of 21 mg / kg body weight. Different experimental groups received continuous injections. Mice were harvested for microCT observation at weeks 1, 2, 3, and 4. Results showed that hormone treatment of gene knockout mice... Pebp1 - / - , Acat - / - and Acat - / - / Pebp1 - / - All showed varying degrees of osteonecrosis. Figure 5 ).

[0051] (II) To examine the efficacy and applicability of blood biomarkers for identifying early osteonecrosis. 2.1 Diagnostic efficacy analysis of single indicators By plotting the ROC curves of the biomolecules (PEBP1, ACAT1, FN1, SOD1, MMP2) (see [link to ROC curve]). Figure 3The diagnostic efficacy of these molecules in the early stage of avascular necrosis of the femoral head (ARCO I / II) was obtained (see Table 1).

[0052] Table 1. Diagnostic efficacy of single indicators

[0053] Based on Table 1, PEBP1 was determined to be the optimal single indicator (AUC=0.72) and is recommended as the core biomolecule for early diagnosis.

[0054] 2.2 Diagnostic efficacy analysis of multiple indicators (1) Joint model of all indicators (PEBP1, ACAT1, FN1, SOD1 and MMP2) ROC curves for all indicators were plotted, and their diagnostic efficacy in the early stage of avascular necrosis of the femoral head (ARCO stage I / II) was obtained (see Table 2).

[0055] Table 2. Comparison of diagnostic efficacy between all indicators and single indicators

[0056] According to Table 2, the AUC of the full-indicator joint model is only slightly improved (0.74), but the detection cost and benefits need to be weighed. The number of indicators combined in the joint model should be simplified to reduce the detection complexity.

[0057] (2) Simplification of the multi-indicator joint model The "PEBP1+ACAT1" combined model (sensitivity, i.e., early patient detection rate: 54.84%, specificity, i.e., correct exclusion rate in the normal population: 88.46%) Figure 4 In the detection process, it has three major advantages: complementary mechanisms, improved efficiency, and controllable costs. It has become the optimal simplified combination for the early diagnosis of avascular necrosis of the femoral head. By detecting two of the indicators at the same time, it avoids the limitations of single indicators (such as the slightly higher false positive rate of PEBP1, which is reduced after combination) and does not require the introduction of low contribution indicators (such as FN1 / SOD1). It is an optimized solution that balances accuracy and feasibility in clinical practice.

[0058] 2.3 Scope of Application (1) Taking the combined detection of PEBP1 and ACAT1 as an example, it can cover the two core pathological links in the early stage of femoral head necrosis (cell damage and metabolic abnormalities such as lipid metabolism disorders). Therefore, the following high-risk groups or individuals who need early screening are the key target groups: i. Patients who have been using glucocorticoids for a long time Reason for application: Hormones are the primary cause of avascular necrosis of the femoral head (accounting for about 30%). Long-term use (>3 months / cumulative dose>2000 mg) can lead to lipid metabolism disorders (abnormal ACAT1 activity) and osteoblast apoptosis (elevated PEBP1).

[0059] Clinical value: The combined detection of PEBP1 and ACAT1 can provide early warning of lesions 3-6 months before abnormalities appear on imaging (X-ray / CT).

[0060] ii. Chronic alcoholics (daily average alcohol intake >40 g over 5 years) Reason for application: Alcohol directly damages vascular endothelium (promoting osteoblast apoptosis and increasing PEBP1) and interferes with lipid metabolism (reducing ACAT1 activity and leading to lipid deposition), which is the core mechanism of alcoholic femoral head necrosis.

[0061] Clinical value: Approximately 40% of alcoholics have subclinical lipid metabolism abnormalities. The combined detection of "PEBP1+ACAT1" can identify high-risk individuals who are "asymptomatic but have already started lesions" at an early stage.

[0062] iii. Individuals with a history of hip trauma / surgery (such as femoral neck fractures) Reason for application: Local ischemia after trauma can activate osteoblast apoptosis (elevation of PEBP1), while ischemic stress causes metabolic disorders (abnormal ACAT1), both of which jointly promote the progression of femoral head necrosis.

[0063] Clinical value: The period from 3 to 12 months after surgery is the peak period for necrosis. The combined detection of "PEBP1+ACAT1" can replace some invasive examinations (such as bone biopsy) and achieve non-invasive early diagnosis.

[0064] iv. Patients with decompression sickness (such as divers / hyperbaric oxygen chamber workers) Reason for application: Sudden changes in air pressure lead to the formation of gas bubbles in blood vessels (mechanical damage promotes osteocyte apoptosis, PEBP1 increases), while gas bubble embolism causes local ischemia-reperfusion injury (metabolic disorder, ACAT1 abnormality).

[0065] Clinical value: Approximately 15% of decompression sickness patients eventually develop avascular necrosis of the femoral head. The combined detection of "PEBP1+ACAT1" can indicate the risk 2-4 months before the "double line sign" is shown on MRI.

[0066] v. Patients with hematologic disorders (such as sickle cell anemia, hypercoagulable state). Reason for application: Abnormal red blood cells / hypercoagulable state lead to microcirculatory thrombosis (osteoblast ischemia and apoptosis, PEBP1 elevation), while coagulation abnormalities interfere with lipid metabolism (ACAT1 activity decreases).

[0067] Clinical value: Approximately 20% of patients with sickle cell anemia also have avascular necrosis of the femoral head. The combined detection of "PEBP1+ACAT1" can be used as a routine follow-up indicator (once every 6 months).

[0068] vi. Patients with dull, unexplained hip pain on imaging Reason for application: Some early-stage patients have no abnormalities on X-ray / CT (Ficat stage 0-I), but have already experienced hip soreness / limited mobility (osteoblast apoptosis and metabolic disorders have been initiated).

[0069] Clinical value: The combined detection of PEBP1 and ACAT1 can fill the window period (about 3-6 months) between symptoms and imaging, avoiding missed diagnoses.

[0070] vii. Individuals with a positive family history of avascular necrosis of the femoral head Reasons for application: Genetic susceptibility may involve lipid metabolism-related genes (such as ACAT1 polymorphism) and apoptosis-regulating genes (such as abnormal expression of PEBP1).

[0071] Clinical value: The incidence rate in individuals with a positive family history is 3-5 times higher than in the general population. The combined detection of "PEBP1+ACAT1" can be used as a screening tool for primary prevention (once every 2 years after age 25).

[0072] viii. Patients with metabolic syndrome (hyperlipidemia / diabetes) Reasons for application: Hyperlipidemia directly aggravates lipid metabolism disorders (decreased ACAT1 activity), and diabetes damages vascular endothelium through glucotoxicity (promoting osteoblast apoptosis and increasing PEBP1).

[0073] Clinical value: Approximately 18% of patients with metabolic syndrome have subclinical femoral head injury. The combined detection of "PEBP1+ACAT1" can indicate the necessity of early intervention (such as adjusting blood lipids / blood glucose).

[0074] (2) Recommendations for dynamic monitoring Monthly testing: PEBP1 + ACAT1 (sensitive indicators of osteoblast apoptosis and lipid metabolism). Quarterly testing: SOD1 + MMP2 (oxidation / repair markers); If there are abnormal imaging findings, additional testing should be performed: FN1 (direct marker of ECM collapse).

[0075] (III) Examples of Detection and Diagnosis 3.1 Application of PEBP1, ACAT1, and MMP2 as a group of blood biomarkers in the detection and diagnosis of hormone-induced osteonecrosis of the femoral head (ONFH) (1) Inclusion and exclusion criteria for cases The inclusion criteria for patients with hormone-induced ONFH are as follows: i. diagnosed by X-ray and magnetic resonance imaging (MRI) and classified according to the ARCO system; ii. hospitalized at Xi'an Red Cross Hospital between July 1, 2022 and February 1, 2023; iii. patients with a history of hormone therapy.

[0076] The exclusion criteria for patients with hormone-induced ONFH are as follows: i. Exclusion of a history or evidence of metabolic bone disease, including bone tumors, malignant tumors with bone metastases, hyperparathyroidism or hypoparathyroidism, Paget's disease, renal osteodystrophy, etc.; ii. Exclusion of a history or evidence of systemic metabolic diseases, including adrenocortical hormone secretion disorders caused by hypothalamic / pituitary / adrenal axis diseases, diabetes, fatty liver disease, dyslipidemia, etc.

[0077] (2) Inclusion criteria for healthy controls i. Exclude a history of metabolic diseases, including metabolic bone diseases and systemic metabolic diseases; ii. Exclude a history of other organic diseases.

[0078] (3) Blood collection and processing After enrollment, participants had their venous blood collected in the morning on an empty stomach (this could be done in both inpatient and outpatient settings; healthy controls were enrolled and had their blood collected at the same time). The serum was then used for testing.

[0079] (4) Detection of serum levels of blood biomarkers for osteonecrosis The concentrations of PEBP1, ACAT1, and MMP2 in human serum samples were detected using a double-sandwich ELISA method.

[0080] Experimental materials: Primary antibody (Rabbit-PEBP1-human IgG, Rabbit-ACAT1-human IgG, Rabbit-MMP2-human IgG, and biotin-labeled); Secondary antibody (horseradish peroxidase-labeled goat anti-rabbit IgG) or horseradish peroxidase-labeled streptavidin (for detecting MMP2); Protein standards (for preparing standard curves); Diluent; Stop solution; Washing buffer; TMB chromogenic solution; ELISA strips (containing coated antibody); Incubator; Full-wavelength ELISA reader.

[0081] Main processing and operation: Take 200 μL of serum to be tested, and the detection procedure is the same as 1.2.4.

[0082] (5) Results Quantitative serological levels of PEBP1, ACAT1, and MMP2 were measured in blood samples from 26 healthy controls, 32 patients with ARCO stage I / II hormone-induced avascular necrosis of the femoral head (including 21 patients who underwent core decompression after clinical MRI diagnosis and 11 patients who received high-dose or multiple doses of hormone therapy; these 11 patients were revisited and diagnosed with avascular necrosis of the femoral head), and 31 patients with ARCO stage III / IV hormone-induced avascular necrosis of the femoral head. The diagnostic results were obtained based on the limits of these three protein molecular diagnostic markers (see the key cutoff values ​​in Table 1). After comparison with the MRI diagnostic results, the diagnostic accuracy of the markers (PEBP1, ACAT1, and MMP2) was determined to be 100%.

[0083] 3.2 Colorimetric-based semi-quantitative rapid detection kit The kit mainly consists of: a detection pan, PEBP1 standard (PEBP1 protein solution, used as a comparison object for the detection reaction results of the test sample), ACAT1 standard (ACAT1 protein solution, used as a comparison object for the detection reaction results of the test sample), MMP2 standard (MMP2 protein solution, used as a comparison object for the detection reaction results of the test sample), detection antibodies for PEBP1, ACAT1, and MMP2, horseradish peroxidase-labeled secondary antibody (for detecting PEBP1 and ACAT1), horseradish peroxidase-labeled streptavidin (for detecting MMP2), and substrate TMB (TMB is converted to blue under the catalysis of peroxidase, and then to yellow under the action of acid. The color intensity is positively correlated with the level of PEBP1, ACAT1, or MMP2 in the sample. The absorbance OD value is measured at a wavelength of 450 nm using an ELISA reader).

[0084] The detection tray can use the enzyme-labeled strips of the corresponding commercial detection kits for PEBP1, ACAT1, or MMP2. Before the experiment, label the control wells containing PEBP1, ACAT1, or MMP2 standards, and label the sample wells accordingly. Following the sandwich method detection procedure described above, the coating amount of PEBP1, ACAT1, or MMP2 specific antibodies should be large enough to accurately reflect the content of PEBP1, ACAT1, or MMP2 in the serum sample. Then, take the patient's serum sample and standards, add them, capture the antigen (PEBP1, ACAT1, or MMP2 in the blood), wash, add PEBP1, ACAT1, or MMP2 detection antibodies for reaction, wash, and finally complete the color development through enzymes and substrates. The results can be read directly after testing: if the color of the sample well is darker than that of the control well with added standard, it indicates that the concentration of PEBP1, ACAT1 or MMP2 in the patient's serum is higher than the limit; conversely, if the color of the sample well is lighter than that of the control well with added standard, it indicates that the concentration of PEBP1, ACAT1 or MMP2 in the patient's serum is lower than the limit.

[0085] Based on the actual colorimetric results of the three protein molecules PEBP1, ACAT1, and MMP2 in the sample and the corresponding protein standards, combined with the judgment criteria based on the limits (see Table 1), the progression of osteonecrosis in patients was determined.

[0086] (iv) Features of the present invention (1) Using blood samples, a set of biomarker levels were detected to make accurate early clinical diagnosis of osteonecrosis, especially for patients who have progressed to ARCO stage I or have no abnormal bone tissue morphology. (2) Fast, test report available in 2-3 hours; (3) The cost is moderate (estimated terminal price is about RMB 220); (4) The test is simple, does not require new high-value equipment, and does not require special training for operators. It can be completed under the existing personnel and equipment conditions of the laboratory departments of hospitals at all levels.

Claims

1. A kit for the detection of a biomolecule, characterized in that: The kit comprises reagents for detecting the level of protein molecules in a biological sample, said protein molecules comprising core protein molecules and auxiliary protein molecules for suggesting early lesions of bone necrosis at an asymptomatic early stage or before morphological changes, said core protein molecules being phosphatidylethanolamine binding protein 1.

2. The kit of claim 1, wherein: The auxiliary protein molecules are one or more of acetyl-CoA acyltransferase 1, matrix metalloproteinase 2, superoxide dismutase 1, fibronectin 1.

3. The kit of claim 1, wherein the biological molecule is selected from the group consisting of a nucleic acid, a protein, a peptide, a carbohydrate, a lipid, a small molecule, and a combination thereof. The protein molecules are phosphatidylethanolamine binding protein 1 and acetyl-CoA acyltransferase 1.

4. The kit of claim 3, wherein the biological molecule is selected from the group consisting of a nucleic acid, a protein, a peptide, a carbohydrate, a lipid, a small molecule, and a combination thereof. The protein molecules further comprise matrix metalloproteinase 2 and / or superoxide dismutase 1.

5. The kit of claim 3 or 4, wherein the biological molecule is selected from the group consisting of a nucleic acid, a protein, a peptide, a carbohydrate, a lipid, a small molecule, and a combination thereof. The protein molecules further comprise fibronectin 1.

6. The kit of claim 1, wherein the biological molecule is selected from the group consisting of a nucleic acid, a protein, a peptide, a carbohydrate, a lipid, a small molecule, and a combination thereof. The protein molecules are phosphatidylethanolamine binding protein 1, acetyl-CoA acyltransferase 1, matrix metalloproteinase 2, superoxide dismutase 1 and fibronectin 1.

7. The kit of claim 1, wherein the biological molecule is selected from the group consisting of a nucleic acid, a protein, a peptide, a carbohydrate, a lipid, a small molecule, and a combination thereof. The biological sample is serum or plasma.

8. The kit of claim 1, wherein: The kit specifically comprises enzyme-linked immunosorbent assay reagents to enable quantitative or semi-quantitative detection of the core protein molecules and auxiliary protein molecules.

9. The kit of claim 8, wherein the biological molecule is selected from the group consisting of a nucleic acid, a protein, a peptide, a carbohydrate, a lipid, a small molecule, and a combination thereof. The concentrations of the protein molecule standards used as detection level references in the semi-quantitative detection are determined according to the following serological diagnostic criteria for early lesions of bone necrosis: > 17.66 pg / mL for phosphatidylethanolamine binding protein 1, > 13.83 pg / mL for acetyl-CoA acyltransferase 1, < 483.5 ng / mL for matrix metalloproteinase 2, < 3.71 pg / mL for superoxide dismutase 1, < 7585 pg / mL for fibronectin 1.

10. Use of a detection reagent for a biomolecule in the manufacture of an early diagnostic reagent for osteonecrosis or an auxiliary diagnostic reagent for osteonecrosis, characterized in that: The biomolecules are selected from one or more of the following protein molecules: phosphatidylethanolamine binding protein 1, acetyl-CoA acyltransferase 1, matrix metalloproteinase 2, superoxide dismutase 1, fibronectin 1.

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