A biomarker and detection kit for diagnosing sarcopenia in osteoporosis

Abstract

The present invention belongs to the field of medical biological detection technology, and specifically relates to a biomarker and a detection kit for diagnosing sarcopenia in osteoporosis. The present invention has found that the NCOA4 level in the serum of osteoporosis patients has a certain degree of correlation with the development of muscular dystrophy, suggesting that serum NCOA4 can be used as a biomarker for sarcopenia in osteoporosis. Accordingly, the present invention provides a detection kit for diagnosing sarcopenia in osteoporosis, and the use of serological detection methods to predict sarcopenia can effectively reduce the lower limit of the detection tolerance range, thereby improving the detection sensitivity of serum NCOA4 and improving the detection accuracy. The present invention predicts the development of sarcopenia by detecting the NCOA4 level in serum, making the detection easy to achieve, and can become a conventional large-scale screening method for sarcopenia, which is convenient for early detection of the occurrence of sarcopenia and intervention.

Description

Technical Field

[0001] The present invention belongs to the technical field of medical biological detection, and specifically relates to a biomarker and a detection kit for diagnosing sarcopenia caused by osteoporosis. Background Art

[0002] With the acceleration of the aging process of society, the prevalence of osteoporosis has increased rapidly, and most of them are accompanied by sarcopenia, which together cause multiple complications, leading to a decline in the quality of life of the elderly and a heavy economic burden on society. It is estimated that there are currently about 50 million people suffering from sarcopenia in the world, and the number of people suffering from this disease is expected to reach 500 million by 2050. Sarcopenia is closely related to activity disorders, falls, low bone density and metabolic disorders, and is one of the important causes and manifestations of the gradual decline of physiological functions in the elderly. Sarcopenia will increase the hospitalization rate and medical expenses of the elderly, seriously affect the quality of life of the elderly, and even shorten the life span of the elderly. However, sarcopenia is a complex disease with the combined effects of environmental and genetic factors, and often has multiple risk factors and pathogenesis, resulting in the current diagnosis and treatment of sarcopenia. The means are limited and ineffective. As the disease progresses, patients with sarcopenia will gradually lose their ability to live and work, or even endanger their lives, which brings heavy psychological pressure and economic burden to patients, and has a huge impact and hindrance to the development of society. Therefore, it is particularly important to deeply explore the pathogenesis of sarcopenia and develop new and effective diagnosis and treatment methods.

[0003] Osteoporosis with sarcopenia is one of the important causes of fractures in the elderly. Sarcopenia increases the risk of falls and fractures, reduces the patient's ability to live in daily life, and is associated with heart disease, respiratory diseases and cognitive impairment; in severe cases, it can lead to motor dysfunction, decreased quality of life, loss of independent living ability, or long-term need for care from others, and increased risk of death. As the disease progresses, there is a progressive decrease in skeletal muscle fiber quality, decreased muscle strength, decreased muscle endurance and metabolic capacity, and increased connective tissue and fat. Sarcopenia lacks specific clinical manifestations. Patients may show weakness, easy falls, difficulty walking, slow gait, thin limbs and weakness, etc. Its diagnosis depends on the evaluation of muscle strength, muscle strength and muscle mass. At present, the treatment of sarcopenia is mainly to prevent the occurrence of the disease, but the existing diagnostic methods for sarcopenia are relatively backward, mainly through imaging to detect muscle mass or evaluate muscle function, and there is no serological detection method. The lack of specificity of existing diagnostic methods and the lack of methods to predict the progression and severity of sarcopenia in advance have brought great difficulties to the prevention of sarcopenia, making the prevention of sarcopenia very difficult. Therefore, it is extremely important to develop reagents and methods for predicting the occurrence and development of sarcopenia.

[0004] NCOA4 (nuclear receptor coactivator 4) was originally considered to be an androgen receptor activator involved in the regulation of downstream gene expression. Later, it was discovered that NCOA4 is a selective cargo receptor for the autophagy-lysosomal pathway, which can specifically bind to ferritin molecules, thereby degrading ferritin and releasing iron ions for cell use. Ferritin degradation regulated by NCOA4 is called iron autophagy. So far, no studies have shown that NCOA4 is associated with muscular atrophy.

[0005] At present, there are a variety of commercial NCOA4 detection kits for detecting NCOA4 levels in cell culture supernatants and serum, such as SignalwayAntibody, Abebio, Abbkine, etc. However, although these kits can detect NCOA4 levels in cell culture supernatants, it is difficult to detect serum. The reason is that the NCOA4 level in serum is low and it is difficult to meet the minimum detection requirements of the kit. Therefore, it is necessary to develop a highly sensitive serum NCOA4 detection method. At present, there are no relevant reagents or kits for predicting the occurrence of sarcopenia in osteoporosis patients by detecting serum NCOA4. Summary of the invention

[0006] In order to overcome the above-mentioned deficiencies of the prior art, the present invention predicts the occurrence of sarcopenia in osteoporosis patients by detecting the NCOA4 level in serum, which is more operational, more suitable for universal screening, and more sensitive in detecting sarcopenia.

[0007] In order to achieve the above object, the technical solution adopted by the present invention is:

[0008] The present invention provides a biomarker for osteoporosis-induced sarcopenia, wherein the biomarker is NCOA4.

[0009] Preferably, the NCOA4 is serum NCOA4.

[0010] Preferably, the NCOA4 can be used to predict the occurrence of sarcopenia at an early stage.

[0011] The present invention constructs an iron-deficient mouse model to study and find that NCOA4 may play a certain role in the process of skeletal muscle atrophy. Further, by detecting the NCOA4 level in the serum of osteoporosis patients, and following up the patients for 2 years, the changes in the skeletal muscle level of the patients were evaluated by means such as DXA. It was found that the NCOA4 level in the serum of osteoporosis patients was correlated to a certain extent with the occurrence and development of muscular dystrophy, indicating that NCOA4 is expected to become a biomarker for predicting the occurrence and development of muscular dystrophy.

[0012] The present invention also provides the use of the above-mentioned biomarkers in the preparation of products for detecting sarcopenia caused by osteoporosis.

[0013] The present invention also provides a detection kit for diagnosing sarcopenia caused by osteoporosis, which includes a 96-well plate pre-coated with anti-human NCOA4 antibodies, a 96-well plate cover, an NCOA4 protein standard, a horseradish peroxidase-labeled antibody, a TMB substrate colorimetric solution, a stop solution, a washing solution, and a score index table corresponding to the NCOA4 detection value.

[0014] Preferably, the detection kit further comprises a standard diluent and a horseradish peroxidase labeled antibody diluent.

[0015] Furthermore, the standard diluent and the horseradish peroxidase labeled antibody diluent are prepared by adding BSA to PBS buffer at a solid-liquid ratio of 0.1 g / 100 mL and mixing thoroughly.

[0016] Preferably, the detection range of the detection kit is 0.1-100 pg / mL.

[0017] Preferably, the configuration method of the 96-well plate pre-coated with anti-human NCOA4 antibody is: first dilute the anti-human NCOA4 antibody to 5ug / mL with PBS buffer at pH=9.6, then add 0.2mL to each well of the 96-well plate, incubate overnight at 4°C, discard the solution in the wells, and wash 3 times with washing solution, each time for 3 minutes.

[0018] Preferably, the stop solution is a 2 mol / L sulfuric acid solution.

[0019] Preferably, the washing solution is prepared by adding 1 mL of Tween solution to 1000 L of PBS buffer and mixing thoroughly.

[0020] Compared with the prior art, the present invention has the following beneficial effects:

[0021] The present invention has found through research that NCOA4-mediated activation of iron autophagy is an important cause of skeletal muscle atrophy. After iron autophagy is activated, NCOA4 interacts with RNF20 and is degraded together by the autophagy-lysosomal pathway, thereby causing a decrease in the histone ubiquitination level of myogenic transcription factors MyoD and MyoG, leading to muscle atrophy. It is suggested that the NCOA4 level in the serum of osteoporosis patients is correlated to a certain extent with the occurrence and development of muscular dystrophy. Detection of serum NCOA4 levels is expected to become a new method for predicting the occurrence of sarcopenia, and can be used as a biomarker for sarcopenia in osteoporosis. The present invention has the following main advantages:

[0022] (1) The present invention is the first to use a serological detection method to predict the occurrence and development of sarcopenia.

[0023] (2) Too much or too little iron ions can cause great harm to the human body, so the occurrence of iron autophagy is precisely regulated by the body. NCOA4, as the core cargo receptor of iron autophagy, will be a relatively sensitive indicator. As a biomarker for sarcopenia in osteoporosis, it can be used to predict the occurrence of sarcopenia at an early stage.

[0024] (3) Detecting the level of NCOA4 in serum to predict the occurrence and development of sarcopenia makes the detection easy to implement and can become a routine large-scale screening method for sarcopenia, facilitating early detection of sarcopenia and providing intervention.

[0025] (4) The prepared sarcopenia detection kit can effectively reduce the lower limit of the detection allowable range, thereby improving the detection sensitivity of serum NCOA4 and improving the detection accuracy.

[0026] (5) The present invention is aimed at the detection of NCOA4 rather than the detection of anti-NCOA4 autoantibodies. Since the production of anti-NCOA4 autoantibodies is related to multiple factors in the body such as B cells, and is not closely related to the occurrence of sarcopenia, it is more accurate to directly detect the level of NCOA4. BRIEF DESCRIPTION OF THE DRAWINGS

[0027] Figure 1 Western blot was used to detect the expression of iron autophagy-related molecules;

[0028] Figure 2 Western blot and immunofluorescence experiments were used to detect the expression of myogenesis-related molecules;

[0029] Figure 3 Western blot was used to detect the expression of myogenesis-related molecules after inhibiting ferroptosis;

[0030] Figure 4 This is the IP experiment result;

[0031] Figure 5 are the results of ChIP experiments (anti-H2Bub1 and anti-RNF20 refer to the antibodies of H2Bub1 and RNF20, respectively, and these two antibodies were used for ChIP experiments; NC represents the normal control group; DFO represents the DFO-treated group);

[0032] Figure 6 The cross-sectional area of ​​muscle bundles and the expression level of NCOA4 in normal group mice and treated group mice were measured by immunofluorescence staining (DAPI is a DNA dye that stains cell nuclei; laminin is a dye that stains the perimysium in muscle fibers to indicate the size of muscle bundles) and the expression levels of NCOA4 and MyHC were detected by Western blot;

[0033] Figure 7 The correlation scatter plot and curve for male osteoporosis patients;

[0034] Figure 8 The correlation scatter plot and curve for female osteoporosis patients;

[0035] Fig. 9 This is the ROC curve for screening sarcopenia by detecting NCOA4. DETAILED DESCRIPTION

[0036] The specific embodiments of the present invention are further described below. It should be noted that the description of these embodiments is used to help understand the present invention, but does not constitute a limitation of the present invention. In addition, the technical features involved in each embodiment of the present invention described below can be combined with each other as long as they do not conflict with each other.

[0037] The experimental methods in the following examples are conventional methods unless otherwise specified, and the experimental materials used in the following examples are commercially available unless otherwise specified.

[0038] Example 1 Study on the association between NCOA4 and muscle atrophy

[0039] C2C12 myoblasts (purchased from the Chinese Academy of Sciences Cell Bank) were cultured in vitro. C2C12 is a mouse-derived myoblast differentiation cell line. It can proliferate rapidly in DMEM high-glucose medium plus 10% fetal bovine serum. In DMEM high-glucose medium plus 2% horse serum, C2C12 cells differentiate and fuse into multinucleated muscle fibers. The cells were plated in a six-well plate under proliferation medium conditions at a cell density of 2.5×10 5 Cells / well. On the second day, the differentiation medium was replaced, and 0, 10, 15, 20, 25, and 30 μmol of DFO (iron chelator) was given to chelate iron ions. The expression of NCOA4 and LC3B, the core molecules of iron autophagy, was detected by Western blot (LC3B-I and LC3B-II are marker molecules of autophagy, and the conversion of LC3B-I to LC3B-II indicates that autophagy is activated). It was found that NCOA4 expression decreased, and the ratio of LC3B-II to LC3B-I increased significantly (such as Figure 1 As shown in Figure 2 ), it was confirmed that ferroptosis was activated. Accordingly, the expression of myosin heavy chain MyHC and myogenic transcription factors MyoD (myogenic differentiation antigen) and MyoG (myogenin) decreased significantly (as shown in Figure 2 ). Figure 2 This indicates that myogenic differentiation gradually weakened.

[0040] Then, iron ions were replenished (ferric citrate, AFC), and the iron autophagy inhibitor DC661, 3MA or NCOA4 knockout was given to inhibit the activation of iron autophagy. It was found that the inhibition of iron autophagy could reverse the inhibitory effect of DFO on muscle differentiation (such as Figure 3 Because NCOA4 is a key cargo molecule in the process of iron autophagy, it can interact with ferritin and be degraded via the autophagy-lysosome pathway.

[0041] Further protein profiling and immunoprecipitation experiments of NCOA4 were performed on DFO-treated C2C12 cells [for specific operations, please refer to the literature "Li, J. et al., TRAF4 positively regulates the osteogenic differentiation of mesenchymal stem cells by acting as an E3 ubiquitin ligase to degrade Smurf2. Cell Death & Differentiation 262652 (2019)"], and it was found that after iron autophagy activation, histone ubiquitinase RNF20 also interacts with NCOA4 and is degraded, indicating that RNF20 may be a potential downstream molecule (such as Figure 4 RNF20 regulates gene expression by regulating the ubiquitination level of histone H2B.

[0042] ChIP experiments were performed on DFO-treated and untreated C2C12 cells [for specific operations, please refer to the literature "Yu, W. et al., SNP-adjacent super enhancer network mediates enhanced osteogenic differentiation of MSCs in ankylosing spondylitis. HUMMOL GENET 30277 (2021)."] and found that after iron autophagy activation, the monoubiquitination level of histone H2B at the promoter position of myogenic transcription factors MyoG and MyoD decreased significantly, resulting in reduced expression of MyoG and MyoD, thereby causing reduced muscle differentiation (such as Figure 5 shown).

[0043] Finally, an iron-deficient diet was used to construct a mouse model of iron autophagy activation. Thirty C57BL / 6 mice aged 3-5 weeks (Experimental Animal Center, Zhongshan Medical College, Sun Yat-sen University) were randomly divided into two groups and fed with a control diet (Dyets, AIN93G) and an iron-deficient diet (Dyets, D115109), respectively. After 8 weeks, the serum iron ion level was detected, and it was found that the serum iron level of iron-deficient mice was significantly decreased. Western blot and immunofluorescence staining experiments were then performed on the gastrocnemius muscle specimens of mice to detect iron autophagy-related molecules (NCOA4, MyHC) and skeletal muscle levels (muscle bundle cross-sectional area). Consistent with in vitro experiments, decreased NCOA4 levels led to the occurrence of muscle atrophy (such as Figure 6 This suggests that iron autophagy may play a certain role in the process of skeletal muscle atrophy, and NCOA4 is a key molecule in iron autophagy. Detection of NCOA4 levels may have a certain diagnostic significance for sarcopenia.

[0044] Example 2 Study on the correlation between NCOA4 level and muscle loss

[0045] Twenty males and 30 females with osteoporosis but without sarcopenia were recruited (as shown in Table 1). The NCOA4 level in the patient's serum was analyzed by ELISA, and different concentration ranges were expressed as corresponding scores (as shown in Table 2). Dual-energy X-ray absorptiometry (DXA) was used to measure the muscle mass level of the skeletal muscles of the limbs of the blood drawers and recorded as muscle mass 1; 2 years later, dual-energy X-ray absorptiometry (DXA) was used to measure the muscle mass level of the same group of patients and recorded as muscle mass 2. Muscle mass 1 minus muscle mass 2 is the occurrence and progression of sarcopenia (2 years). Finally, SPSS statistical software was used to perform Pearson correlation analysis on NCOA4 levels and muscle loss. The test results are shown in Tables 3 and 4 and Figure 5 and Figure 6 .

[0046] The test results in Tables 3 and 4 show that after 2 years of follow-up, 60% of male patients had skeletal muscle mass of less than 7 kg / m 2 67% of women have skeletal muscle mass of less than 6.5 kg / m 2 , indicating the occurrence of sarcopenia. Moreover, the NCOA4 test index was significantly correlated with the amount of muscle loss in 2 years (Pearson coefficients were -0.8630 and -0.8929, respectively, p<0001) (such as Figure 7 , Figure 8 This suggests that NCOA4 levels are strongly correlated with muscle loss in osteoporosis patients and have the potential to predict the risk of sarcopenia in osteoporosis patients at an early stage.

[0047] Table 1 Basic information of osteoporosis patients

[0048]

[0049]

[0050] Note: All osteoporosis patients meet the criteria set by the Osteoporosis Committee of the Chinese Geriatric Society

[0051] Table 2 Score index corresponding to NCOA4 detection values

[0052]

[0053] Table 3 Corresponding scores and limb skeletal muscle mass (kg / m) in male osteoporotic patients 2 )

[0054]

[0055]

[0056] Table 4 Corresponding scores and limb skeletal muscle mass (kg / m) in female osteoporotic patients 2 )

[0057]

[0058]

[0059] Example 3 A kit for detecting sarcopenia in osteoporosis

[0060] (1) Composition of the kit:

[0061] 96-well plate pre-coated with anti-human NCOA4 antibody (H00008031-A01, abnova), 96-well plate film, NCOA4 protein standard (1000pg / tube), standard diluent, horseradish peroxidase-labeled antibody, horseradish peroxidase-labeled antibody dilution, TMB substrate colorimetric solution, stop solution, washing solution, and score index table corresponding to NCOA4 detection values.

[0062] (2) Preparation of the kit:

[0063] 1) Preparation of washing solution: Add 1 mL of Tween solution to 1000 L of PBS buffer and mix thoroughly.

[0064] 1) Antibody coating of 96-well plate: Prepare phosphate buffered saline (PBS) with a pH of 9.6, dilute anti-human NCOA4 to 5 ug / mL, then add 0.2 mL to each well of the 96-well plate, incubate overnight at 4°C, discard the solution in the wells, and wash 3 times with washing solution, 3 minutes each time.

[0065] 3) Preparation of standard diluent and horseradish peroxidase labeled antibody diluent: Add 0.1 g BSA to 100 mL PBS buffer and mix thoroughly.

[0066] 4) Stop solution preparation: Prepare 2 mol / L sulfuric acid solution and let stand at room temperature for later use.

[0067] 5) 96-well plate film was purchased from RayBiotech; NCOA4 protein standard was purchased from abnova; horseradish peroxidase labeled antibody was purchased from Santa Cruz; TMB substrate colorimetric solution was purchased from Sigma; the index table corresponding to the detection value was prepared by our unit, as shown in the following table:

[0068]

[0069] (3) Method for detecting NCOA4 using a kit

[0070] 1) Draw peripheral blood from osteoporosis patients, let it stand at room temperature for 1 hour, aspirate the supernatant and centrifuge at 1000×g for 5 minutes, retain the upper serum and discard the precipitate.

[0071] 2) The NCOA4 protein standard was centrifuged at 10,000 rpm for 1 min, then dissolved in 1 mL of standard diluent and diluted 7 times by serial dilution method (the dilution multiples were: 1000, 500, 250, 125, 62.5, 31.25, 15.625). The concentration of the diluted samples was distributed between 15.6-1000 pg.

[0072] 3) Add 100uL of diluted standard or test sample to each well of a 96-well plate pre-coated with anti-human NCOA4 antibody, apply a film, and incubate on a shaker at room temperature for 2h.

[0073] 4) Discard the liquid, add 200uL of washing solution to each well, and wash three times, each time for 1 min.

[0074] 5) Dissolve and dilute the horseradish peroxidase labeled antibody with horseradish peroxidase labeled antibody diluent at a solid-liquid ratio of 1:100. After dilution, add 100uL to each well of the above 96-well plate and incubate on a shaker at room temperature for 1 hour.

[0075] 6) Add 200uL stop solution to terminate the reaction;

[0076] 7) Add 100uL of TMB substrate colorimetric solution to each well of the above 96-well plate and incubate at room temperature in the dark for 30 minutes.

[0077] 8) Add 50uL of stop solution to each well of the above 96-well plate.

[0078] 9) The optical density of each well was detected at a wavelength of 450 nm using an ELISA instrument, and the actual value (i.e., the NCOA4 detection value) was calculated by fitting the standard curve.

[0079] Example 4 Detection range, specificity, recovery rate and accuracy of the kit

[0080] (1) Detection range: The NCOA4 protein standard was diluted to different concentrations (200, 100, 50, 25, 12.5, 6.25, 3.125, 1.56, 0.78, 0.39, 0.2, 0.1, and 0.05 times, respectively), and then measured according to the NCOA4 detection method in Example 3. The detection range of the kit was 0.1-100 pg / mL (the detection range of the existing detection method is 10-100 pg / mL).

[0081] (2) Specificity: The specificity of the test kit was tested using cargo proteins similar to NCOA4, such as P62, Nbr1, Optn, Ccolco2, and secretory proteins Myostatin, FGF21, IL33, Irisin, IL6, LIF, and TNFα, as well as those frequently found in muscular atrophy. That is, the standard products of the above proteins were used to perform specificity testing according to the NCOA4 detection method in Example 3. The test results showed that NCOA4 had no cross-reaction with P62, Nbr1, Optn, Ccolco2, Myostatin, FGF21, IL33, Irisin, IL6, LIF, and TNFα.

[0082] (3) Recovery rate:

[0083] Peripheral blood was drawn from volunteers, and allowed to stand at room temperature for 1 hour. The supernatant was aspirated and centrifuged at 1000×g for 5 minutes, the upper serum was retained, and the precipitate was discarded. The collected serum was divided into two portions, one of which was added with a quantitative NCOA4 standard; the two portions were analyzed simultaneously according to the steps of Example 3, and the difference between the result obtained from the spiked portion and the result obtained from the unspiked portion was subtracted, and the ratio of the difference to the theoretical value of the added standard substance was the sample spike recovery rate.

[0084] Table 5 Detection recovery rate of the kit

[0085] sample Recovery rate range (%) Average recovery rate (%) Serum (n=50) 97-105 99 Plasma (n=50) 85-91 87 Cell culture supernatant (n=50) 89-94 92

[0086] (4) Accuracy: Five batches of the kit were prepared according to the composition of the kit in Example 3, and then the NCOA4 detection method in Example 3 was used for detection. The detection results showed that the intra-batch difference CV was <5.7%, and the inter-batch difference CV was <4.3%, indicating that the detection accuracy of the kit of the present invention is high.

[0087] Example 5 Application of the kit of the present invention in detecting sarcopenia in osteoporosis

[0088] 22 normal subjects and 22 sarcopenia patients were recruited. The serum NCOA4 levels were detected according to the NCOA4 detection method in Example 3, and then the subjects were divided into normal subjects and patients according to the NCOA4 scores (as shown in Table 6), and the ROC curve was constructed according to the NCOA4 scores.

[0089] like Fig. 9 As shown, the area under the curve is 0.8378, the sensitivity is 86.36%, and the specificity is 68.18%, which indicates that the detection kit of the present invention has a good clinical application effect.

[0090] Table 6 NCOA4 score statistics

[0091]

[0092] The embodiments of the present invention are described in detail above, but the present invention is not limited to the described embodiments. For those skilled in the art, various changes, modifications, substitutions and variations of these embodiments are made without departing from the principles and spirit of the present invention, and still fall within the protection scope of the present invention.

Claims

1. Use of reagents for detecting NCOA4 expression levels in the preparation of products for detecting sarcopenia in osteoporosis.

2. The use according to claim 1, characterized in that The NCOA4 is serum NCOA4.

3. A detection kit for diagnosing sarcopenia in osteoporosis, characterized in that: The kit includes a 96-well plate pre-coated with an anti-human NCOA4 antibody, a 96-well plate cover, an NCOA4 protein standard, a horseradish peroxidase-labeled antibody, a TMB substrate colorimetric solution, a stop solution, a washing solution, and a score index table corresponding to the NCOA4 detection value; when the NCOA4 detection value pg is respectively <0.39, 0.39-0.78, 0.78-1.56, 1.56-3.12, 3.12-6.25, 6.25-12.5, 12.5-25, 25-50, 50-100, and >100, the corresponding NCOA4 scores are 1, 2, 3, 4, 5, 6, 7, 8, 9, and 10.

4. A detection kit for diagnosing sarcopenia caused by osteoporosis according to claim 3, characterized in that: The detection kit also includes a standard diluent and a horseradish peroxide labeled antibody diluent.

5. A detection kit for diagnosing sarcopenia caused by osteoporosis according to claim 4, characterized in that: The standard diluent and the horseradish peroxidase labeled antibody diluent are prepared by adding BSA to PBS buffer at a material-liquid ratio of 0.1 g / 100 mL and mixing thoroughly.

6. A detection kit for diagnosing sarcopenia caused by osteoporosis according to claim 3, characterized in that: The detection range of the detection kit is 0.1-100 pg / mL.

7. A detection kit for diagnosing sarcopenia caused by osteoporosis according to claim 3, characterized in that: The 96-well plate pre-coated with anti-human NCOA4 antibody was prepared as follows: the anti-human NCOA4 antibody was first diluted to 5 μg / mL with PBS buffer at pH 9.6, and then 0.2 mL was added to each well of the 96-well plate. The wells were incubated at 4°C overnight, the solution in the wells was discarded, and the wells were washed three times with washing solution for 3 minutes each time.

8. A detection kit for diagnosing sarcopenia caused by osteoporosis according to claim 3, characterized in that: The stop solution is a 2 mol / L sulfuric acid solution.

9. The detection kit for diagnosing sarcopenia caused by osteoporosis according to claim 3, characterized in that: The washing solution was prepared by adding 1 mL of Tween solution to 1000 L of PBS buffer and mixing thoroughly.

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