Application of FSH as biomarker for diagnosing autoimmune hepatitis
By detecting the FSH protein in the serum of AIH patients and setting the CUTOFF value to 58.81 pg/ml, the accuracy of early diagnosis of AIH is solved. FSH protein, as a biomarker and therapeutic target of AIH, improves the effectiveness of diagnosis and treatment.
Patent Information
- Application Number
- CN202510585469.4
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2025-05-08
- Publication Date
- 2025-08-22
AI Technical Summary
In the prior art, early identification and diagnosis of autoimmune hepatitis (AIH) is difficult, and there are challenges in the identification of specific markers and biochemical characteristics, resulting in patients not being diagnosed in a timely manner in the early stage of the disease, increasing the risk of cirrhosis and acute liver failure.
FSH protein was used as a biomarker, and the FSH content in the serum of AIH patients was detected by ELISA method, Western blot, protein chip, chemiluminescence immunoassay and other methods. The CUTOFF value was set to 58.81 pg/ml to improve diagnostic accuracy, and FSH protein was used as a target for therapeutic drug research.
100% diagnostic accuracy of AIH was achieved, and the FSH protein was significantly higher in the serum of AIH patients than the peers. As a diagnostic tool and therapeutic target, it improved the early recognition and treatment effect of AIH.
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Figure CN120522399A_ABST
Abstract
Description
Technical Field
[0001] The present invention belongs to the technical field of gene diagnosis and treatment of molecular biology, and in particular relates to the application of FSH as a biomarker for diagnosing autoimmune hepatitis. Background Art
[0002] Autoimmune hepatitis (AIH) is a chronic inflammatory liver disease mediated by an abnormal autoimmune response, characterized by persistent liver inflammation and potentially severe damage. In clinical practice, AIH patients often present with positive serum autoantibodies, elevated transaminase and immunoglobulin G (IgG) levels, and features of hepatocellular injury and necrosis on liver histology, particularly interface hepatitis, as a prominent pathological feature. The specific etiology and pathogenesis of AIH remain unclear, but its annual incidence and prevalence are increasing. Currently, the clinical diagnosis of AIH is primarily based on clinical manifestations, biochemical features, and histological evidence. The clinical diagnosis of AIH requires a combination of serum autoantibodies such as anti-SSA / B and anti-LKM antibodies, biochemical profiles (such as elevated serum transaminases, total bilirubin, and γ-GT), and the patient's clinical presentation and histological findings. However, the identification of these markers and characteristics remains challenging, especially in the early stages. Many patients may not have obvious symptoms or only exhibit nonspecific symptoms, leading to failure to timely diagnosis. Approximately 34%-76% of patients have already developed into the cirrhosis stage at the time of initial diagnosis, increasing the risk of acute liver failure and mortality. Therefore, there is an urgent need to explore new diagnostic methods and strategies.
[0003] Current research is focused on exploring the pathogenesis of AIH, including genetic susceptibility, immune system dysfunction, environmental factors, viral infections, and the use of medications that may cause immune system dysfunction. Studies have found that gender is a significant risk factor for the development of AIH, with significantly more female patients than males, with a male-to-female ratio of approximately 1:4 to 1:6. AIH is more common in middle-aged women, but children and adolescents may also be affected. Globally, the peak incidence of AIH occurs between the ages of 50 and 60, a period during which women's follicle-stimulating hormone (FSH) levels experience a dramatic increase, reaching their highest peak in life. This finding reveals a possible association between the peak age of AIH development and peak FSH levels, suggesting that elevated FSH levels may be associated with the development of AIH.
[0004] FSH is a glycoprotein hormone secreted by the pituitary gland that plays a vital role in the maturation and development of ovarian follicles. In recent years, a growing number of studies have revealed that FSH and its receptors also play important roles in tissues other than the reproductive system, such as in hepatocytes, osteoclasts, and cortical neurons, and also play a key role in various diseases in postmenopausal women.
[0005] Autoimmune hepatitis (AIH) shares similarities with other autoimmune diseases, such as rheumatoid arthritis and autoimmune thyroiditis, in that they all originate from a chronic inflammatory process caused by an abnormal autoimmune response. Studies have found that elevated FSH levels are closely associated with rheumatoid arthritis and may lead to cartilage damage. Furthermore, Alzheimer's disease (AD), a common neurodegenerative disease in postmenopausal women, features aggregation of tau protein (a microtubule-associated protein (MAP) specific to nerve cells) as a prominent pathological feature, which worsens with increasing FSH levels. Treatment with FSH antibodies can improve symptoms. These studies, taken together, indicate a correlation between elevated FSH levels and non-infectious chronic inflammatory diseases, suggesting a possible role for FSH in the development of chronic inflammatory diseases.
[0006] Liver histological examination results are an important basis for the clinical diagnosis of AIH, but it is an invasive diagnostic method. On the one hand, it may cause physical and mental damage to patients and is not easily accepted. On the other hand, the reliability of the test results may be affected by differences in physician experience, technology, and sampling quality.
[0007] Early identification and diagnosis of AIH is a major challenge. Because AIH usually progresses slowly, many patients do not have obvious clinical symptoms in the early stages of the disease, which may lead to patients delaying medical treatment or doctors neglecting the disease.
[0008] Identifying specific markers and biochemical features of AIH remains challenging. AIH patients often demonstrate positive serum autoantibodies, elevated transaminase and immunoglobulin G (IgG) levels, but these findings also occur in other inflammatory liver diseases. The specificity of AIH is particularly limited in distinguishing liver damage from other causes. Therefore, the development of more sensitive and specific diagnostic tools is needed to improve the diagnostic accuracy of AIH. Summary of the Invention
[0009] To address the above issues, the present application provides the use of FSH as a biomarker for diagnosing autoimmune hepatitis. The FSH content in the serum of patients with autoimmune hepatitis (AIH) has potential value in diagnosing AIH, and the cutoff value of FSH in the serum of patients with autoimmune hepatitis is 58.81 pg / m.
[0010] In a first aspect, the present invention provides the use of FSH protein or a substance for detecting FSH protein in the preparation of a tool for diagnosing autoimmune hepatitis, wherein the FSH protein is upregulated in the serum of autoimmune hepatitis patients.
[0011] In a preferred example of this aspect, the concentration of FSH protein in the autoimmune hepatitis serum is ≥58.81 pg / mL.
[0012] In another preferred embodiment of this aspect, the tool includes a kit and a chip.
[0013] The aforementioned testing includes protein level testing, including immunoassays, Western blots, protein chips, chemiluminescent immunoassays or surface plasmon resonance (SPR), enzyme-linked immunosorbent assays (ELISA), and detection techniques combining PCR (polymerase chain reaction) and ELISA. To simplify experimental procedures, protein level testing also includes protein detection kits, such as ELISA kits, colloidal gold kits, immunoprecipitation kits, chemiluminescent kits, immunofluorescence kits, and detection kits combining PCR (polymerase chain reaction) and ELISA. The kits typically come with corresponding instructions, which generally include information such as the company logo and name, the kit name, the kit composition, shelf life, intended use, and instructions for use. Users can obtain satisfactory results by following the instructions with little or no optimization.
[0014] Preferably, the immunoassay method is ELISA detection and / or colloidal gold detection.
[0015] The ELISA method uses an ELISA detection kit, which includes: a solid phase carrier coated with FSH monoclonal antibody, an enzyme-labeled antibody, an enzyme substrate, a protein standard, a negative control, a diluent, a washing solution, an enzyme reaction stop solution, etc.
[0016] In a second aspect, the present invention provides the use of FSH protein or a substance for detecting FSH protein in the preparation of a product for evaluating the therapeutic effect of autoimmune hepatitis or judging the prognosis of autoimmune hepatitis, wherein the FSH protein is downregulated in the serum of autoimmune hepatitis after treatment.
[0017] In a third aspect, the present invention provides the use of FSH protein or a substance for detecting FSH protein for screening drugs for preventing or treating autoimmune hepatitis.
[0018] Compared with the prior art, the present invention has the following advantages: This study, published in the journal Nature Medicine, demonstrates for the first time that serum FSH levels in patients with autoimmune hepatitis are significantly higher than those in the same age group. Furthermore, the Youden index reaches its highest value of 100% when the serum concentration reaches 58.81 pg / ml. This results in a cutoff value of 58.81 pg / ml for diagnosing AIH, with 100% accuracy. This study demonstrates that serum FSH levels in patients with autoimmune hepatitis (AIH) have potential diagnostic value. Furthermore, FSH can serve as a target for drug development in the treatment of autoimmune hepatitis. BRIEF DESCRIPTION OF THE DRAWINGS
[0019] Figure 1 Serum FSH levels in AIH patients are significantly higher than those in healthy women of the same age group. A. Statistical analysis of serum FSH levels in AIH patients and healthy women of the same age group. B. ROC curve analysis of serum FSH levels. C. FSH level range for the top ten patients with the highest Youden index.
[0020] Figure 2 .The results showed that the levels of MAP1S (A), IL18 (C) and FSH (E) in the serum of AIH patients were significantly higher than those in healthy women of the same age group. Figure 2 B, D, and F show the correlation analysis results between FSH and MAP1S, FSH and IL18, and MAP1S and IL18, respectively.
[0021] Figure 3 FSH stimulates MAP1S protein expression in the hepatocellular carcinoma cell line HepaRG. A. Western blot analysis shows elevated MAP1S expression in HepaRG cells after treatment with different FSH concentrations (0, 5, 10, 25, 50, and 100 ng / ml). B. Western blot analysis of protein expression (*p < 0.05, **p < 0.01, ***p < 0.005, ****p < 0.001).
[0022] Figure 4High levels of FSH promote MAP1S expression in ovariectomized mice. 12-week-old female C57BL / 6J mice underwent ovariectomy (OVX), with sham-operated mice (Sham) serving as negative controls. A. Images of the uteri of sham and OVX mice; B. Serum FSH levels in sham and OVX mice 12 weeks after ovariectomy; C. Weekly body weights of sham and OVX mice were collected and analyzed (n=6); D. Western blot analysis of MAP1S protein expression in liver tissues of sham and ovariectomized mice; E. Relative quantitative statistical graph of MAP1S (*, p<0.05, **, p<0.01, ***, p<0.005, ****, p<0.001) (n=8) Figure 5 MAP1S is highly expressed in AIH hepatocytes. A. Paraffin sections of liver tissue from a menopausal woman with hepatic hemangioma (HCH) (first column) and AIH (second column) were labeled with MAP1S antibodies. Brown indicates positive MAP1S antibody labeling, while blue indicates hematoxylin-labeled nuclei. Observed under a microscope at 20x (first row) and 40x (second row) magnification, respectively. B. The MAP1S staining index in AIH liver tissue (n=6) was significantly higher than that in HCH (n=4).
[0023] Figure 6 The expression level of IL-18 in liver tissue of AIH patients was significantly higher than that in the normal control group.
[0024] Figure 7 .The cytochrome C content in liver tissue of AIH patients with high MAP1S expression was significantly reduced.
[0025] Figure 8The number of apoptotic cells in liver tissues from AIH patients with high MAP1S expression was significantly higher than that in the MAP1S low expression group and HD patients. A. Representative immunofluorescence analysis results: Apoptotic cells (red), hepatocytes (green), and nuclei (blue) in normal liver tissue (HD, first row), AIH liver tissue with low MAP1S expression (MAP1S-L, second row), and AIH liver tissue with high MAP1S expression (MAP1S-H, third row). B. Statistical analysis: The proportion of apoptotic hepatocytes in total cells (AHIAC) = number of apoptotic hepatocytes / total number of nuclei * 100%; the proportion of apoptotic hepatocytes in total apoptotic cells (AHIAAC) = number of apoptotic hepatocytes / total number of apoptotic cells * 100%; the proportion of apoptotic hepatocytes in total hepatocytes (AHIAH) = number of apoptotic hepatocytes / total number of hepatocytes * 100%. C. Statistical analysis: The ratio of apoptotic non-hepatocytes to total apoptotic cells = 1 - AHIAAC, and the ratio of apoptotic hepatocytes to total apoptotic cells (AHIAAC). DE Statistical analysis of AHIAC, AHIAAC, and AHIAH in the HD, MAP1S-L, and MAP1S-H groups. DETAILED DESCRIPTION
[0026] The preferred embodiments of the present invention are described in detail below with reference to the accompanying drawings so that the advantages and features of the present invention can be more easily understood by those skilled in the art, thereby making a clearer and more precise definition of the protection scope of the present invention.
[0027] Example 1: ELISA to detect serum FSH levels and evaluate its diagnostic value for AIH The inventors used ELISA to detect peripheral serum of 35 AIH patients and 30 age-matched healthy volunteers (HD). Figure 1 AThe results showed that the serum FSH levels in AIH patients (N=35) (Jingmei Biotechnology Co., Ltd., JM-7167H1) were significantly higher than those in the HD group (N=30) (P value less than 0.0001); Figure 1 B. ROC curve analysis showed that the area under the ROC curve (AUC) for FSH was as high as 1, with a statistically significant difference (P < 0.0001). Figure 1 C. When the serum FSH concentration reached 58.81 pg / ml, the Youden index reached its highest value of 1. Therefore, the cutoff value for diagnosing AIH was 58.81 pg / ml, with 100% accuracy. These findings suggest that serum FSH levels have potential diagnostic value for AIH. FSH may also serve as a target for drug development in the treatment of autoimmune hepatitis.
[0028] Example 2: ELISA detection of MAP1S, FSH and IL18 levels in serum The inventors used ELISA to detect peripheral serum of 35 AIH patients and 30 age-matched healthy volunteers (HD). Figure 2 The results in A, C, and E showed that the levels of MAP1S (N=35) (Jingmei Biotechnology Co., Ltd., JM-7167H1), FSH (N=35) (Jingmei Biotechnology Co., Ltd., JM-7167H2), and IL18 (N=35) (Jingmei Biotechnology Co., Ltd., JM-7167H3) in AIH patients were significantly higher than those in the HD group (all P values were less than 0.0001); Figure 2 The correlation analysis results in B, D, and F showed that FSH was positively correlated with MAP1S and IL18, and MAP1S was positively correlated with IL18 (Pearson r was 0.7704, 0.8977, and 0.7454, respectively, and P values were all less than 0.0001), suggesting that in AIH patients, high levels of FSH are closely related to high expression of MAP1S and the production of the inflammatory factor IL-18.
[0029] Example 3: FSH stimulates MAP1S protein expression in the liver cancer cell line HepaRG Considering that AIH mainly occurs in menopausal women aged 50-60 years, and FSH levels in women of this age group reach their peak, we treated HepaRG cells with different concentrations of FSH (HY-P74133, MCE, USA) (0, 10ng / ml, 25ng / ml, 50ng / ml, 100ng / ml, 150ng / ml) for 12 hours, and then detected by immunoblotting. The results showed that FSH could significantly enhance the expression of MAP1S protein in a dose-dependent manner (see Figure 3 A, B). A. Western Blot showed that MAP1S expression increased in HepaRG cells after treatment with different concentrations of FSH (0, 5, 10, 25, 50, 100 ng / ml). B. Western Blot statistical analysis of protein expression (*p<0.05, **, p<0.01, ***, p<0.005, ****, p<0.001) Conclusion: FSH promotes the expression of MAP1S protein in the hepatocellular carcinoma cell line HepaRG.
[0030] Example 4: Increased expression of MAP1S in liver tissue in the ovariectomized (OVX) mouse model We then used a mouse model to further explore the regulatory effects of FSH on MAP1S. To simulate the postmenopausal state and related disease processes in humans, we performed ovariectomy (OVX) on 12-week-old female C57BL / 6J mice (provided by Beijing Weishanglide Biotechnology Co., Ltd.), using sham-operated mice (Sham) as negative controls to simulate the high FSH environment in vivo. Ten weeks after ovariectomy, uterine tissue was collected from the mice. Compared with the Sham group, the uteri of the OVX mice were significantly atrophied ( Figure 4 A); Mouse serum was collected and serum FSH levels were detected by enzyme-linked immunosorbent assay (ELISA) (Jingmei Biotechnology Co., Ltd., JM-02838M1). Compared with the Sham group, the serum FSH level of OVX mice was significantly increased ( Figure 8 B). The body weight of mice was measured every other week. It was found that after ovariectomy, OVX mice gained more weight than the Sham group, and 3 weeks after surgery, the body weight of OVX mice increased significantly ( Figure 4 C), which is consistent with the phenomenon of weight gain in postmenopausal women and consistent with previous studies on castrated mice, demonstrating that the ovariectomized mouse model was successfully established. We collected liver tissue from the sham and OVX mouse groups for protein level determination. The results showed that compared with the sham group, MAP1S expression in the liver of OVX mice was increased (P<0.01) (see Figure 4 D, E).
[0031] Example 5: High expression of MAP1S in AIH liver tissue We labeled paraffin sections of liver tissue adjacent to hepatic hemangioma (HCH) in menopausal women (column 1) and AIH (column 2) with MAP1S antibody (Biorbyt, #orb251641, UK) and performed immunohistochemistry to detect MAP1S expression ( Figure 5 A). Brown indicates positive MAP1S antibody labeling, and blue indicates hematoxylin-labeled cell nuclei. These are the results of observation under a microscope at 20x (first row) and 40x (second row), respectively. The MAP1S staining index in AIH liver tissue (n=6) was significantly stronger than that in HCH (n=4) ( Figure 5 B).
[0032] By immunohistochemical analysis, we found that the expression intensity and positive staining area of MAP1S in the hepatocytes of AIH patients were significantly higher than those in the normal liver tissue (HCH) adjacent to the hemangioma of female patients with hepatic hemangioma of the same age group, with statistical significance (P < 0.05) (see Figure 5 A, B).
[0033] Example 6: MAP1S expression in AIH hepatocytes was significantly stronger than that in HCH The inflammatory cytokine IL-18 is a key effector molecule released by inflammasome activation, and it plays an important role in the activation and regulation of inflammation. Through proteomic analysis, we found that the abundance of IL-18 in the liver tissue of AIH patients (N=19) was 0.6098, which was significantly higher than the abundance of 0.2534 in normal liver tissue (n=6), and the expression level was significantly increased (log2FC>1.2, P=0.0165), indicating that inflammasomes are activated in the liver tissue of patients with autoimmune liver disease, and the level of inflammatory cytokine IL-18 is significantly increased. Figure 6 .
[0034] Method 7: Liver tissue protein profiling to detect the correlation between MAP1S expression and inflammatory factors IL18 and cytochrome C levels Given the high expression of MAP1S in hepatocytes detected by immunohistochemistry, we further analyzed the expression of MAP1S in liver tissues of AIH patients and healthy volunteers based on the results of proteomic analysis. In the livers of the healthy control group, the average abundance of MAP1S protein was 0.5715 (n = 6). Based on this criterion, AIH patients were divided into two groups: one group with a mean of 0.6659 ± 0.02695, higher than 0.5715, was designated as the MAP1S-H group (n = 7), and the other group with a mean of 0.5059 ± 0.02313, lower than 0.5715, was designated as the MAP1S-L group (n = 12). Among them, the MAP1S-H group accounted for 36.84% (7 / 19) of the patients, and the MAP1S-L group accounted for 63.16%. The cytochrome C (CYC) content between the two groups was further analyzed. The results showed that the CYC content in the liver tissue of the MAP1S-H group was 2.132 ± 0.2716, which was significantly lower than that of the MAP1S-L group (3.429 ± 0.3721) and the HD group (3.404), (P = 0.0065, P = 0.0392). There was no significant difference in CYC content between the MAP1S-L and HD groups (P > 0.05) (see Figure 7 The CYC content in the liver tissue of the MAP1S-H group was only 62.18% of that in the MAP1S-L group, indicating that high expression of MAP1S in the liver tissue of AIH patients led to mitochondrial damage and the release of its contents, such as CYC.
[0035] The IL18 levels in the MAP1S high expression group and low expression group were 0.7206 ± 0.1742 and 0.4199 ± 0.3007, respectively, which were higher than those in the healthy control group (0.2534), but there was no significant difference (P = 0.0688). However, the IL18 level in the MAP1S-H group was lower than that in the MAP1S-L group (P = 0.0909) (see Figure 7Given that IL18 is an effector product produced after activation of intracellular inflammasomes, and that patients in the MAP1S high expression group had mitochondrial damage, the release of mitochondrial contents led to liver cell death, thereby causing the release of inflammatory factors. This may be the reason why the IL18 content in the MAP1S high expression group was lower than that in the MAP1S low expression group.
[0036] Example 8: Immunofluorescence detection of apoptotic cells in liver tissue The inventors used the TUNEL assay (Servicebio, G1504, Wuhan, China) to detect apoptotic cells in liver tissues of AIH patients (n=19) and healthy volunteers (HD) (n=6). DAPI (Servicebio, G1012, Wuhan, China) stained cell nuclei with DUNEL (CY3) fluorescein, which emits red fluorescence under laser excitation at a wavelength of 510-560 nm, indicating that the nuclei of apoptotic cells are red. CK18 antibody (Servicebio, GB114832, Wuhan, China, 1:300) was used to label hepatocytes, and FITC-labeled secondary antibody (Servicebio, GB21303, Wuhan, China, 1:300) emitted green fluorescence under laser excitation at a wavelength of 465-495 nm, indicating that the cytoplasm of CK18-positive hepatocytes is green. Figure 8 A. Immunofluorescence analysis of apoptotic cells (red), hepatocytes (green), and nuclei (blue) in normal liver tissue (HD, first row), AIH liver tissue with low MAP1S expression (MAP1S-L, second row), and AIH liver tissue with high MAP1S expression (MAP1S-H, third row). B. Statistical analysis: The proportion of apoptotic hepatocytes in total cells (AHIAC) = number of apoptotic hepatocytes / total number of nuclei * 100%; the proportion of apoptotic hepatocytes in total apoptotic cells (AHIAAC) = number of apoptotic hepatocytes / total number of apoptotic cells * 100%; and the proportion of apoptotic hepatocytes in total hepatocytes (AHIAH) = number of apoptotic hepatocytes / total number of hepatocytes * 100%. C. Statistical analysis: The proportion of apoptotic non-hepatocytes in total apoptotic cells = 1 - AHIAAC, and the proportion of apoptotic hepatocytes in total apoptotic cells (AHIAAC). DE statistical analysis of AHIAC, AHIAAC, and AHIAH in the HD, MAP1S-L, and MAP1S-H groups showed that the rate of hepatocyte apoptosis in liver tissues of AIH patients with high MAP1S expression was significantly higher than that in liver tissues of healthy volunteers and AIH patients with low MAP1S expression, suggesting that high MAP1S expression may be associated with hepatocyte death.
[0037] The above research results indicate that serum FSH levels in AIH patients are significantly higher than those in the HD group. In AIH patients, high FSH levels are closely associated with elevated MAP1S expression and the production of the inflammatory cytokine IL-18. FSH stimulates MAP1S protein expression in the hepatocellular carcinoma cell line HepaRG, and MAP1S expression in liver tissue is increased in ovariectomized mouse models. MAP1S expression in the liver tissue of some AIH patients is significantly higher than that in the healthy control group. AIH patients with high MAP1S expression exhibit mitochondrial damage, with significantly reduced levels of cytochrome C and its related proteins. High MAP1S expression in hepatocytes may cause mitochondrial damage. The rate of hepatocyte apoptosis in liver tissue of AIH patients with high MAP1S expression is significantly higher than that in healthy volunteers and AIH liver tissue with low MAP1S expression, suggesting that high MAP1S expression may be associated with hepatocyte death.
[0038] The above descriptions are merely a few embodiments of the present application and do not constitute any form of limitation to the present application. Although the present application discloses the preferred embodiments as above, they are not intended to limit the present application. Any technical personnel familiar with the present profession, without departing from the scope of the technical solution of the present application, using the technical content disclosed above to make slight changes or modifications are equivalent to equivalent implementation cases and fall within the scope of the technical solution.
Claims
1. Use of FSH protein or a substance for detecting FSH protein in the preparation of a tool for diagnosing autoimmune hepatitis, wherein the FSH protein is upregulated in the serum of autoimmune hepatitis patients.
2. The use according to claim 1, characterized in that The concentration of FSH protein in the autoimmune hepatitis serum is ≥58.81 pg / ml.
3. The use according to claim 1, characterized in that The tools include a test kit and a chip.
4. Use of FSH protein or a substance for detecting FSH protein in the preparation of a product for evaluating the therapeutic effect of autoimmune hepatitis or judging the prognosis of autoimmune hepatitis, wherein the FSH protein is downregulated in the serum or liver tissue of autoimmune hepatitis after treatment.
5. The use according to claim 4, characterized in that The tools include a test kit and a chip.
6. A kit for diagnosing autoimmune hepatitis, characterized in that: The kit includes a biomarker detection reagent, which contains chemical magnetic beads and a chemiluminescent substrate; the biomarker is FSH, and the biomarker is used to detect serum samples.