Serological diagnosis model containing PRO-C3 and application of serological diagnosis model in preparation of detection reagent for fibrosis degree of CHB patient

Abstract

The invention discloses a serological diagnosis model containing PRO-C3 and application of the serological diagnosis model in preparation of a detection reagent for the fibrosis degree of a CHB patient. The serum combined detection method for PRO-C3, PLT and ALP provided by the invention is a sensitive and efficient method for detecting the fibrosis degree of a CHB patient. Therefore, the PRO-C3 combined with the PLT and the ALP can be used as a novel serological diagnosis model for diagnosing the fibrosis degree of the CHB patient. The serological diagnosis model containing PRO-C3 can be used for preparing a detection reagent and / or a kit for detecting the fibrosis degree of a CHB patient by detecting the fibrosis degree of a quantifiable index liver tissue and sensitively and efficiently detecting the remarkable fibrosis, so that the serological diagnosis model containing PRO-C3 can be used for preparing the detection reagent and / or the kit for detecting the fibrosis degree of the CHB patient.

Description

Technical Field

[0001] The present invention belongs to the technical field of biomedicine, and in particular relates to a serological diagnostic model containing PRO-C3 and an application thereof in preparing a detection reagent for the degree of fibrosis in CHB patients. Background Art

[0002] Chronic liver disease (CLD) is a major global health burden, affecting more than 800 million people and causing approximately 2 million deaths each year (Asrani SK, Devarbhavi H, Eaton J, et al. Burden of liver diseases in the world. J Hepatol. 2019, 70, 151-71). Liver fibrosis caused by viral or metabolic liver diseases is a major challenge to global health. Liver fibrosis is associated with CLD progression and is a key factor in CLD prognosis and the risk of developing hepatocellular carcinoma (HCC). Therefore, early diagnosis and treatment of liver fibrosis is key to reducing CLD-related complications and mortality.

[0003] Liver biopsy is the gold standard for diagnosing liver fibrosis, but it has not been widely used in clinical practice due to its invasiveness, non-reproducibility, and sampling errors. Recently, non-invasive tests (NITs) for liver fibrosis have been shown to be a potential alternative to liver biopsy. Serum models represented by the fibrosis index based on four factors (FIB-4) and the aspartate aminotransferase-to-platelet ratio index (APRI) have only shown moderate diagnostic efficacy in the diagnosis of chronic hepatitis B (CHB)-related cirrhosis and significant fibrosis. Therefore, it is urgent to develop a simple, easy-to-use, and highly accurate non-invasive test for liver fibrosis for diagnosing the severity of liver fibrosis.

[0004] Platelets (PLT) are the smallest blood cells in the blood. They are produced by megakaryocytes in the hematopoietic tissue of the bone marrow, and their production is regulated by thrombopoietin. Studies have shown that PLT in patients with liver fibrosis is significantly reduced, and the average platelet volume and distribution width are significantly increased. As the degree of liver fibrosis increases, the number of PLT gradually decreases (Pang Qing et al., Relationship between Platelets and Fibrosis [J], Journal of Practical Hepatology, 2011, 14 (4): 315-317). PLT is used in the conventional index combination model for diagnosing liver fibrosis, and the most common one is APRI.

[0005] Alkaline phosphatase (ALP) is a membrane-bound glycoprotein that catalyzes the hydrolysis of monophosphates in an alkaline environment and can be released into the intestine through bile. When bile is released by obstruction and the bile duct capillary pressure is high, a large amount of ALP can be induced. ALP enters the blood through the lymph and liver sinusoids, leading to elevated serum ALP. Studies have shown that it is an independent factor for predicting significant fibrosis in CHB patients (HU J, ZHANG X, GU J, et al. Serum alkalinephosphatase levels as a simple and useful test in screening for significant fibrosis in treatment-naive patients with hepatitis B e-antigen negative chronic hepatitis B [J]. Eur J Gastroenterol Hepatol, 2019, 31 (7): 817-23.).

[0006] N-terminal type III collagen propeptide (PRO-C3) is the N-terminal propeptide of type III collagen and is a serological biomarker for detecting the formation of type III collagen.

[0007] At present, there are no reports on using PRO-C3 or PRO-C3 combined with clinically relevant indicators as a serological diagnostic model to detect the degree of liver fibrosis in CHB patients. Summary of the invention

[0008] Purpose of the invention: The purpose of the present invention is to address the deficiencies of the prior art and to provide a serological diagnostic model containing PRO-C3 and its application in the preparation of a detection reagent for the degree of fibrosis in CHB patients.

[0009] The present invention has found through research that PRO-C3 combined with PLT and ALP (referred to as PPAscore) is more effective than PRO-C3, PLT, ALP, FIB-4 and APRI in diagnosing significant fibrosis in CHB patients, and the misdiagnosis rate is lower than PRO-C3, PLT, ALP, FIB-4 and APRI, and the missed diagnosis rate is lower than PLT, ALP, FIB-4 and APRI.

[0010] Technical solution: The purpose of the present invention is achieved through the following technical solution:

[0011] The present invention provides a serological diagnosis model containing PRO-C3, wherein the serological diagnosis model is PRO-C3 combined with PLT and ALP.

[0012] Serological markers (including direct markers and indirect markers) are combined into serological diagnostic models to improve the accuracy of fibrosis diagnosis, but there are great differences in the accuracy of these models in diagnosing liver fibrosis, and even opposite results can be obtained. The present invention has been found through research that PRO-C3 combined with PLT and ALP (referred to as PPAscore) is more effective than PRO-C3, PLT, ALP, FIB-4 and APRI in diagnosing significant fibrosis in CHB patients, and the misdiagnosis rate is lower than PRO-C3, PLT, ALP, FIB-4 and APRI, and the missed diagnosis rate is lower than PLT, ALP, FIB-4 and APRI. Therefore, PRO-C3 combined with PLT and ALP can be used as a new serological diagnostic model for diagnosing the degree of fibrosis in CHB patients.

[0013] The present invention also provides the use of the serological diagnostic model containing PRO-C3 in preparing a detection reagent and / or a kit for detecting the degree of fibrosis in CHB patients.

[0014] The detection reagent and / or kit for the degree of fibrosis in CHB patients uses PRO-C3 combined with PLT and ALP as detection targets.

[0015] The CHB patient fibrosis degree detection reagent comprises the sequence shown in SEQ ID NO.1:

[0016] SEQ ID NO. 1: 145'-CPTGPQNYSP-'153.

[0017] The amino acid sequence of the α1 chain of type III collagen is shown in SEQ ID NO.2:

[0018] SEQ ID NO.2:

[0019] MMSFVQKGSWLLLALLHPTIILAQQEAVEGGCSHLGQSYADRDVWKPEPCQICVCDSGSVLCDDIICDDQELDCPNPEIPFGECCAVCPQPPTAPTRPPNGQGPQGPKGDPGPPGIPGRNGDPGIPGQPGSPGSPGPPGICES C PTGPQNYSP

[0020] The detection reagent and / or kit for the degree of fibrosis in CHB patients can detect significant fibrosis of liver tissue (Scheuer score S≥2).

[0021] The detection reagent for the degree of fibrosis in CHB patients includes an enzyme-linked immunosorbent assay reagent.

[0022] The present invention also provides a CHB patient fibrosis degree detection reagent and / or kit, which includes a reagent for detecting PRO-C3 combined with PLT and ALP protein expression levels.

[0023] Beneficial effects:

[0024] The serum combined detection of PRO-C3, PLT and ALP provided by the present invention is a sensitive and efficient method for detecting the degree of fibrosis in CHB patients. It is faster, more convenient, reliable, has clinical guidance significance, and is worth promoting. Therefore, PRO-C3 combined with PLT and ALP can be used as a new serological diagnostic model for diagnosing the degree of fibrosis in CHB patients. The present invention achieves sensitive and efficient detection of significant fibrosis by detecting the degree of fibrosis of liver tissue, which is a quantifiable indicator. Therefore, the serological diagnostic model containing PRO-C3 can be used to prepare a detection reagent and / or a kit for the degree of fibrosis in CHB patients, which is of great value for the detection of the degree of fibrosis in CHB patients, efficacy evaluation, drug development, and research on potential pathogenesis. BRIEF DESCRIPTION OF THE DRAWINGS

[0025] Figure 1 Schematic diagram of the preparation of PRO-C3 standard (AJ);

[0026] Figure 2 This is a comparison of the effectiveness of different serological indicators in diagnosing significant fibrosis in patients with chronic hepatitis B. DETAILED DESCRIPTION

[0027] The technical solution of the present invention is described in detail below through specific embodiments, but the protection scope of the present invention is not limited to the embodiments.

[0028] Although specific terminology is used below for purposes of clarity, these terms are not intended to define or limit the scope of the invention.

[0029] As used herein, the term "liver tissue fibrosis". Liver pathology uses the Scheuer scoring system to evaluate liver tissue fibrosis. The abbreviation for liver tissue fibrosis grade is S. The following Table 1 shows the corresponding pathological diagnostic criteria for specific grades of 0-4. Significant fibrosis is defined as fibrosis stage S2-4; non-significant fibrosis is defined as fibrosis stage S0-1.

[0030] Table 1 Scheuer scoring system

[0031]

[0032] Example 1 Experimental research subjects and specimen collection

[0033] 1. Research subjects

[0034] The subjects of this experiment were 324 CHB patients who underwent liver biopsy from Drum Tower Hospital Affiliated to Nanjing University Medical School and Suzhou Fifth People's Hospital. They were divided into two groups: S0-1 (non-significant fibrosis) and S2-4 (significant fibrosis).

[0035] 2. Specimen collection and storage

[0036] Blood was collected by venipuncture, taking care to avoid hemolysis. Serum was separated from cells within 3 hours of blood collection for PRO-C3 testing. The collected serum samples were immediately frozen and stored below -18°C. (Stored at temperatures below -80°C for up to 12 months). After thawing, serum samples can be stored at or below 20°C for 48 hours and are stable for up to 3 freeze-thaw cycles.

[0037] Example 2 Expression levels of serum PRO-C3, PLT, ALP, ALT, AST, FIB-4, and APRI in different degrees of liver fibrosis

[0038] 1. ELISA method to detect serum PRO-C3 levels

[0039] The following steps were prepared according to the instructions of the PRO-C3 ELISA kit (Nordic Bioscience).

[0040] The kit contains: coating buffer, biotinylated antigen, incubation buffer, controls 1 and 2, horseradish peroxidase-conjugated antibody, washing buffer, standards, and streptavidin-coated microtiter plates.

[0041] (1) Reagent and sample preparation

[0042] Biotinylated antigen solution: dilute the biotinylated antigen with coating buffer: 1:100;

[0043] Serum samples: Dilute serum samples 1:1 with incubation buffer;

[0044] Reference substances: Dissolve reference substances 1 and 2 in 0.5 mL of distilled water.

[0045] Horseradish peroxidase-conjugated antibody: Dilute horseradish peroxidase-conjugated antibody (HRP-Ab) in incubation buffer: 1:100;

[0046] Washing solution: dilute the washing solution with distilled water: 1:50;

[0047] Standards: Prepare the standard curve AJ by diluting the standard with incubation buffer 1:1.5. The standard (AJ) is prepared by diluting the incubation buffer of each plate 1.5 times (see Figure 1 ).

[0048] (2) Detection steps

[0049] ① Add 100 μL of biotinylated antigen solution to each well of the streptavidin-coated microtiter plate to be tested;

[0050] ②Incubate at 18-22°C with shaking for 30 min (300 rpm);

[0051] ③ Wash 5 times with 300 μL diluted washing solution;

[0052] ④ Add 20 μL of standard, serum sample collected in Example 1 and control into the double-to-be-measured wells;

[0053] ⑤ Add 100 μL of diluted HRP-Ab to the detection well;

[0054] ⑥ Incubate at 2-8°C with shaking (300 rpm) for 20 hours;

[0055] ⑦ Wash 5 times with 300 μL washing solution;

[0056] ⑧ Add 100 μL TMB colorimetric solution to the detection wells and incubate at 18-22°C with shaking (300 rpm) in the dark for 15 min;

[0057] ⑨ Add 100 μL of 0.18 mol / L sulfuric acid stop solution into the detection well;

[0058] ⑩ Detection using an ELISA reader: Use 650nm as a reference and read the absorbance at 450nm.

[0059] (3) Results analysis

[0060] Use the parameter fitting model obtained in step (2) to construct a standard curve with the average absorbance of the eight standards AJ (ordinate) and the corresponding PRO-C3 concentration (abscissa). The PRO-C3 analyte concentration of the control group and each sample was determined by interpolation. The concentration is expressed as ng / mL value. For conversion to nM, use the following equation: nM = (ng / mL) / 0.96kDa.

[0061] 2. PLT, aspartate aminotransferase (AST), alanine aminotransferase (ALT) and ALP testing

[0062] The detection methods of PLT, AST, ALT and ALP refer to the health industry standards of the People's Republic of China (standard numbers: WS / T244-2005, WS / T 404.1-2012).

[0063] 3. Baseline data of research subjects

[0064] A total of 324 patients were included in this study, including 167 patients with significant fibrosis (S2-4 group) and 157 patients with non-significant fibrosis (S0-1 group). The median age of the S0-1 group was 40 years old, including 100 males and 57 females; the median age of the S2-4 group was 38 years old, including 106 males and 61 females. There was no statistical difference in gender and age between the two groups (P = 0.60 and 0.97, respectively), see Table 2.

[0065] Table 2 General information of the subjects

[0066]

[0067] Note: PLT: platelet; ALP: alkaline phosphatase; ALT: alanine aminotransferase; AST: aspartate aminotransferase; PRO-C3: N-terminal type III collagen propeptide; FIB-4: fibrosis 4 index; APRI: aspartate aminotransferase to platelet ratio index.

[0068] The present invention uses SPSS19 and MedCalc 19.3 for statistical analysis and GraphPad Prism 9 for drawing. The description method of continuous variables is selected according to their distribution characteristics. The quantitative data that approximately obey the normal distribution are expressed by mean ± standard deviation, and the quantitative data with skewed distribution are described by median and interquartile range. The chi-square test is used for comparison between qualitative data groups, and the Mann-Whitny U test is used for comparison between two independent samples. P<0.05 is statistically significant.

[0069] FIB-4 and APRI calculation formula:

[0070] FIB-4 = (age × AST) ÷ [PLT × (square root of ALT)].

[0071] APRI = AST ÷ (upper limit of normal value of AST) × 100 ÷ PLT.

[0072] As shown in Table 2, the levels of PRO-C3 in the S0-1 and S2-4 groups were 16.3 (11.4, 30.7) and 41.6 (22.5, 60.0), respectively; the levels of PRO-C3, ALP, ALT, AST, FIB-4, and APRI in S2-4 patients were significantly higher than those in S0-1 patients (P<0.05), and the PLT level in S2-4 patients was lower than that in S0-1 patients (P<0.001).

[0073] The analysis results showed that serum PRO-C3, ALP, ALT, AST, FIB-4, and APRI levels increased with the severity of fibrosis, while PLT showed the opposite trend.

[0074] Example 3 Comparison of efficacy in diagnosing the degree of fibrosis

[0075] The present invention screened a combined model of PRO-C3 combined with common clinical indicators (PLT, ALT, ALP, AST, albumin, etc.) for diagnosing significant fibrosis in CHB patients through multivariate logistic regression analysis, and then established a PPAscore (PRO-C3 combined with PLT and ALP model) combined model.

[0076] The established combined model uses the following formula to calculate the predicted value of PPAscore (PRO-C3 combined with PLT and ALP model):

[0077] PPAscore=-1.295+0.042(PRO-C3)-0.007PLT+0.021ALP;

[0078] Among them, PRO-C3 is the concentration value obtained by ELISA to detect serum PRO-C3 level, unit (ng / mL); PLT is the platelet count value measured by industry standard WS / T 244-2005, unit (10 9 / L); ALP is the alkaline phosphatase value measured using the industry standard WS / T404.1-2012, unit (U / L).

[0079] The present invention establishes a receiver operating characteristic (ROC) curve, compares the evaluation values ​​of different indicators for liver fibrosis according to the area under the curve (AUC), and uses the Delong test to compare the differences between the AUCs of different indicators.

[0080] ROC curve and DeLong test were used to evaluate the diagnostic value of 6 serological indicators, including PPAscore, PRO-C3, FIB-4, APRI, PLT and ALP, in identifying significant fibrosis associated with CHB. P<0.05 was statistically significant.

[0081] The results are shown in Tables 3-4 and Figure 2 .

[0082] From Table 3-4, Figure 2 It can be seen that: ROC curve and DeLong test analysis show that the optimal cut-off value of PPAscore is 0.56. Therefore, when PPAscore>0.56, the degree of fibrosis of the subject can be judged as significant fibrosis, and PPAscore≤0.56 is non-significant fibrosis. PPAscore [AUC (95% CI): 0.81 (0.76-0.85)] was superior to PRO-C3 [AUC (95% CI): 0.76 (0.71-0.81)], FIB-4 [AUC (95% CI): 0.62 (0.56-0.67)], APRI [AUC (95% CI): 0.68 (0.63-0.73)], ALP [AUC (95% CI): 0.68 (0.62-0.73)] and PLT [AUC (95% CI): 0.64 (0.59-0.70)] in diagnosing significant fibrosis, P < 0.01. In addition, the positive predictive value of PPAscore in predicting significant fibrosis in CHB patients was higher than that of PRO-C3, PLT, ALP, FIB-4 and APRI, and the negative predictive value was similar to that of PRO-C3 alone, but higher than that of PLT, ALP, FIB-4 and APRI.

[0083] Table 3 Comparison of the value of different serological indices in diagnosing significant fibrosis

[0084]

[0085] Note: PRO-C3: N-terminal propeptide of type III collagen; FIB-4: fibrosis 4 index; APRI: aspartate aminotransferase to platelet ratio index; ALP: alkaline phosphatase; PLT: platelet.

[0086] Table 4 Comparison of AUCs of different indicators

[0087]

[0088] Note: PRO-C3: N-terminal propeptide of type III collagen; FIB-4: fibrosis 4 index; APRI: aspartate aminotransferase to platelet ratio index; ALP: alkaline phosphatase; PLT: platelet.

[0089] In summary, PRO-C3 combined with PLT and ALP (PPAscore for short) is more effective than PRO-C3, PLT, ALP, FIB-4 and APRI in diagnosing significant fibrosis in CHB patients, with lower misdiagnosis rates and lower missed diagnosis rates than PRO-C3, PLT, ALP, FIB-4 and APRI.

[0090] As described above, although the present invention has been shown and described with reference to specific preferred embodiments, it should not be construed as limiting the present invention itself. Various changes in form and details may be made without departing from the spirit and scope of the present invention as defined in the appended claims.

Claims

1. A serological diagnostic model containing PRO-C3, characterized in that: The serological diagnosis model is PRO-C3 combined with PLT and ALP.

2. Use of the serological diagnostic model containing PRO-C3 according to claim 1 in the preparation of a detection reagent and / or kit for the degree of fibrosis in CHB patients.

3. The use according to claim 2, characterized in that: The detection reagent and / or kit for the degree of fibrosis in CHB patients uses PRO-C3 combined with PLT and ALP as detection targets.

4. The use according to claim 3, characterized in that: The CHB patient fibrosis degree detection reagent comprises the sequence shown in SEQ ID NO.1: SEQ ID NO. 1: 145'-CPTGPQNYSP-'153.

5. The use according to claim 2, characterized in that: The CHB patient fibrosis degree detection reagent and / or kit can realize the detection of significant fibrosis of liver tissue.

6. The use according to any one of claims 2 to 4, characterized in that: The detection reagent for the degree of fibrosis in CHB patients includes an enzyme-linked immunosorbent assay reagent.

7. A reagent and / or kit for detecting the degree of fibrosis in CHB patients, characterized in that: It includes reagents for detecting PRO-C3 combined with PLT and ALP protein expression levels.

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