Serum protein marker, kit and detection method for early screening and diagnosis of pan cancer

Abstract

The invention is applicable to the technical field of biomedicine, and provides a serum protein marker, a kit and a detection method for early screening and diagnosis of pan cancer, the serum protein marker is RNASE4 protein, and the amino acid sequence of the serum protein marker is as shown in SEQ ID NO.1. The application finds that the serum concentration of the RNASE4 protein is remarkably related to various cancers for the first time, the RNASE4 protein can be used as an ideal marker for diagnosis of the pan-cancer, and the RNASE4 protein is provided as the pan-cancer marker, the matched kit and the detection method, so that the problems that the existing CA type marker is low in accuracy and sensitivity and easy to interfere are solved; high-specificity and high-sensitivity non-invasive early screening of intestinal cancer, pancreatic cancer, bladder cancer, breast cancer and ovarian cancer is achieved, the area under an ROC curve is larger than or equal to 0.85, detection operation is easy and convenient, and stability is good.

Description

Technical Field

[0001] This invention belongs to the field of biomedical technology, and in particular relates to a serum protein biomarker, reagent kit, and non-diagnostic detection method for early screening and diagnosis of pan-cancer. Background Technology

[0002] As a non-invasive method for tumor detection, liquid biopsy centers on tumor biomarkers. Reliable tumor biomarkers are key factors influencing tumor diagnosis and prognostic assessment, and are closely related to timely tumor prevention and treatment, as well as the evaluation of treatment efficacy. Although the tumor biomarkers used in clinical liquid biopsy currently range from DNA to RNA to proteins, and from untranslated RNA to DNA modifications (such as methylation), the variety is vast. However, apart from a very few that meet clinical requirements, all biomarkers suffer from issues with accuracy, sensitivity, and specificity, or are affected by other physiological and pathological factors. Furthermore, the detection of most tumor biomarkers, especially nucleic acid-based detections, is cumbersome; nucleic acid modification-based detections are not only cumbersome but also susceptible to interference from other factors, and are also expensive.

[0003] Protein biomarkers are relatively stable, but currently used cancer antigen (CA) tumor markers generally suffer from low accuracy and sensitivity, and are susceptible to interference. For example, CEA (carcinoembryonic antigen) is expressed in various cancers, but its diagnostic sensitivity for colorectal cancer and pancreatic cancer is insufficient; CA125 is mainly used for ovarian cancer detection, and its specificity for other cancers is poor. Liquid biopsy, as a non-invasive detection method, urgently needs a broad-spectrum biomarker that can cover multiple cancers. Summary of the Invention

[0004] The purpose of this invention is to provide a serum protein biomarker for early screening and diagnosis of pan-cancer, aiming to solve the problems of low accuracy and sensitivity and susceptibility to interference that are currently used clinical cancer antigen (CA) tumor biomarkers.

[0005] The present invention is implemented as follows: a serum protein biomarker for early screening and diagnosis of pan-cancer, wherein the serum protein biomarker is RNASE4 protein, the amino acid sequence of which is shown in SEQ ID NO.1.

[0006] Furthermore, the concentration of the RNASE4 protein in the serum of patients with colorectal cancer, pancreatic cancer, bladder cancer, breast cancer, and ovarian cancer was significantly higher than that in normal human serum.

[0007] Another objective of this application is to provide a kit comprising the aforementioned serum protein biomarkers for early pan-cancer screening and diagnosis.

[0008] Preferably, the kit further includes: a mouse monoclonal antibody against RNASE4, a rabbit polyclonal antibody against RNASE4, alkaline phosphatase-labeled goat anti-rabbit IgG, RNASE4 standards, and auxiliary reagents.

[0009] Preferably, the auxiliary reagents include blocking solution, sample diluent, washing solution, chromogenic substrate, and stop solution.

[0010] Preferably, the concentration gradient of the RNASE4 standard is 39.1 pg / mL, 78.1 pg / mL, 156.3 pg / mL, 312.5 pg / mL, 625 pg / mL, 1250 pg / mL, and 2500 pg / mL.

[0011] Another objective of this application is to provide a non-diagnostic detection method for serum protein biomarkers used in early pan-cancer screening and diagnosis, comprising:

[0012] The above-mentioned kit was used to detect RNASE4 protein in serum samples;

[0013] The concentration of RNASE4 protein in the serum sample was calculated based on the test results.

[0014] Preferably, the process of detecting RNASE4 protein in serum samples using the above-described kit includes coating, blocking, antigen incubation, antibody incubation, color development, and detection steps, wherein,

[0015] The coating conditions were 4°C overnight, and 100 μL of anti-RNASE4 mouse monoclonal antibody working solution was added to each well.

[0016] The sealing conditions were: incubation at 37°C for 1 hour, with 200 μL of sealing solution added to each well;

[0017] Serum samples were diluted 200-fold with sample diluent, and 100 μL was added to each well and incubated at 37°C for 1.5 hours.

[0018] The antigen incubation conditions were 37°C for 1 hour, and 100 μL of rabbit polyclonal antibody working solution against RNASE4 was added to each well.

[0019] The antibody incubation conditions were 37°C for 1 hour, with 100 μL of alkaline phosphatase-labeled goat anti-rabbit IgG working solution added to each well.

[0020] Preferably, the concentration of RNASE4 protein in the serum sample is calculated using a standard curve, wherein the linear range of the standard curve is 39.1-2500 pg / mL, R 2 ≥0.997.

[0021] This application is the first to discover that serum RNASE4 protein concentration is significantly correlated with multiple cancers, making it an ideal biomarker for pan-cancer diagnosis. By providing RNASE4 protein as a pan-cancer biomarker, along with matching kits and detection methods, this application solves the problems of low accuracy and sensitivity and susceptibility to interference of existing CA-type biomarkers. It achieves high specificity, high sensitivity, and non-invasive early screening for colorectal cancer, pancreatic cancer, bladder cancer, breast cancer, and ovarian cancer, with an area under the ROC curve ≥0.85. The detection operation is simple and has good stability. Attached Figure Description

[0022] Figure 1 Standard curves provided for embodiments of this application Figure 1 ;

[0023] Figure 2 Concentration distribution diagrams of normal human and cancer samples provided for embodiments of this application;

[0024] Figure 3 ROC curve analysis diagrams of normal human and cancer samples provided in the embodiments of this application;

[0025] Figure 4 A comparison chart of R4 ELISA and CEA ELISA provided for embodiments of this application;

[0026] Figure 5 Standard curves provided for embodiments of this application Figure 2 ;

[0027] Figure 6 The R4 ELISA linear quality control chart provided for the embodiments of this application;

[0028] Figure 7 This is a specific quality control chart for R4 ELISA provided in the embodiments of this application. Detailed Implementation

[0029] To make the objectives, technical solutions, and advantages of this invention clearer, the invention will be further described in detail below with reference to the accompanying drawings and embodiments. It should be understood that the specific embodiments described herein are merely illustrative and not intended to limit the invention.

[0030] This application provides a serum protein biomarker for early screening and diagnosis of pan-cancer. The serum protein biomarker is a protein encoded by the RNASE4 gene, and its amino acid sequence is shown in SEQ ID NO.1 (mature protein positions 29-147). The concentration of the RNASE4 protein in the serum of patients with colorectal cancer, pancreatic cancer, bladder cancer, breast cancer, and ovarian cancer is significantly higher than that in normal human serum (P<0.0001).

[0031] RNASE4 protein sequence (SEQ ID NO.1):

[0032] MALQRTHSLLLLLLLTLLGLGLVQPSYGQDGMYQRFLRQHVHPEETGGSDRYCNLMMQRRKMTLYHCKRFNTFIHEDIWNIRSICSTTNIQCKNGKMNCHEGVVKVTDCRDTGSSRAPNCRYRAIASTRRVVIACEGNPQVPVHFDG

[0033] R4 (RNASE4), full name Ribonuclease A family member 4, HGNC ID: HGNC:10047. Also known as RAB1, RNS4. The gene is located on chromosome 14 (human) at 14q11.2, encoding a protein belonging to the pancreatic ribonuclease family molecule 4 (the ribonuclease family contains 13 molecules). It is a secreted protein, highly conserved in mammals, and is involved in angiogenesis and RNA metabolism (94% homology among humans, cattle, mice, and pigs). The human RNASE4 precursor contains 147 amino acids (1-28 are the signal peptide), while the mature protein (29-147) contains approximately 119 amino acids (after signal peptide cleavage). It plays a crucial role in mRNA cleavage and exhibits significant specificity for the 3' side of uridine nucleotides. RNASE4 mRNA is expressed in a variety of cells, especially mucus-secreting cells, enteroendocrine cells, hepatocytes, and blood granulocytes. The main cell types are epithelial cells, endocrine cells, and interstitial cells, with low expression in human brain regions.

[0034] The RNASE4 mRNA sequence is shown in SEQ ID NO.2.

[0035] This application also provides a kit comprising the aforementioned serum protein biomarkers for early pan-cancer screening and diagnosis. Specifically, the kit comprises: a mouse monoclonal antibody against RNASE4 (coating antibody), a rabbit polyclonal antibody against RNASE4 (detection antibody), alkaline phosphatase-labeled goat anti-rabbit IgG (enzyme-labeled secondary antibody), RNASE4 standards (concentration gradients of 39.1 pg / mL, 78.1 pg / mL, 156.3 pg / mL, 312.5 pg / mL, 625 pg / mL, 1250 pg / mL, and 2500 pg / mL), and auxiliary reagents. The auxiliary reagents include blocking buffer, sample diluent, washing buffer, chromogenic substrate, and stop solution.

[0036] In the following specific embodiments of this application, the blocking solution used is 0.5% BSA / PBS solution; the sample diluent used is 0.1% BSA / PBS solution; the washing solution used is 0.05% Tween 20 / PBS; the chromogenic substrate used is 0.05% p-nitrophenyl phosphate solution; and the stop solution used is 3N NaOH solution.

[0037] Blocking buffer: 0.5% BSA / PBS (bovine serum albumin): Weigh 0.25g BSA (bovine serum albumin) and add PBS to a final volume of 50ml. Prepare fresh and use immediately or store at 4°C for three days.

[0038] Standard and sample dilution buffer (0.1% BSA / PBS): Add 0.2g BSA to 80ml PBST buffer, mix well, and then bring the volume up to 200ml with PBS (pH 7.4). Prepare fresh before use.

[0039] Washing buffer (PBST buffer): Pipette 250 μL Tween20 into 500 mL PBS and mix well.

[0040] PNPP-Na2 buffer (pH 9.5, 10 mM / L diethanolamine, 0.5 mM / L MgCl2). First, prepare solution A (0.1 M / L magnesium chloride solution): Weigh 1.015 g of magnesium chloride hexahydrate (MgCl2·6H2O), place it in a 50 ml centrifuge tube, add 40 ml of ultrapure water to dissolve it completely, and then bring the volume to 50 ml. Next, take a clean beaker, add 150 ml of ultrapure water, 1 ml of solution A, and 192 μl of diethanolamine, stir well, adjust the pH of the solution to 9.5 with 1 M HCl (4 ml of 38% concentrated hydrochloric acid diluted to 50 ml with ultrapure water), and then bring the volume to 200 ml with ultrapure water. Filter through a 0.45 μm filter into a sterile glass bottle and store at 4°C for use (within two weeks). This is the PNPP-Na2 buffer.

[0041] Substrate: Take 0.025 g of nitrophenyl phosphate and dilute to 50 ml with PNPP-Na2 buffer. Store at 4°C protected from light and use within one week.

[0042] Termination solution (3N NaOH): Weigh 12g NaOH, dissolve it in 80ml of ultrapure water, and then bring the volume up to 100ml.

[0043] Enzyme-linked immunosorbent assay (ELISA) is a simple and reproducible method for screening tumors by detecting tumor marker proteins in liquids. Commercially available kits include alpha-fetoprotein (AFP) ELISA kits and human epidermal growth factor receptor (EGFR) ELISA kits. Their advantages include high sensitivity and moderate price. However, current kits have drawbacks, such as high specificity only for a single cancer and susceptibility to interference from other factors. Therefore, this application provides a non-diagnostic detection method for serum protein biomarkers for early screening and diagnosis of pan-cancer diseases, comprising the following steps:

[0044] Step S1: Use the kit described above to detect RNASE4 protein in serum samples;

[0045] Step S2: Calculate the concentration of RNASE4 protein in the serum sample based on the test results.

[0046] In step S1, the coating conditions are: overnight at 4°C, with 100 μL of mouse monoclonal antibody working solution against RNASE4 added to each well; the blocking conditions are: incubation at 37°C for 1 hour, with 200 μL of blocking solution added to each well; the serum sample is diluted 200-fold with sample diluent, with 100 μL added to each well and incubated at 37°C for 1.5 hours; the antigen incubation conditions are: incubation at 37°C for 1 hour, with 100 μL of rabbit polyclonal antibody working solution against RNASE4 added to each well; the antibody incubation conditions are: incubation at 37°C for 1 hour, with 100 μL of alkaline phosphatase-labeled goat anti-rabbit IgG working solution added to each well.

[0047] Optionally, coat a 96-well microplate, add 100 μL of mouse monoclonal antibody working solution against RNASE4 to each well, and incubate overnight at 4°C; discard the coating solution, add 200 μL of blocking solution to each well, block at 37°C for 1 hour, wash 4 times with PBST, and add 100 μL of... Serum samples or RNASE4 standards diluted 200-fold were incubated at 37°C for 1.5 hours, followed by washing five times with PBST. 100 μL of rabbit polyclonal antibody working solution against RNASE4 was added, and the sample was incubated at 37°C for 1 hour, followed by washing five times with PBST. 100 μL of alkaline phosphatase-labeled goat anti-rabbit IgG working solution (1:1000 dilution) was added, and the sample was incubated at 37°C for 1 hour, followed by washing five times with PBST. 100 μL of chromogenic substrate was added to each well, and the sample was incubated at 28°C in the dark for 1 hour. 50 μL of stop solution was added, and the OD405 value was measured within 10 minutes. The concentration of RNASE4 protein in the serum sample was calculated based on the standard curve. A positive result was defined as a concentration higher than the cutoff value. The cutoff values ​​were: 150 pg / mL for colorectal cancer, 180 pg / mL for pancreatic cancer, 170 pg / mL for bladder cancer, 160 pg / mL for breast cancer, and 190 pg / mL for ovarian cancer.

[0048] The following detailed description, using specific embodiments, illustrates the serum protein biomarkers, kits, and non-diagnostic detection methods of this application for early screening and diagnosis of pan-cancer. Unless otherwise specified, the experimental methods used in the following embodiments are conventional methods; and the materials and reagents used are commercially available unless otherwise specified.

[0049] Example 1

[0050] The following experimental samples (serum samples) were obtained from Shanghai Xinchao Biotechnology Co., Ltd. There were 13 types of cancer samples: lung cancer, liver cancer, colorectal cancer, prostate cancer, kidney cancer, brain cancer, pancreatic cancer, bladder cancer, esophageal cancer, stomach cancer, breast cancer, ovarian cancer, and thyroid cancer. Except for ovarian cancer samples (56 samples), the remaining samples consisted of 50 cases (including healthy individuals). In this application, we found significant differences between the healthy individuals and five of the cancers (colorectal cancer, pancreatic cancer, bladder cancer, breast cancer, and ovarian cancer), enabling relatively accurate differentiation. The company also provided some information about the samples, including gender, age, tissue type, tumor differentiation degree, and whether the individual had other infectious diseases.

[0051] The kit used in this embodiment consists of:

[0052] Coating antibody: Mouse anti-human RNASE4 monoclonal antibody (Nanjing Genscript Biotech Co., Ltd.), diluted to 1 μg / mL with 0.05M carbonate buffer (pH 9.6);

[0053] Antibody detection: Rabbit anti-human RNASE4 polyclonal antibody (in-house pAb), diluted to 0.5 μg / mL with PBST containing 1% BSA;

[0054] Enzyme-labeled secondary antibody: alkaline phosphatase-labeled goat anti-rabbit IgG (Aicoin Biotechnology Co., Ltd.), diluted 1:1000;

[0055] Standard: Recombinant RNA SE4 protein (Hu Guofu Laboratory, Tufts University, USA), concentration gradient 39.1-2500 pg / mL;

[0056] Auxiliary reagents: blocking solution (0.5% BSA / PBS solution), sample diluent (0.1% BSA / PBS solution), washing solution (0.05% Tween 20 / PBS), chromogenic substrate (0.05% p-nitrophenyl phosphate solution), and stop solution (3N NaOH solution).

[0057] All cancer samples were tested using the same testing method, which included the following steps:

[0058] Step 1: Coat a 96-well microplate, add 100 μL of mouse monoclonal antibody working solution against RNASE4 to each well, and incubate overnight at 4°C;

[0059] Step 2: Discard the coating solution, add 200 μL of blocking solution to each well, block at 37°C for 1 hour, and wash 4 times with PBST;

[0060] Step 3: Add 100 μL of serum sample diluted 200 times or RNASE4 standard, incubate at 37°C for 1.5 hours, and then wash 5 times with PBST;

[0061] Step 4: Add 100 μL of rabbit polyclonal antibody working solution against RNASE4, incubate at 37°C for 1 hour, and wash 5 times with PBST.

[0062] Step 5: Add 100 μL of alkaline phosphatase-labeled goat anti-rabbit IgG working solution (1:1000 dilution), incubate at 37°C for 1 hour, and wash 5 times with PBST;

[0063] Step 6: Add 100 μL of chromogenic substrate to each well, incubate at 28°C in the dark for 1 hour, add 10 μL of stop solution, and detect the OD405 value within 10 minutes;

[0064] Step 7: Calculate the concentration of RNASE4 protein in the serum sample based on the standard curve. When the concentration is higher than the critical value, it is considered positive.

[0065] The standard curve was plotted as follows: The standard concentration was plotted on the horizontal axis (X), and the corresponding OD405 value on the vertical axis (Y). A linear regression was used to fit the standard curve, and the trend line equation was calculated to ensure R0. 2 ≥0.997.

[0066] Sample concentration calculation: Based on the sample OD405 value, substitute it into the standard curve equation to calculate the original concentration, and then multiply it by the dilution factor of 9200 times to obtain the actual concentration of RNASE4 protein in serum.

[0067] Results Interpretation: The cutoff value for each cancer was determined based on the ROC curve; a sample concentration ≥ the cutoff value was considered positive, and < the cutoff value was considered negative.

[0068] Standard preparation:

[0069] Take one RNASE4 standard with a concentration of 2.5 ng / uL, dispensed in 2 μL tubes, and stored at -80℃. Serially dilute the standard with standard diluent to 2500 pg / mL, 1250 pg / mL, 625 pg / mL, 312.5 pg / mL, 156.3 pg / mL, 78.1 pg / mL, 39.1 pg / mL, and 0 pg / mL.

[0070] Standard curve determination: Prepare standard solutions of varying concentrations as described above. Add 100 μL / well to a 96-well microplate, performing three replicates for each concentration gradient. Follow the experimental protocol to obtain the OD405 values ​​for each concentration gradient. Import the data into an Excel spreadsheet, calculate the mean, background-reduced value, and SD value for each group, and plot a line graph using the background-reduced value and corresponding concentration values. The horizontal axis represents sample concentration, and the vertical axis represents absorbance. Set the trend line equation and R-squared to obtain the standard curve, as shown below. Figure 1 As shown. Representative standard curve: linear range 39.1-2500 pg / ml, R0 2 =0.997.

[0071] The sample data (OD value, trend line equation Y value) obtained from the above experimental operations were converted into concentration values. The concentration value was obtained by multiplying the trend line equation X value of the standard curve by the dilution factor (200-fold dilution). The data were grouped by cancer type and imported into a Prim's table. The distribution of results for the five cancer types and normal human samples is shown in the figure below. Ordinary one-way ANOVA, Kruskal-Wallis test, and Brown-Forsythe and Welch ANOVA were performed on the table data. All tests were consistent and confirmed that the differences between groups were extremely significant (P < 0.0001).

[0072] The analysis of variance showed the following: F-value = 8.428: the variation between groups was significantly greater than the variation within groups, indicating that at least one group's mean was significantly different from the other groups. P-value < 0.0001 (****): the result was highly significant, rejecting the null hypothesis that there was no difference in the mean between groups. R 2 =0.1273: The treatment variable explained 12.73% of the total variation, which is a moderately low effect size, and may require further analysis with other factors. Conclusion: There was a significant difference in the means between groups (P<0.05).

[0073] Based on the P-values ​​obtained above for cancer samples compared to normal individuals, the results are summarized in Table 1 below. The results show that there are significant differences between normal human samples and these five types of cancer samples.

[0074] Table 1

[0075]

[0076] Furthermore, its sensitivity and specificity were analyzed using ROC curves, such as... Figure 3 As shown in Table 2 below, the area under the ROC curve for the normal human sample and the five cancer samples all reached above 0.85, indicating good discrimination and the ability to distinguish cancer from normal people relatively accurately.

[0077] Table 2

[0078]

[0079]

[0080] Example 2: Comparison of R4 ELISA with CEA, a commonly used clinical tumor marker

[0081] Normal human samples and samples from various cancers were tested using R4 ELISA and CEA ELISA, respectively. The distribution of the sample test results is as follows. Figure 4 Unmatched t-test analysis (also known as independent samples t-test, a statistical method used to compare whether there is a significant difference between the means of two independent samples. When the variances between sample groups are unequal, a Welch-corrected t-test can be used to improve the robustness of the analysis) was performed to analyze the differences between cancer samples and normal samples. The results showed that there was a significant difference between normal individuals and cancer patients in the R4 ELISA; in the CEA ELISA, there was a significant difference between normal individuals and liver cancer patients, and no or insignificant differences with other cancers. Compared with CEA testing, the R4 ELISA can better distinguish between normal individuals and cancer patients.

[0082] The samples were sourced from Shanghai Xinchao Biotechnology Co., Ltd., and the sample information is shown in the table below:

[0083] Sample types normal people liver cancer pancreatic cancer lung cancer Kidney cancer Bladder cancer Stomach cancer Colorectal cancer NT Number of examples 39 10 11 11 8 4 10 19 4

[0084] Example 3: Quality Control of R4 ELISA Detection

[0085] The R4 ELISA standard curve was obtained, and the results are as follows: Figure 5 As shown. The R-squared value is 0.9983, and the trend line equation is: Y = 0.0008656 * X + 0.2663. The lowest detection limit LLD = Mean + 2 * SD = 0.283699.

[0086] R4 ELISA quality control recovery rate:

[0087] Recovery rate is presented as a percentage. When the recovery rate is between 80% and 120%, it indicates that the test method meets the standard.

[0088] The serum sample to be tested was diluted 600-fold with sample diluent to serve as the working diluent for diluting the standards. A standard protein with a known concentration of 900 ng / ml was first diluted to 10 ng / ml with the working diluent (2u R4 frozen protein + 178ul working diluent), and then diluted to two concentration groups: 600 pg / ml and 1000 pg / ml. 100ul / well, 12 replicates per concentration, totaling 1200ul. The assay was repeated 5 times. Two 1.5ml EP tubes were labeled B1 (600 pg / ml) and B2 (1000 pg / ml) for preparation, as shown in the table below:

[0089] serial number Final concentration pg / ml Additive 1 Volume ul Additive 2 Volume ul B1 600 Working diluent 1222 10ng / ml standard 78 B2 1000 Working diluent 1170 10ng / ml standard 130

[0090] The test results are shown in Table 3 below, indicating that the method has a good recovery rate.

[0091] Table 3

[0092]

[0093] R4 ELISA Quality Control - Intra-plate and Inter-plate Precision:

[0094] Interplate: The repeatability of known concentration samples across different plates. Interplate %CV of less than 15% is considered good repeatability.

[0095] Intra-plate: The repeatability of samples with known concentrations within the same plate. Intra-plate repeatability %CV of less than 10% is considered good repeatability.

[0096] Three samples were randomly selected for intra- and inter-plate precision testing: Sample 1 was a normal individual (sample number 42); Sample 2 was a liver cancer patient (sample number B2); and Sample 3 was a lung cancer patient (sample number B34). For intra-plate testing, each sample was repeated 12 times; for inter-plate testing, each sample was repeated 5 times. As shown in Table 4, the CV (%) obtained from the experiment were all below 10%, indicating good reproducibility of the method.

[0097] Table 4

[0098]

[0099] R4 ELISA Quality Control - Linearity:

[0100] Two samples were selected for linear detection, with the X-axis representing the dilution factor and the Y-axis representing the absorbance value. For example... Figure 6 As shown, sample 1 corresponds to the gray line, and sample 2 corresponds to the black line. Circles represent a spiked concentration of 350 pg / ml, and squares represent a spiked concentration of 500 pg / ml. Both samples were tested using two different spiked concentrations, with three replicates performed at dilutions of 3, 5, and 9 times. The results showed good linearity.

[0101] R4 ELISA Quality Control - Specificity:

[0102] The extent to which the antibody reacts with non-target proteins. An acceptable result is cross-reactivity of less than 5%. Members of highly homologous protein families are typically used as test molecules. In this method, R4 (from the same family), R5 (sharing a promoter and five exons) is used to evaluate cross-reactivity. Results are as follows: Figure 7 As shown, the antibody used in this ELISA does not cross-react with R5.

[0103] The embodiments described above are merely illustrative of several implementations of the present invention, and while the descriptions are specific and detailed, they should not be construed as limiting the scope of the present invention. It should be noted that those skilled in the art can make various modifications and improvements without departing from the concept of the present invention, and these modifications and improvements all fall within the scope of protection of the present invention. Therefore, the scope of protection of this patent should be determined by the appended claims.

[0104] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. Any modifications, equivalent substitutions, and improvements made within the spirit and principles of the present invention should be included within the protection scope of the present invention.

Claims

1. A serum protein biomarker for early screening and diagnosis of pan-cancer, characterized in that, The serum protein marker is RNASE4 protein, whose amino acid sequence is shown in SEQ ID NO.

1.

2. The serum protein biomarker for early screening and diagnosis of pan-cancer according to claim 1, characterized in that, The concentration of RNASE4 protein in the serum of patients with colorectal cancer, pancreatic cancer, bladder cancer, breast cancer, and ovarian cancer was significantly higher than that in normal individuals.

3. A reagent kit, characterized in that, The kit includes serum protein biomarkers for early pan-cancer screening and diagnosis as described in any one of claims 1-2.

4. The reagent kit according to claim 3, characterized in that, include: Mouse monoclonal antibody against RNASE4, rabbit polyclonal antibody against RNASE4, alkaline phosphatase-labeled goat anti-rabbit IgG, RNASE4 standard, and auxiliary reagents.

5. The reagent kit according to claim 4, characterized in that, The auxiliary reagents include blocking solution, sample diluent, washing solution, chromogenic substrate, and stop solution.

6. The reagent kit according to claim 4, characterized in that, The concentration gradients of the RNASE4 standard were 39.1 pg / mL, 78.1 pg / mL, 156.3 pg / mL, 312.5 pg / mL, 625 pg / mL, 1250 pg / mL, and 2500 pg / mL.

7. A non-diagnostic detection method for serum protein biomarkers used in early screening and diagnosis of pan-cancer, characterized in that, include: The RNASE4 protein in serum samples was detected using the kit described in any one of claims 3-6; The concentration of RNASE4 protein in the serum sample was calculated based on the test results.

8. The method for detecting serum protein biomarkers for early screening and diagnosis of pan-cancer according to claim 7, characterized in that, The process of detecting RNASE4 protein in serum samples using the kit described in any one of claims 3-6 includes coating, blocking, antigen incubation, antibody incubation, color development, and detection steps, wherein... The coating conditions were 4°C overnight, and 100 μL of anti-RNASE4 mouse monoclonal antibody working solution was added to each well. The sealing conditions were: incubation at 37°C for 1 hour, with 200 μL of sealing solution added to each well; Serum samples were diluted 200-fold with sample diluent, and 100 μL was added to each well and incubated at 37°C for 1.5 hours. The antigen incubation conditions were 37°C for 1 hour, and 100 μL of rabbit polyclonal antibody working solution against RNASE4 was added to each well. The antibody incubation conditions were 37°C for 1 hour, with 100 μL of alkaline phosphatase-labeled goat anti-rabbit IgG working solution added to each well.

9. The method for detecting serum protein biomarkers for early screening and diagnosis of pan-cancer according to claim 7, characterized in that, The concentration of RNASE4 protein in serum samples was calculated using a standard curve, the linear range of which was 39.1-2500 pg / mL. 2 ≥0.997.

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