Diagnostic reagent and method for early discovery of breast nodules based on serum GRP detection

Abstract

The invention provides a diagnostic reagent and method for early discovery of breast nodules based on serum GRP detection. The reagent comprises a GRP standard substance, a GRP primary antibody, a buffer solution, a developing solution and a stop solution, the GRP level in serum is detected through enzyme-linked immunosorbent assay (ELISA), and the reagent is used for screening breast benign nodules and early breast cancer. By collecting a serum sample, detecting the GRP concentration and comparing the GRP concentration with a healthy control group, existence of breast nodules is judged, and the kit has high sensitivity and specificity, is particularly suitable for early-stage cases without obvious anomalies in imaging, and provides an efficient tool for non-invasive diagnosis of breast diseases. The diagnostic reagent is easy and convenient to operate, short in detection time and suitable for clinical large-scale screening, the method is combined with correlation analysis of the GRP level and the nodule volume, benign nodules, early breast cancer and advanced breast cancer can be effectively distinguished, a basis is provided for disease prognosis evaluation, the accuracy of early discovery of breast nodules is remarkably improved, and the clinical application prospect is wide. And an important reference is provided for formulating a treatment scheme.

Description

Technical Field

[0001] This invention relates to the field of disease detection reagent technology, specifically to a diagnostic reagent and method for early detection of breast nodules based on serum GRP detection. Background Technology

[0002] Solid breast nodules are a relatively common type of breast disease. Although traditional diagnostic methods, such as palpation, color Doppler ultrasound, and X-ray examination, can provide some diagnostic evidence, they have limitations in terms of sensitivity, specificity, and early detection. Especially when the nodules are small or located in concealed areas, traditional diagnostic methods often fail to detect them promptly and accurately determine their benign or malignant nature. Therefore, early detection of breast nodules and accurate differentiation between benign and malignant nodules are crucial for developing treatment plans and predicting disease progression.

[0003] Gastrin-releasing peptide (GRP) is a 27-amino acid neuropeptide widely found in the central nervous system, gastrointestinal tract, and lung tissue. This peptide participates in regulating various physiological processes, including gastric acid secretion, gastrointestinal motility, cell proliferation, and nerve signal transmission, by binding to the BB2 receptor on the cell membrane. It is also closely related to the development of various diseases, including tumors and inflammation. The autocrine hypothesis of tumor growth posits that a cell can simultaneously produce a growth factor and its corresponding homologous receptor; the interaction between these factors and receptors promotes cell proliferation. Gastrin-releasing peptide (GRP) is considered a typical autocrine growth factor, with initial evidence coming from the detection of GRP and its homologous receptor in small cell lung cancer (SCLC) and the anti-proliferative effects exhibited by GRP antibodies. However, GRP has also been shown to be a potent mitogen in several other cancers, including colon cancer, pancreatic cancer, prostate cancer, and breast cancer, and its involvement in mitotic signaling pathways has been extensively studied.

[0004] Recent studies show that GRP not only has autocrine mitotic effects but also paracrine and endocrine functions, and can act as a morphogenetic agent and angiogenetic agent. As a non-invasive biomarker, GRP shows great potential in early cancer screening, differential diagnosis, and prognostic prediction. Nevertheless, current research on the expression characteristics and clinical value of GRP in the circulatory system (e.g., serum) of patients with breast nodules is insufficient, and there is a lack of standardized detection methods based on serum GRP, making it difficult to meet the clinical need for early and accurate diagnosis. Summary of the Invention

[0005] The purpose of this section is to outline some aspects of embodiments of the present invention and to briefly describe some preferred embodiments. Simplifications or omissions may be made in this section, as well as in the abstract and title of this application, to avoid obscuring the purpose of these documents; however, such simplifications or omissions should not be construed as limiting the scope of the invention.

[0006] In view of the problems existing in the prior art, the inventors have proposed the present invention.

[0007] Therefore, the purpose of this invention is to overcome the shortcomings of the prior art and provide a diagnostic reagent and method for early detection of breast nodules based on serum GRP detection.

[0008] To solve the above-mentioned technical problems, the present invention provides the following technical solution: a diagnostic reagent for early detection of breast nodules based on serum GRP detection, the reagent comprising GRP standard, GRP primary antibody, buffer, chromogenic solution and stop solution, wherein the amino acid sequence of GRP is:

[0009] >sp|P07492|GRP_HUMANGastrin-releasingpeptideOS=HomosapiensOX=9606GN=GRPPE=1SV=2

[0010] MRGRELPLVLLALVLCLAPRGRAVLPAGGGTVLTKMYPRGNHWAVGH

[0011] LMGKKSTGESSS

[0012] VSERGSLKQQLREYIRWEEAARNLLGLIEAKENRNHQPPQPKALGNQQPSWDSEDSSNFK

[0013] DVGSKGKVGRLSAPGSQREGRNPQLNQQ;

[0014] The reagent is used to detect the level of GRP in the serum of the subject by enzyme-linked immunosorbent assay (ELISA) and is used for the early diagnosis or prognostic evaluation of benign breast nodules or early breast cancer.

[0015] As a preferred embodiment of the diagnostic reagent for early detection of breast nodules based on serum GRP detection according to the present invention, wherein: the concentration range of the GRP standard is 0-800 mg / L, and the purity is not less than 95%; the GRP primary antibody is rabbit anti-human GRP polyclonal antibody, and the dilution ratio is 1:1000-1:5000; the buffer is phosphate buffered saline (PBS) with pH 7.2-7.4; the chromogenic solution is tetramethyl-forbidden TMB; and the stop solution is 2M sulfuric acid solution.

[0016] As a preferred embodiment of the diagnostic reagent for early detection of breast nodules based on serum GRP detection described in this invention, the reagent is used to detect GRP from the serum of the subject. The serum sample is stored at -80°C after collection, and the sample volume is 50-100 μL. Repeated freeze-thaw cycles are avoided to ensure detection accuracy.

[0017] As a preferred embodiment of the diagnostic reagent for early detection of breast nodules based on serum GRP detection according to the present invention, the reagent is used to prepare a kit for diagnosing breast nodules. The kit further includes an enzyme-labeled secondary antibody and a sample diluent, wherein the enzyme-labeled secondary antibody is goat anti-rabbit IgG-HRP, and the dilution ratio is 1:5000-1:10000, and the sample diluent is a PBS solution containing 0.05% Tween-20.

[0018] A diagnostic method for early detection of breast nodules based on serum GRP detection includes the following steps:

[0019] a) Collect serum samples from the subjects, with a sample size of 500-1000 μL, and store at -80℃;

[0020] b) Using the diagnostic reagents described above, the serum GRP level was detected by ELISA, including preparing a standard curve, adding the sample, incubation, washing the plate, adding the enzyme, color development, and terminating the reaction.

[0021] c) Compare the detected GRP level with the level in the healthy control group. If the GRP concentration is higher than the mean plus two standard deviations of the healthy control group (P<0.05), it is judged as a positive breast nodule.

[0022] d) Combine the nodule volume measured by color Doppler ultrasound to analyze the correlation between GRP level and nodule volume, and differentiate between benign nodules, early breast cancer, or late breast cancer.

[0023] As a preferred embodiment of the diagnostic method for early detection of breast nodules based on serum GRP detection described in this invention, the ELISA detection step includes:

[0024] a) Add 50-60 μL LGRP standard or sample to a 96-well microplate, add 100 μL LGRP primary antibody, and incubate at 37°C for 1-2 hours;

[0025] b) Wash the plate 4-6 times with 200μL PBS, add 100μL enzyme-labeled secondary antibody, and incubate at 37℃ for 30-60 minutes;

[0026] c) Wash the plate 4-6 times again, add 100 μL TMB colorimetric solution, and react at room temperature in the dark for 10-20 minutes.

[0027] d) Add 50 μL of stop solution and measure the absorbance (OD value) at a wavelength of 450 nm. Calculate the GRP concentration based on the standard curve.

[0028] As a preferred embodiment of the diagnostic method for early detection of breast nodules based on serum GRP detection according to the present invention, the following are provided: the healthy control group consists of healthy individuals without any breast nodules and with GRP levels below 90 mg / L; the benign nodule group consists of patients whose postoperative pathological diagnosis of benign nodules is 400-600 mg / L; the early breast cancer group consists of patients without tissue or organ metastasis and with GRP levels of 250-450 mg / L; and the advanced breast cancer group consists of patients with tissue or organ metastasis and with GRP levels of 100-250 mg / L.

[0029] As a preferred embodiment of the diagnostic method for early detection of breast nodules based on serum GRP detection according to the present invention, the method determines the early warning threshold of breast nodules by analyzing the negative correlation between GRP level and nodule volume, wherein the GRP level of benign nodules and early breast cancer is negatively correlated with nodule volume, and the correlation coefficient r is in the range of -0.50 to -0.70 (P<0.01).

[0030] As a preferred embodiment of the diagnostic method for early detection of breast nodules based on serum GRP detection as described in this invention, the method evaluates diagnostic efficacy through receiver operating characteristic (ROC) curves, wherein the AUC value of the benign nodule group compared with the healthy control group is not less than 0.88, the AUC value of the early breast cancer group compared with the healthy control group is not less than 0.70, the sensitivity is not less than 75%, and the specificity is not less than 80%.

[0031] As a preferred embodiment of the diagnostic method for early detection of breast nodules based on serum GRP detection described in this invention, the method is applicable to screening high-risk groups of breast diseases for whom imaging examinations have not revealed significant nodules. Early diagnosis is achieved by detecting GRP levels, with a positive predictive value of not less than 70%.

[0032] The beneficial effects of this invention are as follows: By detecting serum GRP levels, the sensitivity and specificity of early diagnosis of breast nodules are significantly improved, especially in cases where imaging examinations show no obvious abnormalities, effectively identifying benign nodules and early breast cancer. The diagnostic reagent uses the ELISA method, which is simple to operate, has a short detection time, and is low in cost, making it suitable for large-scale clinical screening. It fills the gap in existing technologies for non-invasive diagnosis and provides a reliable tool for regular monitoring of high-risk groups. Furthermore, by analyzing the correlation between GRP levels and nodule volume, it provides new evidence for disease classification and prognostic evaluation. The high expression characteristics of GRP in benign nodules and early breast cancer, as well as its negative correlation with malignancy, make it not only suitable for early screening but also for assisting in assessing the risk of disease progression, improving the accuracy of breast nodule diagnosis, and providing important support for optimizing treatment plans and improving patient prognosis. Attached Figure Description

[0033] To more clearly illustrate the technical solutions of the embodiments of the present invention, the drawings used in the following description of the embodiments will be briefly introduced. Obviously, the drawings described below are only some embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort. Wherein:

[0034] Figure 1 The fold change in GRP expression between breast cancer tissue and normal breast tissue.

[0035] Figure 2 To investigate the relationship between GRP expression levels in breast cancer tissues and patient survival.

[0036] Figure 3 To determine the level of GRP in peripheral blood in patients with different breast nodules.

[0037] Figure 4 The relationship between peripheral blood GRP levels and nodule volume in patients with benign nodules.

[0038] Figure 5 The relationship between peripheral blood GRP levels and tumor volume in early-stage breast cancer patients.

[0039] Figure 6 The relationship between peripheral blood GRP levels and tumor volume in patients with advanced breast cancer.

[0040] Figure 7 ROC curves comparing patients with benign breast nodules with healthy controls.

[0041] Figure 8 ROC curves comparing early-stage breast cancer patients with healthy controls.

[0042] Figure 9ROC curves comparing patients with advanced breast cancer with healthy controls. Detailed Implementation

[0043] To make the above-mentioned objects, features and advantages of the present invention more apparent and understandable, the specific embodiments of the present invention will be described in detail below with reference to the examples in the specification.

[0044] Many specific details are set forth in the following description in order to provide a full understanding of the invention. However, the invention may also be practiced in other ways different from those described herein, and those skilled in the art can make similar extensions without departing from the spirit of the invention. Therefore, the invention is not limited to the specific embodiments disclosed below.

[0045] Secondly, the term "an embodiment" or "embodiment" as used herein refers to a specific feature, structure, or characteristic that may be included in at least one implementation of the present invention. The phrase "in one embodiment" appearing in different places throughout this specification does not necessarily refer to the same embodiment, nor is it a single embodiment or an embodiment selectively excluded from other embodiments.

[0046] Example 1

[0047] Reference Figure 1 , Figure 3 , Figure 4-6 This embodiment provides a diagnostic reagent and method for early detection of breast nodules based on serum GRP detection.

[0048] Specifically, the preparation of diagnostic reagents based on serum GRP detection and their application in breast nodule screening were studied to verify their diagnostic efficacy.

[0049] Reagent preparation: Diagnostic reagents include the following components:

[0050] GRP standards (concentrations of 0, 50, 100, 200, 400, and 800 mg / L, purity >95%);

[0051] GRP primary antibody (rabbit anti-human GRP polyclonal antibody, 1:1000 dilution, concentration 1 mg / mL);

[0052] Buffer solution (PBS, pH 7.4, containing 0.01M phosphate);

[0053] Colorimetric reagent (TMB, concentration 0.4 mg / mL);

[0054] Stop solution (2M sulfuric acid);

[0055] Enzyme-labeled secondary antibody (goat anti-rabbit IgG-HRP, 1:5000 dilution);

[0056] Sample diluent (PBS containing 0.05% Tween-20).

[0057] Reagents should be stored at 4°C, while standards and primary antibodies should be stored at -20°C and brought to room temperature before use.

[0058] Biomarker screening: Through comprehensive gene expression database analysis, the expression level of GRP in breast cancer tissue was significantly higher than that in normal breast tissue, about 6 times that of normal tissue, suggesting the potential of GRP as a diagnostic biomarker for breast nodules.

[0059] Sample Collection: 264 participants were recruited, including:

[0060] Healthy control group: 80 healthy women, with no nodules detected by breast ultrasound, aged 30-60 years;

[0061] Benign nodule group: 70 patients who were pathologically diagnosed with benign nodules after surgery, with nodule volume ranging from 100 to 5000 mm;

[0062] Early-stage breast cancer group: 60 patients with non-metastatic breast cancer, with tumor volume ranging from 500 to 12000 mm. 3 ;

[0063] Advanced breast cancer group: 54 patients with lymph node or organ metastases, tumor volume 10,000-60,000 mm. 3 .

[0064] Collect 800 μL of serum from each case, centrifuge (3000 rpm, 10 minutes), collect the supernatant, and store at -80℃. Avoid repeated freeze-thaw cycles before testing.

[0065] Testing process:

[0066] Preparation of standard curve: Add 50 μL of GRP standard (concentration 0-800 mg / L) and 100 μL of GRP primary antibody (1:1000) to a 96-well microplate, and incubate at 37°C for 1.5 hours.

[0067] Wash the plate 5 times with 200 μL PBS (pH 7.4) to remove unbound material.

[0068] Add enzyme: Add 100 μL of enzyme-labeled secondary antibody (1:5000) and incubate at 37°C for 45 minutes.

[0069] Wash the plate five more times, add 100 μL of TMB colorimetric solution, and react at room temperature in the dark for 15 minutes.

[0070] Add 50 μL of stop solution and measure the OD value at a wavelength of 450 nm.

[0071] Standard curve: Linear regression of OD value on GRP concentration, y = 0.0024x + 0.048 (R²) 2 =0.99).

[0072] Sample testing: Take 50 μL of serum, dilute it 1:2 and add it to the ELISA plate. Repeat the above steps and calculate the GRP concentration.

[0073] Results analysis: GRP levels in the healthy control group were significantly lower than those in the benign nodule and breast cancer groups. GRP levels were higher in the benign nodule and early-stage breast cancer groups, and lower in the late-stage breast cancer group. GRP levels were negatively correlated with nodule volume in the benign and early-stage groups, but positively correlated in the late-stage group, indicating its potential in disease classification. The established diagnostic threshold effectively distinguished positive cases, with a low false-positive rate, high repeatability, and short testing time, making it suitable for clinical application.

[0074] Example 2

[0075] Reference Figure 3 , Figure 4-6 , Figure 7-9 This embodiment validates the performance of the diagnostic kit using clinical samples and evaluates its application effect in the early detection of breast nodules.

[0076] Reagent Kit Assembly: Based on the reagents of Example 1, assemble a diagnostic reagent kit comprising:

[0077] 96-well microplate (pre-coated with antibody protective membrane);

[0078] GRP standard (0-800 mg / L, 6 concentration points);

[0079] GRP primary antibody (1:1000, 5 mL);

[0080] Enzyme-labeled secondary antibody (1:5000, 5 mL);

[0081] PBS buffer (pH 7.4, 50 mL);

[0082] TMB colorimetric solution (10 mL);

[0083] 2M sulfuric acid stop solution (5mL);

[0084] Sample diluent (50 mL).

[0085] The kit comes with an instruction manual that specifies storage conditions (4℃, standards -20℃) and sample processing requirements.

[0086] Sample collection: 180 participants were recruited.

[0087] Healthy control group: 50 participants, aged 25-55 years, with no nodules confirmed by ultrasound;

[0088] Benign nodule group: 50 patients, pathologically confirmed, nodule volume 200-4500 mm. 3 ;

[0089] Early-stage breast cancer group: 40 patients, no metastasis, tumor volume 800-10000 mm 3 ;

[0090] Advanced breast cancer group: 40 patients with metastases, tumor volume 12000-55000 mm. 3 .

[0091] Serum was collected in the same manner as in Example 1, 1000 μL per case, and stored at -80°C.

[0092] Testing process:

[0093] Sample pretreatment: Dilute serum 1:2, mix well, and take 60 μL.

[0094] Standard curve: Prepared according to Example 1, concentration 0-800 mg / L, OD value fitting y = 0.0026x + 0.045(R²) 2 =0.98).

[0095] Detection: Add 60 μL of sample and standard to the ELISA plate, add 100 μL of primary antibody (1:1500), incubate at 37℃ for 2 hours; wash the plate 5 times; add 100 μL of secondary antibody (1:6000), incubate for 40 minutes; after washing the plate, add 100 μL of TMB, develop color for 12 minutes; add 50 μL of stop solution, and measure the OD value.

[0096] Repeatability testing: Each sample was tested 3 times, with low intra-batch variation.

[0097] Results Analysis: The results showed that serum GRP levels in the benign nodule group and early breast cancer group were significantly higher than those in the healthy control group, while levels in the late breast cancer group were lower, showing a negative correlation with disease malignancy. Diagnostic efficacy assessment showed that the area under the ROC curve (AUC) was higher in the benign nodule group compared to the healthy control group, indicating high sensitivity and specificity; the AUC value in the early breast cancer group was slightly lower, but it could still effectively distinguish healthy individuals; the AUC value in the late breast cancer group was the lowest, suggesting its limitations in late-stage diagnosis. Analysis of GRP levels and nodule volume further confirmed a negative correlation between benign nodules and early breast cancer, while a positive correlation was observed in the late-stage group. Application of the kit in cases with unclear imaging results showed that GRP detection can effectively identify potential lesions, has strong positive predictive ability, and the total testing time after optimization is <1.8 hours, making it suitable for clinical promotion.

[0098] Example 3

[0099] Reference Figure 2 , Figure 3 , Figure 4-6 , Figure 7-9 This embodiment verifies the effectiveness of the diagnostic method in early screening and prognostic evaluation in high-risk groups of breast diseases.

[0100] Method Implementation: The detection process was optimized using the kit from Example 2.

[0101] Sample size: 50 μL, diluted 1:3;

[0102] Primary antibody diluted 1:2000, incubated for 1.2 hours;

[0103] Secondary antibody diluted 1:7000, incubated for 30 minutes;

[0104] The color development time was 10 minutes. The standard curve was the same as in Example 2, with a high degree of fit.

[0105] Sample collection: 200 high-risk individuals (with a family history or history of breast hyperplasia), including:

[0106] Healthy control group: 60 participants, no nodules;

[0107] Benign nodule group: 60 patients, pathologically confirmed, volume 150-4000 mm. 3 ;

[0108] Early-stage breast cancer group: 40 patients, no metastasis, breast cancer volume 600-9000 mm 3 ;

[0109] Advanced breast cancer group: 40 patients with metastases, breast cancer volume 15,000-50,000 mm. 3 .

[0110] Collect 600 μL of serum and store at -80℃.

[0111] Detection and Diagnosis: The detection procedure was the same as in Example 2, with a positive threshold set. Results showed that GRP levels in the benign nodule group and early-stage breast cancer group were significantly higher than in the control group, while levels were lower in the late-stage group. ROC analysis indicated that the benign nodule group had the highest diagnostic efficacy compared to the healthy control group, followed by the early-stage breast cancer group, and the lowest in the late-stage group. GRP levels were negatively correlated with nodule volume in the benign and early-stage groups, and positively correlated in the late-stage group. A 12-month follow-up showed that early-stage breast cancer patients with high GRP levels experienced slower disease progression, while late-stage patients with low GRP levels had a higher risk of metastasis. A negative correlation trend between GRP levels and survival suggested its potential as a prognostic indicator.

[0112] This embodiment confirms the method's screening capability in high-risk populations, particularly its significant effectiveness in the early diagnosis of cases with unclear imaging findings. GRP levels reflect disease status and provide a basis for clinical classification. Prognostic assessment shows that changes in GRP levels are closely related to disease progression and patient survival, supporting the development of personalized treatment plans. The method is simple to operate, highly efficient, and suitable for regular monitoring of high-risk populations.

[0113] This invention utilizes the unique biological characteristics of GRP as a biomarker to provide a novel perspective for the early screening of breast diseases. GRP, as a neuropeptide, plays a crucial role in cell proliferation and signal transduction, exhibiting significantly high expression, particularly in benign breast nodules and early-stage breast cancer. This differential expression reflects GRP's function as an autocrine growth factor in the early stages of tumorigenesis, promoting cell division through binding to cell surface receptors. This biological mechanism provides the theoretical basis for this invention, making serum GRP levels an important indicator for distinguishing healthy individuals from patients with breast nodules. Especially when imaging examinations have not yet revealed obvious abnormalities, GRP detection can capture early pathological changes, providing a valuable window for clinical diagnosis.

[0114] The diagnostic reagents and methods of this invention are designed with full consideration of the practical needs of clinical applications. Enzyme-linked immunosorbent assay (ELISA) is used as the detection method, a technology that has been widely validated in the medical field and has the advantages of simple operation, low cost, and reliable results. The reagent components include GRP standards, specific antibodies, and a matching buffer, ensuring high sensitivity and specificity of the detection. Through a standardized detection procedure, the method can rapidly quantify serum GRP levels and accurately determine the presence of breast nodules by comparing with a healthy control group. Furthermore, the method incorporates correlation analysis of nodule volume, revealing a negative correlation between GRP levels and nodule volume in benign nodules and early breast cancer. This correlation not only enhances diagnostic resolution but also provides a basis for early disease warning, enabling clinicians to take intervention measures in the early stages of pathological changes and prevent the disease from progressing to a more serious stage.

[0115] Three embodiments validate the technical feasibility and clinical applicability of the present invention from different perspectives, covering application scenarios such as reagent preparation, kit validation, and high-risk population screening. The design of the embodiments reflects the incremental optimization of the technical solution; for example, by adjusting the antibody dilution ratio and incubation time, the detection cycle was shortened while maintaining the stability of the results. This optimization reflects a focus on clinical efficiency, enabling the method to be implemented efficiently in busy medical environments. The embodiments also demonstrate the applicability of the method in different populations, particularly in high-risk groups, where GRP detection can identify minute lesions that are difficult to detect with imaging, providing crucial support for early diagnosis. Simultaneously, the correlation between GRP levels and disease progression provides new insights into prognostic assessment; physicians can predict the risk of disease progression in patients based on changes in GRP expression, thereby developing personalized treatment plans.

[0116] Compared to traditional imaging examinations, this invention utilizes serum GRP testing, which requires no complex equipment or invasive procedures and only a small serum sample, reducing the burden on patients and making it particularly suitable as a routine screening tool. Furthermore, the method's high diagnostic efficacy for benign nodules and early breast cancer compensates for the shortcomings of existing technologies in early diagnosis, especially when imaging results are unclear, providing a reliable basis for clinical decision-making. By combining GRP level and nodule volume analysis, the method can not only confirm the presence of lesions but also further differentiate their nature, providing multidimensional information for subsequent treatment selection.

[0117] In summary, this invention successfully addresses the challenge of early diagnosis of breast nodules by translating the biological characteristics of GRP into a clinical diagnostic tool. Its design concept is based on a deep understanding of the molecular mechanisms of the disease, combined with mature ELISA technology, achieving a smooth translation from laboratory to clinical practice. The method's non-invasiveness, high efficiency, and low cost make it suitable for large-scale deployment, while its support for prognostic assessment further expands its clinical value. This invention not only provides an efficient means for early screening of breast nodules but also offers a new perspective on disease management, demonstrating significant potential for clinical application.

[0118] It should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention and are not intended to limit it. Although the present invention has been described in detail with reference to preferred embodiments, those skilled in the art should understand that modifications or equivalent substitutions can be made to the technical solutions of the present invention without departing from the spirit and scope of the technical solutions of the present invention, and all such modifications or substitutions should be covered within the scope of the claims of the present invention.

Claims

1. A diagnostic reagent for early detection of breast nodules based on serum GRP detection, characterized in that: The reagents include GRP standards, GRP primary antibody, buffer solution, chromogenic solution, and stop solution, wherein the amino acid sequence of the GRP is: >sp|P07492|GRP_HUMANGastrin-releasingpeptideOS=HomosapiensOX=9606GN=GRPPE=1SV=2 MRRGRELPLVLLALVLCLAPRGRAVLPLPAGGGTVLTKMYPRGNHWAVGHLMGKKSTGEESSS VSERGSLKQQLREYIRWEEAARNLLGLIEAKENRNHQPPQPKALGNQQPSWDSEDSSNFK DVGSKGKVGRLSAPGSQREGRNPQLNQQ; The reagent is used to detect the level of GRP in the serum of the subject by enzyme-linked immunosorbent assay (ELISA) and is used for the early diagnosis or prognostic evaluation of benign breast nodules or early breast cancer.

2. The diagnostic reagent for early detection of breast nodules based on serum GRP detection as described in claim 1, characterized in that: The concentration range of the GRP standard is 0-800 mg / L, and the purity is not less than 95%. The GRP primary antibody is rabbit anti-human GRP polyclonal antibody with a dilution ratio of 1:1000-1:5000. The buffer is phosphate buffered saline (PBS) with a pH of 7.2-7.

4. The chromogenic solution is tetramethyl-forbidden TMB. The stop solution is 2M sulfuric acid solution.

3. The diagnostic reagent for early detection of breast nodules based on serum GRP detection as described in claim 1, characterized in that: The reagent is used to detect GRP from the serum of the subject. The serum sample is stored at -80℃ after collection, and the sample volume is 50-100μL. Repeated freeze-thaw cycles should be avoided to ensure the accuracy of the test.

4. The diagnostic reagent for early detection of breast nodules based on serum GRP detection as described in claim 1, characterized in that: The reagent is used to prepare a kit for diagnosing breast nodules. The kit further includes an enzyme-labeled secondary antibody and a sample diluent, wherein the enzyme-labeled secondary antibody is goat anti-rabbit IgG-HRP with a dilution ratio of 1:5000-1:10000, and the sample diluent is a PBS solution containing 0.05% Tween-20.

5. A diagnostic method for early detection of breast nodules based on serum GRP detection, characterized in that: Includes the following steps: a) Collect serum samples from the subjects, with a sample size of 500-1000 μL, and store at -80℃; b) Using the diagnostic reagent of claim 1, the serum GRP level is detected by ELISA, including preparing a standard curve, adding the sample, incubation, washing the plate, adding the enzyme, color development and terminating the reaction; c) Compare the detected GRP level with the level in the healthy control group. If the GRP concentration is higher than the mean plus two standard deviations of the healthy control group (P<0.05), it is judged as a positive breast nodule. d) Combine the nodule volume measured by color Doppler ultrasound to analyze the correlation between GRP level and nodule volume, and differentiate between benign nodules, early breast cancer, or late breast cancer.

6. The diagnostic method for early detection of breast nodules based on serum GRP detection as described in claim 5, characterized in that: The ELISA detection steps include: a) Add 50-60 μL LGRP standard or sample to a 96-well microplate, add 100 μL LGRP primary antibody, and incubate at 37°C for 1-2 hours; b) Wash the plate 4-6 times with 200μL PBS, add 100μL enzyme-labeled secondary antibody, and incubate at 37℃ for 30-60 minutes; c) Wash the plate 4-6 times again, add 100 μL TMB colorimetric solution, and react at room temperature in the dark for 10-20 minutes. d) Add 50 μL of stop solution and measure the absorbance (OD value) at a wavelength of 450 nm. Calculate the GRP concentration based on the standard curve.

7. The diagnostic method for early detection of breast nodules based on serum GRP detection as described in claim 5, characterized in that: The healthy control group consisted of healthy individuals without any breast nodules and with GRP levels below 90 mg / L; the benign nodule group consisted of patients whose postoperative pathology confirmed benign nodules and with GRP levels of 400-600 mg / L; the early breast cancer group consisted of patients without tissue or organ metastasis and with GRP levels of 250-450 mg / L; and the advanced breast cancer group consisted of patients with tissue or organ metastasis and with GRP levels of 100-250 mg / L.

8. The diagnostic method for early detection of breast nodules based on serum GRP detection as described in claim 5, characterized in that: The method determines the early warning threshold of breast nodules by analyzing the negative correlation between GRP levels and nodule volume. Among them, the GRP levels of benign nodules and early breast cancer are negatively correlated with nodule volume, and the correlation coefficient r is in the range of -0.50 to -0.70 (P<0.01).

9. The diagnostic method for early detection of breast nodules based on serum GRP detection as described in claim 5, characterized in that: The method assesses diagnostic efficacy using receiver operating characteristic (ROC) curves, where the AUC value of the benign nodule group compared with the healthy control group is not less than 0.88, the AUC value of the early breast cancer group compared with the healthy control group is not less than 0.70, the sensitivity is not less than 75%, and the specificity is not less than 80%.

10. The diagnostic method for early detection of breast nodules based on serum GRP detection as described in claim 5, characterized in that: The method is applicable to screening high-risk groups of breast disease who have not been found to have significant nodules on imaging examinations. It achieves early diagnosis by detecting GRP levels, with a positive predictive value of not less than 70%.

Images