Detection reagents, kits, and applications for detecting natural antibodies against breast cancer.

Abstract

This invention relates to antigenic peptide compositions for detecting natural antibodies against breast cancer, kits comprising the antigenic peptide composition, methods for detecting the concentration of natural antibodies against breast cancer in human plasma using the antigenic peptide or the kit, and the application of the antigenic peptide or the kit to detect natural antibodies against breast cancer in human plasma. This invention utilizes four linear antigenic peptides that are completely complementary to the target natural antibodies against breast cancer to achieve qualitative and quantitative detection of natural antibodies against breast cancer in human plasma, and contributes to the research and treatment of breast cancer.

Description

Technical Field

[0001] This invention belongs to the field of clinical application of immunological technology, and relates to detection reagents, kits and application methods for detecting natural antibodies against breast cancer. Background Technology

[0002] Malignant tumors are one of the leading killers threatening human health. According to the latest data released by the World Health Organization, approximately 20 million new cancer patients are diagnosed globally each year, and more than 9 million die from cancer. China ranks first in the world in the number of new cancer cases, with nearly 5 million new cancer cases annually. Previously, lung cancer was the most common cancer worldwide; however, the latest data from 2020 shows that breast cancer has 2.26 million new cases annually, followed by lung cancer with 2.2 million new cases annually. Lung cancer remains the leading cause of death, with an average of 1.8 million people dying from it each year. Although breast cancer patients have a relatively longer survival period, its mortality rate has risen to fifth place, with as many as 680,000 patients dying from breast cancer each year. In fact, breast cancer is the most common malignant tumor among women, with the highest incidence rate among female cancer patients. In 2020, there were 420,000 new cases of breast cancer in Chinese women. Although early diagnosis and treatment technologies are constantly improving, and the mortality rate of breast cancer is showing a downward trend, the problem of chemotherapy resistance remains a bottleneck in the treatment of advanced-stage breast cancer, and solving the problem of breast cancer drug resistance is a key link in improving the survival rate.

[0003] Chemotherapy plays an increasingly important role in cancer treatment. However, while chemotherapeutic drugs kill tumor cells, they may also induce tumor stem cells to express drug resistance proteins, primarily ATP-binding cassette transporters (ABC transporters). These transporters mediate the transmembrane transport of various substrate molecules across the cell membrane. Based on sequence homology and transmembrane domain topology, ABC transporters are divided into seven subfamilies (ABC AG). To date, 49 members of the ABC family have been identified in humans, with drug transport-related proteins mainly concentrated in the ABCB, ABCC, and ABCG subfamilies. Studies have found that the pump function of ABC transporters is related to reducing drug accumulation within tumor cells, a major reason for drug resistance in tumor cells, and an important defense mechanism against chemotherapeutic drugs. ABCC5 belongs to the ABC transporter superfamily; this transporter can bind adenosine triphosphate (ATP) and use energy to drive the transmembrane transport of various molecules. Further research shows that ABCC5 is highly expressed in breast cancer cells, which may be an important reason for breast cancer cells' resistance to chemotherapeutic drugs.

[0004] Currently, monoclonal antibodies remain a hot topic in clinical targeted cancer therapy, such as the widely used trastuzumab and bevacizumab. The former targets HER2 (herpetic endothelial growth factor receptor type 2), while the latter targets VEGF-A (vascular endothelial growth factor A). These two monoclonal antibodies have become important tools for treating advanced cancers. However, due to significant side effects, they can only be used as third-line treatments. Furthermore, prolonged use of monoclonal antibodies can stimulate the immune system to produce anti-antibodies, weakening the therapeutic effect.

[0005] Trastuzumab is primarily used to treat breast cancer. It is a recombinant DNA-derived humanized monoclonal antibody that targets HER2, selectively acting on the extracellular site of HER2 to prevent human epidermal growth factor from binding to HER2, thereby blocking cancer cell growth. Alternatively, it can stimulate the body's own immune cells to destroy cancer cells, a process known as antibody-dependent cell-mediated cytotoxicity (ADCC). Trastuzumab has become an important clinical treatment for advanced breast cancer. However, trastuzumab only has a short-term therapeutic effect, and significant drug resistance develops after several months. Furthermore, trastuzumab can cause serious side effects, such as cardiotoxicity and gastrointestinal reactions. Therefore, finding safer and more effective drugs to treat advanced breast cancer has become a hot research topic in the field of clinical oncology.

[0006] Recent research reports on natural antibodies suggest that over 50% of antibodies in human blood are natural antibodies, primarily produced automatically by B1 lymphocytes without the need for specific antigen stimulation. Natural antibodies participate in various physiological regulations and immune function stability, acting as a bridge between innate and specific immune systems. Notably, some natural antibodies possess immune surveillance functions, promptly eliminating malignant cells formed in the body and maintaining internal homeostasis. Therefore, maintaining a certain level of natural antibodies can play a role in cancer prevention. Based on this, it can be inferred that individuals lacking or testing negative for natural antibodies may have a significantly higher risk of developing tumors than the general population. Establishing methods for detecting natural anticancer antibodies will help in studying the patterns of tumor development and finding effective treatments for tumors. Recently, Qingdao Hailanshen Biotechnology Co., Ltd. introduced human plasma natural anticancer antibody detection technology. The project research found that 5%–10% of healthy individuals have plasma rich in natural anticancer antibodies. Laboratory studies have demonstrated that plasma rich in natural anticancer antibodies from healthy individuals can significantly inhibit the growth and proliferation of various cancer cells, including primary liver cancer, lung cancer, stomach cancer, pancreatic cancer, nasopharyngeal carcinoma, and oral cancer. Furthermore, the study also found that some healthy individuals have high levels of natural antibodies against drug-resistant proteins in their plasma. If plasma rich in these natural antibodies is screened from a biopharmaceutical company's plasma bank and routinely transfused to patients with advanced-stage cancer undergoing chemotherapy, it may prevent cross-resistance and improve the effectiveness of chemotherapy. This is of great significance for prolonging the survival time and improving the quality of life of patients with advanced-stage cancer. Summary of the Invention

[0007] This invention provides a set of linear antigenic peptides for detecting the concentration of natural anti-breast cancer antibodies in human plasma, detection reagents and kits containing these peptides, and methods and applications for detecting natural anti-breast cancer antibodies in human plasma using the antigenic peptides. The antigenic peptides of this invention can be used to prepare enzyme-linked immunosorbent assay (ELISA) antibody detection kits and to detect the concentration of natural anti-breast cancer antibodies in human plasma. They can also be used to screen for natural anti-breast cancer antibodies in human plasma samples, extract and prepare anti-cancer gamma globulin from human plasma rich in natural anti-breast cancer antibodies, and for use in tumor-bearing animal model studies. The antigenic peptides are derived from the ABCC5 and HER2 protein sequences associated with human breast cancer, with two sequences from the ABCC5 transporter protein (SEQ ID NO:1 and SEQ ID NO:2) and two sequences from the HER2 receptor protein (SEQ ID NO:3 and SEQ ID NO:4). The italicized and underlined regions shown in Tables 1 and 2 represent the amino acid sequences constituting the antigenic peptides. These antigenic peptides can specifically bind to ABCC5 and HER2 natural antibodies in human plasma, respectively.

[0008] Table 1. Human ABCC5 protein sequence

[0009]

[0010] Table 2. Human HER2 protein sequence

[0011]

[0012] This invention utilizes immunoinformatics methods and epitope mapping technology to analyze human leukocyte antigen II (HLA-II) epitopes and B cell epitopes on the ABCC5 and HER2 protein sequences, identify amino acid sequences with high affinity, and then design HLA-II restricted epitopes and linear antigen peptides that can be recognized by the vast majority of human antigen-presenting cells.

[0013] This invention provides antigenic peptides for qualitative or quantitative detection of the concentration of anti-breast cancer natural antibodies in human plasma. The antigenic peptides can be single peptides or complex peptides, and kits for detecting anti-breast cancer natural antibodies in human plasma can be prepared. These antigenic peptides can specifically bind to anti-breast cancer natural antibodies in human plasma.

[0014] It is generally accepted that antigen-antibody binding primarily occurs between antigenic determinants (i.e., antigenic epitopes) and antibody binding sites. Therefore, the closer the two are to complete complementarity in spatial structure and conformation, the more stable, specific, and efficient the antigen-antibody binding.

[0015] Based on the biological characteristics of protein epitopes, this invention performs immunoinformatics prediction and simulation for multiple epitopes. After analyzing various parameters related to antigenicity, four linear antigenic peptides that are completely complementary to the target antibodies in human plasma in terms of spatial structure and conformation are designed. Their amino acid sequences are shown in Table 3.

[0016] Table 3. Linear antigen peptide sequences for detecting natural antibodies ABCC5 and HER2 in human plasma

[0017]

[0018] In this invention, "natural antibody" and "autoantibody" are used interchangeably, referring to a mixture of various antibodies (monoclonal and / or polyclonal antibodies) that naturally exist in the body and can recognize antigenic epitopes of target substances (e.g., target proteins, such as inflammatory cytokines). The "anti-breast cancer natural antibody" as defined in this invention is a mixture of various antibodies (monoclonal and / or polyclonal antibodies) that naturally exist in the human body and can recognize antigenic epitopes of breast cancer-related proteins, such as human ATP-binding cassette transporter C5 (ABCC5) and / or human epidermal growth factor receptor 2 (HER2). For example, it can be a natural IgG antibody that can recognize antigenic epitopes of breast cancer-related proteins, such as ATP-binding cassette transporter C5 (ABCC5) and / or human epidermal growth factor receptor 2 (HER2). In some embodiments, the "anti-breast cancer natural antibody" of this invention is also referred to as anti-ABCC5 and / or anti-HER2 natural antibody, meaning an anti-ABCC5 natural antibody or an anti-HER2 natural antibody, or an anti-ABCC5 and anti-HER2 natural antibody. In some embodiments of the present invention, the term "anti-breast cancer natural antibody" refers to a mixture of antibodies (monoclonal and / or polyclonal antibodies) capable of recognizing any one, two, three, or four polypeptide sequences selected from SEQ ID NO:1-4. Plasma rich in anti-breast cancer natural antibodies may have a therapeutic or preventive effect on breast cancer. Furthermore, detecting the level of anti-breast cancer natural antibodies in plasma can predict the risk of developing breast cancer; individuals with low or negative levels of anti-breast cancer natural antibodies in their plasma may have a higher risk of developing breast cancer. Individuals with low or negative levels of anti-breast cancer natural antibodies in their plasma can prevent breast cancer by regularly receiving plasma rich in anti-breast cancer natural antibodies.

[0019] The above-mentioned antigenic peptides are synthesized via solid-phase chemical methods and can be used alone or in combination to prepare ELISA antibody detection kits for detecting the concentration of natural anti-breast cancer antibodies in human plasma according to a set procedure. Detection kits containing one, two, three, or four antigenic peptides can be prepared into simple and easy-to-use convenient kits for practical applications. These kits are vacuum-sealed in non-metallic materials such as glass or medical plastics and can be stored for more than 6 months at 4 degrees Celsius. In short, one antigenic peptide or a mixture of two, three, or four antigenic peptides is coated onto a maleimide-activated 96-well microplate, dried in an oven at 40-45 degrees Celsius, and then vacuum-sealed in non-metallic packaging materials to prepare the kit. Preferably, the mixture of two, three, or four antigenic peptides is a mixture containing two, three, or four antigenic peptides with a mass-to-volume concentration ratio of 1:1, 1:1:1, or 1:1:1:1. Preferably, any one, two, three, or four antigenic peptides are products with a purity >95%.

[0020] Therefore, according to one embodiment of the present invention, a detection reagent for detecting natural antibodies against ABCC5 and HER2 is provided, comprising any one, two, three, or four of the following four antigenic polypeptides:

[0021] H-GLSLDASMHSQLRILDSKFRRTRPLECG–OH(SEQ ID NO:1);

[0022] H-DYHHGLSALKPIRTTSKHQHPVDNAGLFSCD-OH (SEQ ID NO: 2);

[0023] H-DLLALLPPGAASTQVCTGTDMKLRLPAS-OH (SEQ ID NO: 3); and

[0024] H-KVARCPSGVKPDLSYMPIWKFPDEEGAH-OH (SEQ ID NO: 4).

[0025] In some embodiments, the detection reagent consists of any one, any two, any three, or all four antigenic polypeptides selected from the four antigenic polypeptides.

[0026] In some embodiments, any one, any two, any three, or all four antigenic polypeptides are high-purity products, preferably chemically synthesized products with a purity >95%.

[0027] In some embodiments, any one, two, three, or all four antigenic polypeptides can be mixed, and when mixed, the two, three, or four antigenic polypeptides can be mixed in equal mass ratios. Therefore, in some embodiments, the detection reagent is a mixture of the two, three, or four antigenic polypeptides, for example, a mixture solution. In some embodiments, the ratio of any two, three, or four antigenic polypeptides in the detection reagent is 1:1, 1:1:1, or 1:1:1:1 (mass ratio).

[0028] According to another aspect of the present invention, a kit comprising the above-described detection reagents is provided.

[0029] In some embodiments, the kit includes a micro-detection plate, wherein the detection reagent is encapsulated within the wells of the micro-detection plate.

[0030] In some embodiments, the micro-detection plate coated with the above-described detection reagents is dried and then vacuum-sealed using non-metallic medical packaging material. In some embodiments, the micro-detection plate is a maleimide-activated 96-well micro-detection plate.

[0031] In some preferred embodiments, the non-metallic medical packaging material is glass or medical plastic.

[0032] In some embodiments, the kit further includes a positive control and / or a negative control. In some embodiments, the positive control and / or negative control are coated on a microplate. The positive control, also known as a positive standard, may be, for example, an anti-human ABCC5 antibody or an anti-human HER2 antibody or a mixture of both. The negative control may be, for example, any antibody that does not contain breast cancer-related proteins, such as a reagent containing an anti-human ABCC5 antibody or an anti-human HER2 antibody or both, such as an albumin solution.

[0033] According to another aspect of the present invention, a method is provided for detecting anti-breast cancer natural antibodies in a test sample using the above-described detection reagent or kit. In some embodiments, the method includes detecting the level of anti-ABCC5 and / or anti-HER2 natural antibodies in the test sample via an antigen-antibody binding reaction using the above-described detection reagent. The method is preferably performed in vitro, and its purpose is to obtain test data itself rather than for diagnostic purposes.

[0034] In some embodiments, the method includes causing an antigen-antibody binding reaction between the detection reagent and natural antibodies against ABCC5 and / or anti-HER2 in the test sample, and determining the level of natural antibodies against ABCC5 and / or anti-HER2 in the test sample. Techniques for detecting or determining antibody levels in a test sample using antigenic peptides via antigen-antibody binding reactions are well known in the art, such as enzyme-linked immunosorbent assay (ELISA). In a preferred embodiment, the level of natural antibodies against ABCC5 and / or anti-HER2 in the test sample is detected or determined by enzyme-linked immunosorbent assay (ELISA) in the above method.

[0035] In a more preferred embodiment, the enzyme-linked immunosorbent assay (ELISA) is a sandwich ELISA.

[0036] In some embodiments, the method includes the following steps: (1) mixing and incubating the above-mentioned detection reagent with the test sample, negative control, and positive control respectively under conditions suitable for antigen-antibody binding reaction between the above-mentioned detection reagent and the anti-ABCC5 and / or anti-HER2 natural antibodies in the test sample, then adding enzyme-labeled secondary antibody and incubating, followed by adding a chromogenic reagent, terminating the reaction after color development, and detecting the optical density (OD) value; (2) determining the level of anti-ABCC5 and / or anti-HER2 natural antibodies. The level can be a relative level, such as the relative level of anti-ABCC5 and / or anti-HER2 natural antibodies in the test sample relative to the positive control.

[0037] In some embodiments, the relative levels of anti-ABCC5 and / or anti-HER2 natural antibodies are determined using the positive sample ratio (PSR). In some embodiments, the PSR is calculated using the following formula:

[0038] PSR = [OD value of test sample – NC OD value] / [OD value of positive control – NC OD value]

[0039] NC represents the negative control. The antibody concentration in the sample (e.g., plasma) is expressed as the mean and standard deviation of the PSR.

[0040] In some implementations, thresholds for positive and negative samples of anti-ABCC5 and / or anti-HER2 natural antibodies can be set. The thresholds for positive and negative samples of anti-ABCC5 and / or anti-HER2 natural antibodies are determined using percentile methods, based on test results from a normal population. A sample test result above the positive threshold is considered positive, indicating a high level of anti-ABCC5 and / or anti-HER2 natural antibodies in the sample. In this case, the positive sample is plasma rich in anti-ABCC5 and / or anti-HER2 natural antibodies and can be used to treat breast cancer. The positive sample threshold can be determined using, for example, the 95th percentile method, the 90th percentile method, etc. A sample test result below the negative threshold is considered negative, indicating a low level of anti-ABCC5 and / or anti-HER2 natural antibodies in the sample, and a potentially higher risk of breast cancer. The negative sample threshold can be determined using, for example, the 5th percentile, the 10th percentile, etc. In some implementations, the normal population can be normal adults. The normal population can also be referred to as a healthy population, meaning a person who has never had breast cancer as determined by clinical examination. It should be understood that the determination of positive and negative sample thresholds is to screen samples with relatively high or low levels of anti-ABCC5 and / or anti-HER2 natural antibodies. Therefore, the thresholds are not limited to a specific normal population or a specific percentile. For example, existing normal population test results can be used, or samples from a new normal population can be used for testing. The number of individuals in the normal population can be arbitrarily chosen, as long as their test results are statistically significant; for example, it can be no less than 50, 100, or 200 individuals. Different thresholds can be set according to different needs and purposes under different circumstances.

[0041] Those skilled in the art will readily recognize the conditions suitable for antigen-antibody binding reactions between the detection reagent and the anti-ABCC5 and / or anti-HER2 natural antibodies in the sample to be tested, for example, based on conventional antigen-antibody reaction conditions and / or routine experiments.

[0042] In some embodiments, the mixing incubation in step (1) above includes coating the above-mentioned detection reagent onto a maleimide-activated micro-detection plate and adding the sample to be tested into the well of the micro-detection plate.

[0043] In some embodiments, the enzyme-labeled secondary antibody is a horseradish peroxidase-labeled goat anti-human IgG antibody; in some embodiments, the chromogenic agent is 3,3',5,5'-tetramethylbenzidine (TMB). In some embodiments, the reaction is terminated using a sulfuric acid solution, preferably at a concentration of 10-12% (volume / volume ratio). In some embodiments, the detection wavelength for optical density (OD) is 450 nm, and the reference wavelength is 630 nm.

[0044] In some embodiments, the sample is human plasma. In some embodiments, the sample is plasma from a healthy or normal person. In some embodiments, the sample is more preferably plasma from a single individual. The healthy or normal person can be someone who has never had breast cancer, as determined by a clinical examination. In some embodiments, the individual plasma is plasma from a single healthy or normal individual.

[0045] In a more preferred embodiment, step (1) above includes:

[0046] Dissolve the four antigenic peptides listed in Table 1 in 67% acetic acid to prepare a 5 mg / ml stock solution and store them in a -20°C freezer. They can be stored individually or mixed in equal volumes before storage.

[0047] Dilute the individual stock solution or mixture with a coating buffer to prepare a working solution of 10–50 μg / ml. The coating buffer is a 0.1 M phosphate buffer containing 0.15 M sodium chloride and 10 mM EDTA, with a pH between 7.0 and 7.4.

[0048] A 96-well microplate activated with maleimide was coated with working solution and incubated overnight at 4°C. The plate was then washed three times with washing buffer, which was a 0.1M phosphate buffer containing 0.15M sodium chloride and 0.1% TWEEN-20, with a pH between 7.0 and 7.4.

[0049] The plasma sample to be tested was prepared in duplicate, with two negative control (NC) wells (containing negative control solutions without anti-ABCC5 and anti-HER2 antibodies, such as bovine serum albumin, which can reflect the experimental index values ​​of the four polypeptide antigens described in Table 3 in the anti-ABCC5 and HER2 natural antibody negative reaction system) and two positive control (PC) wells (containing a mixture of anti-human ABCC5 and HER2 antibodies, which can reflect the experimental index values ​​of the four polypeptide antigens described in Table 3 in the anti-ABCC5 and HER2 natural antibody positive reaction system).

[0050] Dilute the plasma sample to be tested 1:100-200 with the analytical solution, which is the same as the antigen coating solution, i.e., 0.1M phosphate buffer containing 0.15M sodium chloride and 10mM EDTA, with a pH between 7.0 and 7.4. Add 100 μl to each well; incubate at 20-25℃ for 1-2 hours, and then wash the plate 3 times.

[0051] Dilute horseradish peroxidase-labeled goat anti-human IgG antibody (used to verify whether the substance being tested in the plasma is a specific antibody) with analytical buffer (i.e., 0.1M phosphate buffer containing 0.15M sodium chloride and 10mM EDTA, pH between 7.0 and 7.4) at a ratio of 1:10000 to 1:50000, add 100 μl to each well, and incubate at 20-25°C for 1-2 hours.

[0052] After washing the plate three times with washing buffer (i.e., 0.1M phosphate buffer containing 0.15M sodium chloride and 0.1% TWEEN-20, pH 7.0-7.4), add 100 μl of 3,3',5,5'-tetramethylbenzidine (TMB) to each well and incubate at room temperature in the dark for 20-30 minutes.

[0053] Add 50 μl of stop solution (12% sulfuric acid solution, 12% H2SO4) to each well, and then use an ELISA reader to detect the optical density (OD) value at a wavelength of 450 nm and a reference wavelength of 630 nm. The detection process is completed within 10 minutes after adding the stop solution, thereby enabling relative quantitative analysis of the levels of anti-ABCC5 and HER2 natural antibodies in the plasma of different individuals.

[0054] In some implementation schemes, the levels of anti-ABCC5 and / or anti-HER2 natural antibodies in the plasma of breast cancer patients can be detected in vitro, and the data obtained from the detection of each individual can be analyzed during population random sampling analysis.

[0055] In another aspect of the invention, the use of the above-described detection reagents or kits in detecting anti-ABCC5 natural antibodies and / or anti-HER2 natural antibodies in samples is provided. In some embodiments, the use is in vitro and for non-diagnostic purposes.

[0056] In another aspect of the invention, the use of the above-described detection reagent in the preparation of a reagent for detecting natural antibodies against ABCC5 and / or natural antibodies against HER2 in a sample is also provided.

[0057] In a preferred embodiment, the sample is human plasma, more preferably plasma from a single individual.

[0058] In a more preferred embodiment, the individual plasma is plasma from a healthy individual.

[0059] In another aspect of the invention, the use of the above-described detection reagents or kits in screening human plasma rich in natural antibodies against ABCC5 and / or natural antibodies against HER2 is also provided.

[0060] Unless otherwise specified, the antigenic polypeptide sequence described in this invention is from the N-terminus to the C-terminus from left to right.

[0061] Based on the above solutions, this invention provides a high-precision, easy-to-operate, and cost-effective detection technology for anti-ABCC5 and / or anti-HER2 natural antibodies. Furthermore, it provides a semi-quantitative (or relative quantitative) analysis and application scheme for anti-ABCC5 and / or anti-HER2 natural antibodies, thereby laying an important foundation for the development and application of technologies based on anti-ABCC5 and HER2 natural antibodies from healthy human plasma, such as predicting the risk of breast cancer and developing novel immunotherapy strategies with low side effects.

[0062] The detection system of this invention is a precise relative quantitative method that can detect anti-ABCC5 natural antibodies and / or anti-HER2 natural antibodies in plasma at a reasonable cost. It will play a key role in the application of plasma rich in anti-ABCC5 natural antibodies and / or anti-HER2 natural antibodies and gamma globulin rich in anti-ABCC5 natural antibodies and / or anti-HER2 natural antibodies in the field of low-toxicity anti-breast cancer treatment.

[0063] In summary, this invention provides a simple method for detecting natural antibodies against ABCC5 and / or HER2 in human plasma. This method can be used for qualitative or relatively quantitative detection of anti-ABCC5 and / or anti-HER2 antibody levels, assisting in the quantitative determination of anti-ABCC5 and / or anti-HER2 antibody levels in different individuals, and distinguishing between plasma rich in anti-ABCC5 and / or anti-HER2 antibodies (strongly positive) and plasma with low or no anti-ABCC5 and / or anti-HER2 antibodies (negative). It can be inferred that plasma rich in anti-ABCC5 and / or anti-HER2 antibodies may have a preventive effect against breast cancer. Because the antigenic peptide synthesis method of this invention is relatively simple and cost-effective, it lays an important foundation for the next step of applying plasma rich in anti-ABCC5 and / or anti-HER2 antibodies to prevent and treat breast cancer and assessing the risk of breast cancer in individuals with negative plasma anti-ABCC5 and / or anti-HER2 antibodies. Furthermore, individuals with negative plasma anti-ABCC5 and / or anti-HER2 antibodies detected by this invention can be clinically followed up to obtain a definitive conclusion, achieving the goal of early prevention and treatment. The detection method for anti-ABCC5 and / or anti-HER2 natural antibodies of the present invention can also be used for breast cancer biology research to explore the mechanisms of breast cancer development, tumor cell immune "escape" mechanisms, and immune surveillance mechanisms.

[0064] Attached Figure Description

[0065] Figure 1 These are the results of breast cancer cell viability experiments, where A represents the results for the breast cancer cell line BT-474, and B represents the results for the breast cancer cell line SK-BR-3.

[0066] Other features and advantages of the invention will be set forth in the following description of embodiments, and will be apparent in part from the description, or may be learned by practicing the invention. The main objects and other advantages of the invention may be realized and obtained by means of the embodiments and those particularly pointed out in the claims. Example

[0067] The following specific implementation plan uses a 1:1:1:1 mixture of the four antigenic polypeptides listed in Table 3 as the detection reagent to illustrate the technical solution of the present invention.

[0068] 1. Screening for anti-ABCC5 and HER2 natural antibodies in plasma from healthy individuals

[0069] The levels of anti-ABCC5 and HER2 natural antibodies in plasma samples from healthy blood donors were screened at a blood bank in Guangdong Province. The four polypeptide antigens used in this experiment (see Table 3) were chemically synthesized using solid-phase chromatography with a purity of 95%. The specific procedures were as follows:

[0070] (1) Before operation, each antigenic polypeptide was dissolved in 67% acetic acid to prepare a 5.0 mg / ml storage solution, then mixed with equal weight volumes and stored in a -20°C refrigerator.

[0071] (2) At the start of the operation, the antigen mixture was first diluted with coating solution to a working solution of 20 micrograms / ml. The coating solution was 0.1M phosphate buffer containing 0.15M sodium chloride and 10mM EDTA, and the pH value was measured to be 7.2.

[0072] (3) The 96-well detection plate (Thermo Scientific, USA) activated with maleimide was coated with working solution and incubated overnight at 4 degrees Celsius for 15 hours. The plate was then washed three times with washing buffer, which was 0.1M phosphate buffer containing 0.15M sodium chloride and 0.1% TWEEN-20. The pH value was measured to be 7.2.

[0073] (4) Then, the sample was added and analyzed step by step as follows:

[0074] a) All plasma samples to be tested were tested in duplicate, with two negative control wells (NC, reference material being bovine serum albumin (provided by Sigma-Aldrich)) and two positive control wells (PC, reference material being a mixture of anti-human ABCC5 and HER2 antibodies, provided by Sigma-Aldrich).

[0075] b) Dilute the plasma 1:200 with the analytical solution, which is the same as the antigen coating solution, namely 0.1M phosphate buffer containing 0.15M sodium chloride and 10mM EDTA, with a pH of 7.2. Add 100 μl to each well and incubate at room temperature for 1.5 hours.

[0076] c) After washing the plate three times with the aforementioned washing buffer (i.e., 0.1M phosphate buffer containing 0.15M sodium chloride and 0.1% TWEEN-20, with a pH of 7.2), dilute the horseradish peroxidase-labeled goat anti-human IgG (provided by Sigma-Aldrich) with analytical buffer at a dilution ratio of 1:30000, add 100 μl to each well, and incubate at room temperature for 1.5 hours.

[0077] d) After washing the plate three times with the aforementioned washing solution (i.e., 0.1M phosphate buffer containing 0.15M sodium chloride and 0.1% TWEEN-20, with a pH of 7.2), add 100 μl of 3,3',5,5'-tetramethylbenzidine (TMB) (provided by Life Technologies) to each well and incubate at room temperature in the dark for 25 minutes.

[0078] e) Add 50 μl of stop solution (12% sulfuric acid solution, 12% H2SO4) to each well, and then use an ELISA reader to detect the optical density (OD) value at a wavelength of 450 nm and a reference wavelength of 630 nm. The detection should be completed within 10 minutes after adding the stop solution. Subsequent steps will be based on this result to perform quantitative comparative analysis of ABCC5 and HER2 natural IgG antibodies for each individual.

[0079] 3. Plasma with positive and negative natural IgG antibodies

[0080] When analyzing the data obtained from the aforementioned tests, the positive sample ratio (PSR) was used to determine the levels of ABCC5 and HER2 natural IgG antibodies in plasma. The PSR calculation formula is: PSR = [OD value of the sample – OD value of the sample] NC [Value] / [Positive control OD value – OD] NC [Value], where NC is the negative control for each sample.

[0081] By screening 100 plasma samples from healthy individuals, two plasma samples with the highest and two with the lowest PSR values ​​were selected. These samples were then mixed separately during filtration and sterilization to obtain high-antibody mixed plasma (positive plasma) and low-antibody mixed plasma (negative plasma). After aliquoting, the plasma samples were stored in a -20°C freezer and thawed at room temperature before being used for cell culture.

[0082] 4. Breast Cancer Cell Viability Assay

[0083] Since trastuzumab is also a monoclonal antibody drug targeting HER2, it was chosen for comparison. BT-474 and SK-BR-3 breast cancer cell lines (provided by the National Experimental Cell Resource Sharing Service Platform of the National Biomedical Experimental Cell Resource Bank) were selected for viability experiments. BT-474 and SK-BR-3 cells were cultured in 96-well plates using IMDM medium (provided by GIBCO) containing 10% fetal bovine serum (BT-474 cells were supplemented with 0.01 mg / ml insulin), with 3000 cells / 100 μL seeded per well. Nine replicates were made for each plasma-treated cell group. Cells were cultured at 37°C and 5% CO2 for 24 hours. The supernatant was then removed, and 100 μL of IMDM medium containing 20% ​​human negative and positive plasma were added to each well. Another group of cells was treated with 20% negative plasma and trastuzumab (provided by Roche). Cells were cultured for another 48 hours. The reagent blank wells contained only 100 μL of cell culture medium. To determine cell viability, 10 μL of CCK-8 cell counting medium (provided by Sigma-Aldrich) was added, and after incubation for 2 hours, the optical density (OD) value was measured at 450 nm using a 96-well trigger reader, and cell viability was calculated. Cell viability = [Experimental group plasma OD value – Blank OD value] / [Negative plasma OD value – Blank OD value]. See Table 4 and... Figure 1 As shown, plasma from healthy individuals rich in anti-breast cancer antibodies significantly inhibited the activity of both BT-474 and SK-BR-3 cells, while trastuzumab only inhibited the activity of BT-474 cells and its effect was less than that of human positive plasma.

[0084] Table 4. Effects of plasma from healthy individuals rich in anti-breast cancer antibodies on the viability of BT-474 and SK-BR-3 cells.

[0085]

[0086] Note: Plasma group 1 is negative plasma, plasma group 2 is negative plasma + trastuzumab, plasma group 3 is positive plasma, each group has 9 replicates, and the t-test has 16 degrees of freedom.

[0087] The above description is only a preferred embodiment of the present invention, but the scope of protection of the present invention is not limited thereto. Any changes or substitutions that can be conceived by those skilled in the art within the scope of the technology disclosed in the present invention should be included within the scope of protection of the present invention. sequence list <110> Qingdao Hailanshen Biotechnology Co., Ltd. <120> Detection reagents, kits, and applications for detecting natural antibodies against breast cancer. <160> 4 <170> SIPOSequenceListing 1.0 <210> 1 <211> 27 <212> PRT <213> Artificial Sequence <400> 1 Gly Leu Ser Leu Asp Ala Ser Met His Ser Gln Leu Arg Ile Leu Asp 1 5 10 15 Ser Lys Phe Arg Arg Thr Arg Pro Leu Glu Cys 20 25 <210> 2 <211> 31 <212> PRT <213> Artificial Sequence <400> 2 Asp Tyr His His Gly Leu Ser Ala Leu Lys Pro Ile Arg Thr Thr Ser 1 5 10 15 Lys His Gln His Pro Val Asp Asn Ala Gly Leu Phe Ser Cys Asp 20 25 30 <210> 3 <211> 28 <212> PRT <213> Artificial Sequence <400> 3 Asp Leu Leu Ala Leu Leu Pro Pro Gly Ala Ala Ser Thr Gln Val Cys 1 5 10 15 Thr Gly Thr Asp Met Lys Leu Arg Leu Pro Ala Ser 20 25 <210> 4 <211> 28 <212> PRT <213> Artificial Sequence <400> 4 Lys Val Ala Arg Cys Pro Ser Gly Val Lys Pro Asp Leu Ser Tyr Met 1 5 10 15 Pro Ile Trp Lys Phe Pro Asp Glu Glu Gly Ala His 20 25

Claims

1. A diagnostic reagent for detecting natural antibodies against breast cancer, said reagent comprising the following four antigenic polypeptides: H-GLSLDASMHSQLRILDSKFRRTRPLECG-OH; H-DYHHGLSALKPIRTTSKHQHPVDNAGLFSCD-OH; H-DLLALLPPGAASTQVCTGTDMKLRLPAS-OH; and H-KVARCPSGVKPDLSYMPIWKFPDEEGAH-OH.

2. The detection reagent according to claim 1, wherein the detection reagent is composed of the four antigenic polypeptides.

3. The detection reagent according to claim 1 or 2, wherein the mass ratio of the four antigenic polypeptides is 1:1:1:

1.

4. A kit for detecting natural antibodies against breast cancer, characterized in that: The kit comprises the detection reagent as described in any one of claims 1-3.

5. The kit according to claim 4, wherein the kit comprises a micro-detection plate, and the detection reagent is encapsulated within the wells of the micro-detection plate.

6. A method for detecting natural anti-breast cancer antibodies in vitro for non-diagnostic purposes, comprising detecting natural anti-breast cancer antibodies in a sample using the detection reagent according to any one of claims 1-3 or the kit according to claim 4 or 5.

7. The detection method according to claim 6, comprising the following steps: (1) Under conditions suitable for antigen-antibody binding reaction between the detection reagent and the anti-breast cancer natural antibody in the sample to be tested, the detection reagent is mixed and incubated with the sample to be tested, the negative control and the positive control respectively, then enzyme-labeled secondary antibody is added and incubated, then chromogenic reagent is added, the reaction is terminated after color development and the optical density value (OD) is detected. (2) Determine the level of natural anti-breast cancer antibodies in the sample.

8. The use of the detection reagent according to any one of claims 1-3 in the preparation of a reagent for detecting natural antibodies against breast cancer in a sample.

9. The use of the detection reagent according to any one of claims 1-3 or the kit according to claim 4 or 5 in the in vitro detection of natural antibodies against breast cancer for non-diagnostic purposes.

10. The use of the detection reagent according to any one of claims 1-3 or the kit according to claim 4 or 5 in screening human plasma rich in natural antibodies against breast cancer.

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