Kit and method for detecting breast cancer

Abstract

The present invention belongs to the field of biotechnology, and specifically relates to a kit and a detection method for detecting breast cancer. The kit includes an enzyme-labeled plate coated with a capture antibody L4-CAP, a biotin-labeled detection antibody L4-SIG, a protein standard, HRP, a sample diluent, a washing solution, a color developing solution, and a stop solution; the heavy chain amino acid sequence of the capture antibody L4-CAP is shown in SEQ ID NO.1, and the light chain amino acid sequence is shown in SEQ ID NO.2; the heavy chain amino acid sequence of the detection antibody L4-SIG is shown in SEQ ID NO.3, and the light chain amino acid sequence is shown in SEQ ID NO.4. The kit for detecting breast cancer of the present invention has strong specificity and can specifically detect the URCL4 protein content in serum. As an auxiliary diagnostic method for breast cancer with high URCL4 protein expression, it has good application prospects.

Description

Technical Field

[0001] The present invention belongs to the field of biotechnology, and in particular relates to a kit and a detection method for detecting breast cancer. Background Art

[0002] With the development of society and the accelerated pace of life, more and more people are facing the risk of cancer. Among them, the incidence of breast cancer has been increasing year by year, with over 1.2 million women diagnosed with breast cancer each year, making it one of the greatest threats to women's health. Early symptoms of breast cancer are not obvious, and diagnosis is often already in the advanced stages. Among all types of breast cancer, TNBC is the most dangerous. Although it accounts for less than 20% of breast cancers, it lacks ER, PR, and HER-2 receptors, making it insensitive to traditional trastuzumab and targeted therapies. This makes it difficult to treat clinically and carries a high risk of metastasis and recurrence after surgery.

[0003] Currently, there are three main technologies for early diagnosis of breast cancer. 1) Clinical examination: Regular clinical examinations are an effective method for early detection of breast cancer. 2) Imaging diagnostics: Imaging examinations primarily use X-rays, supplemented by ultrasound and MRI scans. 3) Biological target early detection: This includes screening for breast cancer-related genes and measuring serum levels of relevant tumor markers, which can be used as indicators for diagnosing breast cancer. A key challenge in the early diagnosis and treatment of breast cancer is the lack of sensitive biomarkers to identify tumors in their early stages.

[0004] Based on the above objectives, the present invention provides a kit and a detection method for detecting breast cancer. Summary of the Invention

[0005] The first object of the present invention is to provide a kit for detecting breast cancer.

[0006] In order to achieve the above object, the technical solution adopted by the present invention is:

[0007] A kit for detecting breast cancer, comprising an ELISA plate coated with a capture antibody L4-CAP, a biotin-labeled detection antibody L4-SIG, a protein standard, HRP, a sample diluent, a washing solution, a color development solution, and a stop solution;

[0008] The capture antibody L4-CAP is a monoclonal antibody L4-CAP against URCL4 protein, whose heavy chain amino acid sequence is shown in SEQ ID NO.1, and the light chain amino acid sequence is shown in SEQ ID NO.2; the detection antibody L4-SIG is a monoclonal antibody L4-SIG against URCL4 protein, whose heavy chain amino acid sequence is shown in SEQ ID NO.3, and the light chain amino acid sequence is shown in SEQ ID NO.4.

[0009] Furthermore, the amino acid sequence of the URCL4 protein is shown in SEQ ID NO.5.

[0010] Furthermore, the kit is an enzyme-linked immunosorbent assay kit.

[0011] Furthermore, the protein standard is a recombinant URCL4 protein antigen; the sample diluent is PBS; the washing solution is PBST; the color developing solution is TMB color developing solution; and the stop solution is sulfuric acid.

[0012] Furthermore, the concentration of the developing solution is 2 mg / L-4 mg / L; the concentration of the stop solution is 2M-4M.

[0013] The second object of the present invention is to provide a detection method for a kit for detecting breast cancer.

[0014] The detection method of the above-mentioned kit for detecting breast cancer is as follows:

[0015] (1) Take out the enzyme-labeled plate coated with the capture antibody L4-CAP, wash the plate with washing solution, add the sample to be tested and the gradient diluted protein standard, incubate at room temperature for 1-3 hours, wash with washing solution and spin dry;

[0016] (2) Add the biotin-labeled detection antibody L4-SIG to the system of step (1), incubate at room temperature for 1-3 hours, wash with washing solution and spin dry;

[0017] (3) Add HRP to the system after the reaction in step (2), incubate at room temperature for 1-3 hours, wash with washing solution and spin dry;

[0018] (4) Add a color developing solution to the system after the reaction in step (3), develop the color at room temperature in the dark for 5-10 minutes, and then add a stop solution to terminate the reaction;

[0019] (5) Use an enzyme-labeled instrument for dual-wavelength detection, measure the OD value at the maximum absorption wavelength of 450 nm and the reference wavelength of 630 nm, and calculate the results using the standard curve.

[0020] Furthermore, the sample to be detected is serum.

[0021] Compared with the prior art, the beneficial effects of the present invention are mainly:

[0022] This study experimentally demonstrated that URCL4 protein is abnormally overexpressed in breast cancer patients. Inhibiting URCL4 expression can inhibit breast cancer cell proliferation, suggesting that URCL4 protein could serve as a diagnostic biomarker for breast cancer. An ELISA kit for detecting breast cancer markers was also provided. This kit specifically detects URCL4 protein levels in human serum samples and has promising potential as an auxiliary diagnostic tool for breast cancers with high URCL4 protein expression. BRIEF DESCRIPTION OF THE DRAWINGS

[0023] Figure 1 This is a schematic diagram of the results of screening for differentially expressed mRNAs associated with breast cancer;

[0024] Figure 2 Schematic diagram of Western blotting results of URCL4 protein levels in breast cancer serum and healthy subjects' serum;

[0025] Figure 3 This is the standard curve of the kit for specifically detecting serum URCL4 protein content;

[0026] Figure 4 Transwell assay was used to detect the invasion and migration abilities of MCF-7 cells. DETAILED DESCRIPTION

[0027] The technical solution of the present invention will be further described below in conjunction with specific embodiments. However, it should be understood by those skilled in the art that the following examples are only intended to illustrate the present invention and should not be construed as limiting the present invention. Specific conditions not specified in the examples are to be followed according to conventional conditions or the conditions recommended by the manufacturer. All reagents or instruments used, unless otherwise specified, are conventional products obtained from commercial channels.

[0028] Example 1

[0029] (1) Screening of differentially expressed mRNAs related to breast cancer:

[0030] The whole transcriptome of sera from 30 breast cancer patients and 30 healthy subjects was sequenced by high-throughput sequencing to analyze the changes in mRNA expression profiles in the sera of breast cancer patients and healthy subjects, and mRNAs with a large differential expression in the sera of breast cancer patients and healthy subjects were screened as candidate molecules.

[0031] The results are as follows Figure 1As shown, after GEO2R and R language screening of differentially expressed genes, KEGG and GO functional enrichment analysis, the five mRNA candidate molecules with the largest differential expression folds related to breast cancer were finally screened out, namely URCL4, COL9A3, SOX10, MMP28, and MMP19. Furthermore, URCL4 was screened out as the candidate molecule with the largest differential expression folds in the serum of breast cancer patients.

[0032] (2) Verify the relationship between URCL4 expression and breast cancer:

[0033] Serum samples from 5 breast cancer patients and 5 healthy controls were analyzed by Western blotting. Figure 2 shown.

[0034] Figure 2 Western blot results of URCL4 protein levels in serum of breast cancer patients and healthy controls. Figure 2 It can be observed that the expression level of URCL4 protein is higher in breast cancer patients (lanes 1-5) and lower in healthy people (lanes 6-10), indicating that URCL4 protein has the potential to serve as a breast cancer marker.

[0035] Example 2

[0036] The preparation of URCL4 protein monoclonal antibodies specifically includes the following steps:

[0037] (1) Immunization of experimental animals: Healthy female mice of about 8 weeks old were selected and recombinant URCL4 protein (C1orf74) was used as the immunogen to immunize the mice. The subcutaneous multi-point injection method was used for immunization. For the first immunization, the recombinant URCL4 protein was mixed with an equal volume of complete Freund's adjuvant. For subsequent immunizations, the recombinant URCL4 protein was mixed with incomplete Freund's adjuvant. Secondary immunizations were performed in the third and fifth weeks after the first immunization. The entire immunization process was performed three times, with 200 μg injected each time. After the third immunization, the mouse serum was collected and the mouse serum antibody titer was analyzed using ELISA. The mice with the highest titer were selected for subsequent experiments.

[0038] (2) Cell fusion and screening: Take the female mouse with the highest antibody titer in step (1), kill the immunized mouse by cervical dislocation, and soak it in a beaker filled with 75% alcohol for 2 minutes. Remove the spleen of the mouse in a sterile clean bench and grind it on a 200-mesh sterile sieve to obtain a spleen cell suspension. Collect sp2 / 0 cells with good growth status and in the logarithmic growth phase. The cell number ratio of spleen cells to myeloma cells SP2 / 0 is 5:1. Fuse them in a 37℃ water bath for 2 minutes, using 50% polyethylene glycol as the fusion reagent. Culture the fused cells in HAT selective medium. Collect the cell culture supernatant after one week. Screen the positive hybridoma cells by ELISA. Subclone the screened positive hybridoma cells three times by limiting dilution method. Select the monoclonal cell with the strongest positive reaction for expansion culture.

[0039] (3) Preparation of URCL4 protein monoclonal antibody: 2×10 6 The hybridoma cells obtained in step (2) are inoculated into the peritoneal cavity of female mice sensitized with paraffin oil. The abdomen of the mouse is observed. When the abdomen is obviously bulging, the mouse ascites is collected. The collected ascites of different mice are purified by immunochromatography to obtain purified monoclonal antibodies.

[0040] (4) Analysis of monoclonal antibody sequences: The activity of the purified monoclonal antibodies was accurately measured. The two most active monoclonal antibodies were named L4-CAP and L4-SIG, and their variable region sequences were determined. Monoclonal antibody L4-CAP and monoclonal antibody L4-SIG include a heavy chain and a light chain, respectively. The heavy chain amino acid sequence of monoclonal antibody L4-CAP is shown in SEQ ID NO.1, and the light chain amino acid sequence is shown in SEQ ID NO.2. The heavy chain amino acid sequence of monoclonal antibody L4-SIG is shown in SEQ ID NO.3, and the light chain amino acid sequence is shown in SEQ ID NO.4.

[0041] Table 1 Sequence Listing

[0042]

[0043] Example 3

[0044] Preparation of ELISA kit:

[0045] The ELISA kit of the present invention comprises an enzyme labeling plate coated with a capture antibody L4-CAP, a biotin-labeled L4-SIG detection antibody, a recombinant URCL4 protein standard, horseradish peroxidase (HRP), a sample diluent (PBS), a washing solution (PBST), a color developing solution (TMB), and a stop solution (sulfuric acid).

[0046] The protein standard is a recombinant URCL4 protein antigen, and the amino acid sequence of the URCL4 protein is shown in SEQ ID NO.5.

[0047] SEQ ID NO.5:

[0048] MLLLDLMSSPSPQLLVAAAQQTLGMGKRRSPPQAICLHLAGEVLAVARGLKPAVLYDCNCAGASELQSYLEELKGLGFLTFGLHILEIGENSLIVSPEHVCQHLEQVLLGTIAFVDVSSCQRHPSVCSLDQLQDL KALVAEIITHLQGLQRDLSLAVSYSRLHSSDWNLCTVFGILLGYPVPYTFHLNQGDDNCLALTPLRVFTARISWLLGQPPILLYSFSVPESLFPGLRDILNTWEKDLRTRFRTQNDFADLSISSEIVTLPAVAL;

[0049] Preparation of an ELISA plate coated with the capture antibody L4-CAP: Use L4-CAP as the capture antibody and a 96-well ELISA plate as the solid phase support. Dilute the capture antibody to 2 μg / mL in coating buffer (50 mM carbonate buffer) to prepare a coating solution. Add 200 μL of coating solution to the ELISA plate, seal the plate, and coat overnight at 4°C. Discard the coating solution, wash six times with washing solution, add 200 μL / well of blocking solution, block at room temperature for 3 hours, discard the liquid, dry the plate, and store at 4°C.

[0050] Preparation of biotinylated L4-SIG detection antibody: Use L4-SIG as the detection antibody and an NHS-biotin derivative dissolved in PBS. Mix the L4-SIG detection antibody with biotin at a molar ratio of 1:10 in carbonate buffer (pH 9.0) and allow to react overnight at 4°C. Place the reaction mixture into a dialysis bag and dialyze against PBS to ensure complete removal of unreacted biotin. After dialysis, remove the bag and replace with PBS. Add glycerol to the solution, aliquot, and store at -20°C.

[0051] To prepare the standard curve for the kit: Remove the ELISA plate coated with the capture antibody L4-CAP and wash three times with PBST. Add 100 μL of recombinant URCL4 protein standards at different dilutions (50 pg / mL, 100 pg / mL, 200 pg / mL, 300 pg / mL, 400 pg / mL, 500 pg / mL, 600 pg / mL, 700 pg / mL, 800 pg / mL, 900 pg / mL, and 1000 pg / mL). Incubate at room temperature for 1 hour, wash three times with PBST, and spin dry. Add 100 μL of biotin-labeled detection antibody L4-SIG to the reaction wells, incubate at room temperature for 1 hour, wash three times with PBST, and spin dry. Add 100 μL / well of horseradish peroxidase (HRP), incubate at room temperature for 1 hour, wash three times with PBST, and spin dry. Then, add 100 μL of 2 mg / L TMB colorimetric solution to the reaction wells, develop at room temperature in the dark for 10 minutes, and terminate the reaction by adding 100 μL of 2 M sulfuric acid. Use a microplate reader for dual-wavelength detection, measuring the OD values ​​at the maximum absorption wavelength of 450 nm and the reference wavelength of 630 nm. A standard curve was plotted with the standard concentration (pg / mL) as the horizontal axis and OD 450 - OD 630 as the vertical axis. The results are shown in Figure 2. Figure 3 The standard curve of the kit used in this application for the detection of serum URCL4 protein content is y=0.0049x+0.0997, R 2 = 0.9972, and the detection range of the kit is 100-900 pg / mL.

[0052] Test Example 1

[0053] Transwell invasion and migration assay:

[0054] (1) Human breast cancer MCF-7 cells were placed in DMEM medium containing 10% FBS, penicillin (100 U / mL) and streptomycin (100 μg / mL) and cultured in an incubator at 37°C and 5% CO2.

[0055] (2) Human breast cancer MCF-7 cells in the logarithmic growth phase were cultured at a rate of 6×10 5 The cells were seeded into 6-well plates and collected when the cell confluence reached 70%.

[0056] (3) Take 5 μL of transfection reagent si-URCL4 and si-NC and mix them with 120 μL of Opti-MEN serum-free medium respectively, and let it stand at room temperature for 5 minutes to obtain diluted si-URCL4 and si-NC reagents; the sequences of si-URCL4 and si-NC are shown in Table 2.

[0057] Table 2 si-URCL4 and si-NC sequences

[0058]

[0059] (4) Dilute the transfection reagent Lipo-2000 to 1 μg / μL. Mix the diluted si-URCL4 and si-NC reagents from step (3) with the diluted transfection reagent and incubate at room temperature for 30 min to form a transfection mixture.

[0060] (5) The breast cancer MCF-7 cells collected in step (2) were washed with preheated PBS, and 1 mL of serum-free culture medium was added to each well of a 6-well plate. The transfection mixture obtained in step (4) was then added and slowly mixed. The cells were placed in an incubator at 37°C and 5% CO2 and cultured for 6 h. The original culture medium was discarded and replaced with DMEM culture medium containing 10% FBS, penicillin (100 U / mL), and streptomycin (100 μg / mL). After culture, the transfection efficiency was observed under an inverted fluorescence microscope. Successfully transfected human breast cancer cells si-URCL4 and si-NC were obtained and recorded as the si-URCL4 group and si-NC group, respectively.

[0061] (6) Pre-construct a matrix in the chamber with Matrigel glue and RPMI-1640 culture medium. After the matrix solidifies overnight, resuspend the cells in the si-URCL4 group and si-NC group and count them. Add 200 μL of cell suspension to the chamber and 500 μL of complete culture medium to the lower chamber. Place the chamber seeded with cells in a cell culture incubator at 37°C and 5% CO2 for constant temperature culture for 24 hours. Then wash with PBS and fix the chamber in paraformaldehyde solution. Add 1% crystal violet staining solution and stain at room temperature for 15 minutes. After staining, wash off the floating color, wipe off the cells in the upper chamber with a cotton swab, and dry at room temperature. Take out the film in the chamber, mount it on a slide, and cover it with a coverslip. Observe and take pictures under a microscope to ensure that the cells that migrate to the bottom of the chamber can be clearly observed. The experimental results were quantified and analyzed using Image J to count the number of migrated cells. The results are as follows: Figure 4 .

[0062] The results are as follows Figure 4 As shown in the figure, compared with the si-NC group, the number of cells passing through the bottom of the chamber in the si-URCL4 group was significantly reduced, indicating that after URCL4 gene silencing, the invasion and migration abilities of breast cancer MCF-7 cells were significantly reduced. This indicates that URCL4 gene silencing has a significant inhibitory effect on the invasion and migration abilities of breast cancer MCF-7 cells, and the URCL4 gene may serve as a potential therapeutic target for breast cancer.

[0063] Test Example 2

[0064] Blood sample testing:

[0065] Fifteen serum samples from healthy individuals (numbered 1-15) and 15 serum samples from breast cancer patients (numbered 16-30) were collected. The purified serum samples were added to an ELISA plate coated with the capture antibody L4-CAP and incubated at room temperature for 1 hour. The plate was washed three times with PBST and then dried. 100 μL of biotin-labeled detection antibody L4-SIG was added to the reaction wells, incubated at room temperature for 1 hour, washed three times with PBST, and dried. Horseradish peroxidase (HRP) was added at 100 μL / well and incubated at room temperature for 1 hour. The plate was washed three times with PBST and dried. Then, 100 μL of 2 mg / L TMB colorimetric solution was added to the reaction wells. The reaction was developed at room temperature in the dark for 10 minutes, and 100 μL of 2 M sulfuric acid was added to terminate the reaction. Dual-wavelength detection was performed using a microplate reader, with OD values ​​measured at the maximum absorbance wavelength of 450 nm and the reference wavelength of 630 nm. The URCL4 protein content in the serum was calculated based on the standard curve. Three replicates were performed and the average values ​​were calculated. The results are shown in Table 3.

[0066] Table 3 Clinical sample test results

[0067]

[0068] As shown in Table 3, the kit of the present invention can accurately detect serum URCL4 protein levels. The blood of healthy volunteers contains trace amounts of URCL4 protein, while the serum URCL4 protein level in breast cancer patients ranges from 600 to 900 pg / mL. These results further demonstrate that serum URCL4 protein can be used as a biomarker for distinguishing healthy individuals from breast cancer patients. This demonstrates that the kit of the present invention can detect breast cancer using serum as a sample, meeting the needs of both basic research and clinical diagnosis.

[0069] In summary, this study experimentally demonstrated that URCL4 protein is abnormally overexpressed in breast cancer patients and can be used as a breast cancer biomarker. The kit for detecting breast cancer markers provided by the present invention has strong specificity and can specifically detect URCL4 protein levels in serum. It can be used as an auxiliary diagnostic tool for breast cancers with high URCL4 protein expression and has promising application prospects.

[0070] Finally, it should be noted that the above embodiments are intended only to illustrate the technical solutions of the present invention and are not intended to limit the same. The basic principles and main features of the present invention have been described above using specific implementation schemes. Modifications or substitutions may be made based on the present invention, but such modifications or substitutions do not detract from the essence of the corresponding technical solutions from the scope of protection claimed by the present invention.

Claims

1. A kit for detecting breast cancer, characterized in that: The kit includes an ELISA plate coated with a capture antibody L4-CAP, a biotin-labeled detection antibody L4-SIG, a URCL4 protein standard, HRP, a sample diluent, a washing solution, a color development solution, and a stop solution; The capture antibody L4-CAP is a monoclonal antibody L4-CAP against URCL4 protein, whose heavy chain amino acid sequence is shown in SEQ ID NO.1, and the light chain amino acid sequence is shown in SEQ ID NO.2; the detection antibody L4-SIG is a monoclonal antibody L4-SIG against URCL4 protein, whose heavy chain amino acid sequence is shown in SEQ ID NO.3, and the light chain amino acid sequence is shown in SEQ ID NO.

4.

2. The kit for detecting breast cancer according to claim 1, wherein The amino acid sequence of the URCL4 protein is shown in SEQ ID NO.

5.

3. The kit for detecting breast cancer according to claim 1, wherein The kit is an enzyme-linked immunosorbent assay kit.

4. The kit for detecting breast cancer according to claim 1, wherein The URCL4 protein standard is a recombinant URCL4 protein antigen; the sample diluent is PBS; the washing solution is PBST; the color developing solution is TMB color developing solution; and the stop solution is sulfuric acid.

5. The kit for detecting breast cancer according to claim 4, characterized in that The concentration of the color developing solution is 2 mg / L-4 mg / L; the concentration of the stop solution is 2M-4M.

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