Detection kit and detection method for pretumor marker

Abstract

The invention belongs to the technical field of in-vitro diagnosis, and particularly relates to a detection kit and a detection method for a pretumor marker. The kit comprises an elisa plate coated with a captured antibody, an HRP labeled antibody, an antigen standard substance, a washing solution, a developing solution and a stop solution, the capture antibody and the HRP labeled antibody are both prepared from a monoclonal antibody of the anti-SLC31A1 protein. According to the detection method of the kit for detecting the pretumor marker, the SLC31A1 protein content in human serum is detected through the kit, the specificity is high, a large batch of samples can be rapidly detected in a high-throughput mode, the cancer risk of a to-be-detected crowd can be effectively judged, and therefore relevant patients can find and treat the pretumor marker in advance, treatment prognosis is improved, and the death rate is reduced.

Description

Technical Field

[0001] The present invention belongs to the technical field of in vitro diagnosis, and particularly relates to a detection kit and a detection method for pre-tumor markers. Background Art

[0002] Breast cancer is a malignant tumor that occurs in the mammary gland epithelial cells, ranking first among the causes of cancer deaths in women globally. Breast cancer is a highly heterogeneous tumor formed by the coordinated action of multiple genes in multiple stages. Its clinical and pathological characteristics are affected by many factors, and the mechanisms of the occurrence and development of breast cancer cannot be fully revealed at present. Early studies on breast cancer pointed out that factors such as reproductive factors, breastfeeding factors, age of menarche, age of menopause, and age of onset may all affect the incidence and progression of breast cancer. Despite the progress made in surgery, endocrine therapy, chemotherapy, and radiotherapy, the expected five-year relative survival rate of patients with metastatic breast cancer is only 29%, and the median progression-free survival is less than 8 months.

[0003] In recent years, due to the continuous improvement of the diagnostic level, the increasing detection rate of early breast cancer, and the continuous improvement of surgical methods, the overall level of its treatment has been significantly improved. However, various current treatment methods still face problems such as poor sensitivity to chemotherapy drugs, low specificity, and lack of effective second-line drugs. Therefore, the most effective way is to be able to detect breast cancer in a timely manner during its early development process to avoid patients missing the best treatment time. However, existing tumor markers generally have disadvantages such as poor sensitivity and low specificity, resulting in unreliable screening and early detection. Therefore, there is an urgent need to develop a safe, reliable, highly sensitive, and highly specific detection kit for early screening and auxiliary diagnosis of breast cancer, so that more patients can benefit.

[0004] Currently, with the large-scale development of molecular epidemiology research, solute carrier family 31 member 1 (SLC31A1) has been screened out as the most potential copper death-related gene in breast cancer, which is closely related to the occurrence and prognosis of breast cancer. Compared with normal tissues, the expression level of SLC31A1 in breast cancer samples is higher. Compared with low expression, high expression of SLC31A1 is associated with poor prognosis of breast cancer patients. Therefore, the copper death-related gene SLC31A1 is considered a potential predictor for the pre-diagnosis of breast cancer. Summary of the Invention

[0005] The first object of the present invention is to provide a detection kit for pre-tumor markers, which can effectively improve the clinical diagnosis of breast cancer.

[0006] The second object of the present invention is to provide a detection method for a detection kit for pre-tumor markers, and this method is simple and easy to operate.

[0007] To achieve the above object, the technical solution adopted by the present invention is as follows:

[0008] A detection kit for pre-tumor markers, comprising an enzyme-labeled plate coated with a capture antibody, an HRP-labeled antibody, an antigen standard, a washing solution, a chromogenic solution, and a termination solution; both the capture antibody and the HRP-labeled antibody are prepared from monoclonal antibodies against the SLC31A1 protein; the heavy chain amino acid sequence of the capture antibody is as shown in SEQ ID NO.1, and the light chain amino acid sequence is as shown in SEQ ID NO.2; the heavy chain amino acid sequence of the HRP-labeled antibody is as shown in SEQ ID NO.3, and the light chain amino acid sequence is as shown in SEQ ID NO.4.

[0009] Further, the tumor is breast cancer.

[0010] Further, the antigen standard is the SLC31A1 protein antigen; the washing solution is PBST; the chromogenic solution is TMB; the termination solution is 1 mol / L sulfuric acid.

[0011] Further, the preparation process of the monoclonal antibody against the SLC31A1 protein is as follows:

[0012] (1) Using the recombinant SLC31A1 protein as an antigen to immunize mice, fusing the spleen cells of the immunized mice with myeloma cells to form hybridoma cells;

[0013] (2) Screening hybridoma cells secreting monoclonal antibodies against SLC31A1 by ELISA, subcloning and expanding the positive cell line, then inoculating it into mice, collecting ascites and purifying, detecting the activity of the purified monoclonal antibody against SLC31A1, and naming the two monoclonal antibodies with the highest activity as SLC31A1-1 and SLC31A1-2.

[0014] Further, the preparation method of the HRP-labeled antibody is as follows: adding a NaIO4 solution to the HRP solution, stirring at room temperature in the dark, purifying, adding the monoclonal antibody SLC31A1-2 to the purified solution and reacting at room temperature in the dark, continuing to add a NaBH4 solution and reacting at 4°C for a period of time, dialyzing overnight to obtain the HRP-labeled antibody.

[0015] Further, the preparation steps of the enzyme-labeled plate coated with the capture antibody are as follows: diluting the monoclonal antibody SLC31A1-1 with an antibody coating solution and adding it to the micro-wells of the enzyme-labeled plate, sealing the plate with a sealing film, coating overnight at 4°C, adding a blocking solution and blocking at room temperature for a period of time, discarding the liquid in the plate wells of the enzyme-labeled plate, drying and sealing, and storing at 4°C.

[0016] A detection method for a detection kit for pre-tumor markers, the detection steps are as follows:

[0017] (1) Centrifuge the sample to be detected and take the supernatant.

[0018] (2) Take out the enzyme-labeled plate coated with the capture antibody, restore it to room temperature, wash the plate with the washing solution and centrifuge it dry, add the sample processed in step (1), incubate at room temperature and then wash the plate.

[0019] (3) Add the HRP-labeled antibody to the system in step (2), incubate at room temperature for a period of time, wash the plate with the washing solution and centrifuge it dry.

[0020] (4) Add the chromogenic solution to the system after the reaction in step (3), develop color at room temperature in the dark for a period of time, and add the stop solution to terminate the reaction.

[0021] (5) Use an enzyme-labeled instrument for dual-wavelength detection, subtract the OD value at 630 nm from the OD value measured at 450 nm, and calculate the result through the standard curve.

[0022] Further, the test sample is blood.

[0023] Compared with the prior art, the beneficial effects of the present invention mainly lie in that the present invention discloses a detection kit and a detection method for pre-tumor markers. By detecting the content of SLC31A1 in human serum samples, the kit can quickly and highly-throughput detect a large number of samples, has strong specificity, can effectively judge the cancer risk of the population to be tested, so as to facilitate early detection and early treatment of related patients, improve the treatment prognosis and reduce the mortality rate. BRIEF DESCRIPTION OF THE DRAWINGS

[0024] Figure 1 It is a standard curve for the detection of the pre-breast cancer marker SLC31A1. DETAILED DESCRIPTION OF THE EMBODIMENTS

[0025] The following further describes the technical solutions of the present invention in conjunction with specific embodiments. However, those skilled in the art should understand that the following embodiments are only used to illustrate the present invention and should not be regarded as a limitation of the present invention. The specific conditions not specified in the embodiments are carried out according to the conventional conditions or the conditions recommended by the manufacturer. The reagents or instruments used, unless otherwise specified, are all conventional products obtained through commercial channels.

[0026] Example 1

[0027] Obtaining of monoclonal antibody against SLC31A1 protein:

[0028] I. Antigen immunization

[0029] (1) Using the recombinant SLC31A1 protein as an antigen, 7 SPF-grade female BALB / c mice weighing approximately 22 g were selected and immunized by subcutaneous injection. At the first immunization, 50 μg / mouse (100 μL) of the recombinant SLC31A1 protein antigen was mixed with an equal volume of Freund's complete adjuvant and emulsified, and then injected intradermally at multiple points on the back of the mice.

[0030] (2) Then, immunizations were carried out again at the second, fourth, and sixth weeks respectively. For each booster immunization, 25 μg / mouse (100 μL) of the recombinant SLC31A1 protein antigen was mixed with an equal volume of Freund's incomplete adjuvant and injected subcutaneously into the back of the mice.

[0031] (3) One week after the last immunization, a boost immunization was performed by injecting 25 μg / mouse (100 μL) of the recombinant SLC31A1 protein antigen via the tail vein. Three days later, blood was collected, and the antibody titers of the sera of each mouse were measured by the indirect ELISA method.

[0032] II. Cell fusion and establishment of hybridoma cell lines

[0033] (1) Mouse spleen cells with a serum titer greater than 1:12000 were selected for cell fusion. Three days after fusion, the abdominal cavity of the mouse was opened under sterile conditions to remove the spleen. The spleen was rinsed with 5 mL of DPBS containing 2% FBS, and the spleen cells were collected using a grinder. After centrifugation at 900 rpm for 9 min to discard the supernatant, the cells were resuspended in 10 mL of DPBS without FBS to prepare a spleen cell suspension.

[0034] (2) The spleen cells were mixed with the pre-resuscitated myeloma cells SP2 / 0 at a cell number ratio of 10:1, and 50% polyethylene glycol was used as the fusogen for a 2-minute fusion in a 37°C water bath.

[0035] (3) The fused cells were dispensed into 96-well cell culture plates containing feeder cells (spleen cells from non-immunized mice) and cultured in a 37°C, 5% CO2 incubator using HAT (containing 20% FBS) for 7 days of selective culture and screening. The cell culture supernatant was collected, and positive hybridoma cell lines were screened out by the indirect ELISA method.

[0036] (4) Positive cell wells were cloned by the limiting dilution method until the positive rate of the cell supernatant of the monoclonal wells was 100%. Three positive cell lines were selected for subcloning and expansion culture to establish a monoclonal hybridoma cell line secreting specific antibodies against the SLC31A1 protein.

[0037] III. Antibody preparation and purification

[0038] (1) Inject 0.5 mL of liquid paraffin per mouse into the peritoneal cavity. After 7 days, inject 0.5 mL of hybridoma cells resuspended in RPMI-1640 complete medium (containing 5×10 6 cells) into the peritoneal cavity of each mouse. Observe the production of ascites in the abdominal cavity of mice every day. After 8 days, draw ascites from the mice with a syringe.

[0039] (2) Purify the antibody by immunochromatography: Centrifuge the freshly collected ascites at 2000 rpm for 15 min to remove cell components. Filter the supernatant through a 0.45 μm filter, dilute it with an equal volume of Buffer A (0.05 mol / L Boricocid, 4.0 mol / L NaCl, pH 9.0), and equilibrate the column with Buffer A. Add an appropriate amount of ascites diluted with Buffer A to wash the column until there are no impurity proteins.

[0040] (3) Then elute with Buffer B (0.05 mol / L Sodium phosphate, 0.05 mol / L Sodium citrate, 0.3 mol / L NaCl, pH 3.0). Collect the eluted antibody, immediately add 5% volume of 1 mol / L Tris-HCl, pH 8.0 solution for neutralization, and then analyze the purity of the collected antibody by SDS-PAGE. Detect the activity of the purified anti-SLC31A1 monoclonal antibody.

[0041] (4) Name the two monoclonal antibodies with the highest activity as SLC31A1-1 and SLC31A1-2, adjust the concentration to 10 mg / mL, and determine the sequences of the variable regions of their heavy and light chains. The amino acid sequence of the heavy chain of monoclonal antibody SLC31A1-1 is shown in SEQ ID NO.1, and the amino acid sequence of the light chain is shown in SEQ ID NO.2. The amino acid sequence of the heavy chain of monoclonal antibody SLC31A1-2 is shown in SEQ ID NO.3, and the amino acid sequence of the light chain is shown in SEQ ID NO.4.

[0042] SEQ ID NO.1:

[0043] EVSLVHSGGDLTKPRGSLKLSCSPSTPTFTSTGHESMKWPGHKALEWIATINDGSNNALANE

[0044] TFKGNASFRADTAGTTSILQLSSLRLKTEWDTAMYYCVTVATGPADYANTGTTLTVVT

[0045] SEQ ID NO.2:

[0046] DVLMSGTPKSLPVSPGDRASITCKSSQSLLTRDGNTYLHKFLQKPGGSYWPLIYETGVPSSHVVPAFSDYTLRINSVQHEDLGDYFTQLYSGPYGFGCGTKHEIK;

[0047] SEQ ID NO.3:

[0048] GVKLVQGSGELHKPGLTVHLTCLPSGPSHSDYYMSWVKQTPEKGLEWVARIRTKRYNPSYL TISLLKDRVSRDTSNSQLGLSEDEEQLRASAVYYCAITYWGFFDVWLAGSTVRVLS;SEQ ID NO.4:

[0049] AIVMTQSPSSFSVSLGDSASITCKSSGSLANNSHWYYLHPGNGGLALLTTASCRLASVPDSG VPDRFSGSGSSGTSNVGSGAVGDYFLGVYFCSQSTFGCGTPLEIKG。

[0050] Example 2

[0051] Preparation of a kit for detecting pre - cancer markers:

[0052] (1) Preparation of HRP - labeled antibody: The monoclonal antibody SLC31A1 - 2 prepared in Example 1 was dialyzed in 50 mM carbonate buffer for 24 h, with the buffer changed twice during this period. After dialysis, its concentration was adjusted to 2 mg / mL. Weighed 5 mg of HRP dry powder and dissolved it in 1 mL of ddH2O. Added 200 μL of freshly prepared 20 mg / mL NaIO4 solution to the above solution and stirred gently in the dark at room temperature for 20 min to oxidize the glycosyl group of HRP into aldehyde group. The above solution was put into a dialysis bag and dialyzed against 1 mM sodium acetate buffer at pH 4.4 overnight at 4 °C. Added an appropriate amount of 0.2 M carbonate buffer at pH 9.5 to adjust the pH value to 9.0, and then immediately added an equal volume of the monoclonal antibody SLC31A1 - 2 and stirred gently in the dark at room temperature for 2 h. Then added 20 μL of freshly prepared 20 mg / mL NaIO4 solution, mixed well and reacted at 4 °C for 3 h to obtain a stable enzyme - labeled antibody. Then it was dialyzed overnight at 4 °C in 0.15 M PBS at pH 7.4 to remove unbound antibodies and other impurities to obtain a purified enzyme - labeled antibody.

[0053] (2) Preparation of the enzyme-labeled plate coated with capture antibody: Select the monoclonal antibody SLC31A1-1 prepared in Example 1 as the capture antibody. Use an enzyme-labeled 96-well plate as the solid-phase carrier. Dilute the capture antibody with antibody coating solution (carbonate buffer, 0.05 mol / L) to 1 μg / mL, add 100 μL per well to the micro-wells of the enzyme-labeled plate, seal the plate with a sealing film, and place it at 4 °C for overnight coating. Discard the liquid in the wells, wash the plate 3 times with ELISA washing solution, add 200 μL per well of 1% BSA blocking solution, and place it at room temperature for 2 h for blocking. Discard the liquid in the plate wells, pat dry, seal, and store at 4 °C.

[0054] (3) Take out the enzyme-labeled plate coated with capture antibody 40 min before sample detection, restore it to room temperature, wash the plate 3 times with PBST washing solution and spin dry.

[0055] (4) Add 100 μL of SLC31A1 protein antigens at different dilution ratios (50 pg / mL, 100 pg / mL, 150 pg / mL, 200 pg / mL, 300 pg / mL, 400 pg / mL, 500 pg / mL, 600 pg / mL, 800 pg / mL) and set a blank control, incubate at room temperature for 2 h, wash the plate 3 times and spin dry.

[0056] (5) Add 100 μL of HRP-labeled antibody working solution to the reaction wells, incubate at room temperature for 40 min, wash the plate 3 times and spin dry.

[0057] (6) Add 100 μL of chromogenic substrate TMB to the reaction wells, develop color at room temperature in the dark for 10 min, and add 100 μL of 1 mol / L sulfuric acid to terminate the reaction.

[0058] (7) Use an enzyme-labeled instrument for detection, measure the OD values at the maximum absorption wavelength of 450 nm and the reference wavelength of 630 nm, and subtract the OD value at 630 nm from the OD measurement value at 450 nm. Plot a standard curve with the standard product concentration as the abscissa (pg / mL) and OD 450 - OD630 as the ordinate. The results are as Figure 1 shown.

[0059] Experimental Example 1

[0060] Blood sample test:

[0061] Collect 15 healthy human blood samples (numbered 1 - 15) and 15 breast cancer patient blood samples (numbered 16 - 30). After blood collection, let it coagulate at room temperature, then centrifuge at 3000 rpm for 15 min to take the supernatant, and take 100 μL each and add it to an EP tube as the antigen for standby. The detection method is as follows:

[0062] (1) Take the enzyme-labeled plate coated with capture antibody, wash the plate 3 times with PBST washing buffer and spin dry;

[0063] (2) Add 100 μL of blood sample supernatant from patients 1 to 30, incubate at room temperature for 2 h, wash the plate three times, and spin dry;

[0064] (3) Add 100 μL of HRP-labeled antibody working solution to the reaction wells, incubate at room temperature in the dark for 40 min, wash the plate three times and spin dry;

[0065] (4) Add 100 μL of the chromogenic substrate TMB to the reaction wells, develop the color at room temperature in the dark for 10 min, and then add 100 μL of 1 mol / L sulfuric acid to terminate the reaction. Use a microplate reader for dual-wavelength detection, measure the OD value at the maximum absorption wavelength of 450 nm and the reference wavelength of 630 nm, and subtract the OD value at 630 nm from the OD value at 450 nm as the final result. Draw a standard curve with the standard concentration as the horizontal axis (pg / mL) and OD 450-OD 630 as the vertical axis. The results are shown in Table 1.

[0066] Table 1 Blood sample test results

[0067]

[0068]

[0069] The test results showed that the OD values in the serum samples of healthy individuals were low, and the SLC31A1 content calculated based on the linear regression curve was too low to be accurate, so the result was judged as not detected. However, the SLC31A1 content in the serum samples of breast cancer patients was significantly higher than that of healthy patients. These results together indicate that the gene SLC31A may serve as a potential predictive factor for breast cancer diagnosis.

[0070] Experimental Example 2

[0071] Kit stability study:

[0072] The capture antibody-coated ELISA plate, HRP-labeled antibody, and SLC31A1 antigen standard were placed at 37°C for 30 days, with SLC31A1 antigen detection performed every 10 days to test their stability. Furthermore, the capture antibody-coated ELISA plate, HRP-labeled antibody, and SLC31A1 antigen standard were stored in a 4°C refrigerator for two years, with SLC31A1 antigen detection performed at 6, 12, and 24 months, respectively.

[0073] The detection method is:

[0074] (5) Take the capture antibody-coated ELISA plate, wash it three times with PBST washing buffer and spin dry;

[0075] (6) Add 100 μL of SLC31A1 at different dilution ratios (800 pg / mL, 600 pg / mL, 500 pg / mL, 400 pg / mL, 300 pg / mL, 200 pg / mL), and set up a blank control. Incubate at room temperature for 2 h, wash the plate 3 times and centrifuge to dry;

[0076] (7) Add 100 μL of the HRP-labeled antibody working solution to the reaction wells, incubate at room temperature for 40 min, wash the plate 3 times and centrifuge to dry;

[0077] (8) Add 100 μL of the chromogenic substrate TMB to the reaction wells, develop color in the dark at room temperature for 10 min, and add 100 μL of 1 mol / L sulfuric acid to terminate the reaction. Use an enzyme-linked immunosorbent assay (ELISA) reader for dual-wavelength detection, measure the OD values at the maximum absorption wavelength of 450 nm and the reference wavelength of 630 nm, and subtract the OD value at 630 nm from the OD value at 450 nm as the final result. Plot a standard curve with the standard product concentration on the x-axis (pg / mL) and OD 450 - OD 630 on the y-axis. The results are shown in Tables 2 and 3.

[0078] Table 2 Investigation of the stability of the kit after storage at 37°C

[0079]

[0080]

[0081] Table 3 Investigation of the stability of the kit after storage at 4°C

[0082]

[0083] The above results together indicate that within 30 days of exposure at 37°C and 2 years of low-temperature storage, the OD value gradient of the standard curve of the kit is good, indicating good stability of the kit.

[0084] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention, rather than to limit them. The basic principles and main features of the present invention have been described in the above specific implementation manners. Based on the present invention, some modifications or replacements can be made, but these modifications or replacements do not cause the essence of the corresponding technical solutions to deviate from the scope of protection required by the present invention.

Claims

1. A detection kit for pre-tumor markers, characterized in that, It includes an enzyme-labeled plate coated with a capture antibody, an HRP-labeled antibody, an antigen standard, a washing solution, a color development solution, and a stop solution; The heavy chain amino acid sequence of the monoclonal antibody SLC31A1-1 against the SLC31A1 protein in the capture antibody 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 monoclonal antibody SLC31A1-2 against the SLC31A1 protein in the HRP-labeled antibody is shown in SEQ ID NO.3, and the light chain amino acid sequence is shown in SEQ ID NO.

4.

2. The detection kit for a pre-tumor marker according to claim 1, characterized in that, The antigen standard is SLC31A1 protein antigen; the washing solution is PBST; the color developing solution is TMB; and the stop solution is 1 mol / L sulfuric acid.

3. The detection kit for pre-tumor markers according to claim 1, characterized in that: The preparation process of the monoclonal antibody against SLC31A1 protein is as follows: (1) Recombinant SLC31A1 protein was used as an antigen to immunize mice, and spleen cells from the immunized mice were fused with myeloma cells to form hybridoma cells; (2) Hybridoma cells secreting SLC31A1 monoclonal antibodies were screened by ELISA. The positive cell lines were subcloned and expanded, then inoculated into mice. Ascites was collected and purified. The activity of the purified SLC31A1 monoclonal antibodies was tested, and the two most active monoclonal antibodies were named SLC31A1-1 and SLC31A1-2.

4. The detection kit for a pre-tumor marker according to claim 1, wherein The HRP-labeled antibody is prepared as follows: NaIO4 solution is added to the HRP solution, stirred at room temperature in the dark, purified, the monoclonal antibody SLC31A1-2 is added to the purified solution, reacted at room temperature in the dark, NaBH4 solution is added, reacted for a period of time at 4°C, and dialyzed overnight to obtain the HRP-labeled antibody.

5. The detection method of a detection kit for a pre-tumor marker according to claim 1, characterized in that, The preparation steps of the capture antibody-coated ELISA plate are as follows: diluting the monoclonal antibody SLC31A1-1 with an antibody coating solution and adding it to the microwells of the ELISA plate; sealing the plate with a sealing film; placing it at 4°C for overnight coating; adding a blocking solution and blocking it at room temperature for a period of time; discarding the liquid in the wells of the ELISA plate; drying and sealing the plate; and storing it at 4°C.

6. The detection method of a detection kit for a pre-tumor marker according to any one of claims 1-5, characterized in that, The detection steps are: (1) Centrifuge the sample to be tested and take the supernatant; (2) taking out the capture antibody-coated ELISA plate, returning it to room temperature, washing the plate with a washing solution and drying it, adding the sample treated in step (1), incubating at room temperature, and then washing the plate; (3) Add the HRP-labeled antibody to the system of step (2), incubate at room temperature for a period of time, wash the plate with washing solution and spin dry; (4) adding a color developing solution to the system after the reaction in step (3), developing the color for a period of time at room temperature in the dark, and then adding a stop solution to terminate the reaction; (5) Use a microplate reader for dual-wavelength detection, subtract the OD value at 630 nm from the OD value at 450 nm, and calculate the results using a standard curve.

7. The detection method of a detection kit for a pre-tumor marker according to claim 6, wherein, The test sample is blood.

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