A kit for early cancer screening and auxiliary diagnosis and its application

Abstract

The present invention discloses a kit for early screening and auxiliary diagnosis of cancer and its application, which belongs to the field of in vitro diagnosis. The kit for early screening and auxiliary diagnosis of cancer provided by the present invention includes a capture antibody magnetic bead complex, a fluorescent microsphere detection antibody complex, a magnetic bead cleaning solution, a magnetic bead preservation solution, an antigen diluent, a calibrator and a quality control product; the capture antibody magnetic bead complex and the fluorescent microsphere detection antibody complex are prepared from monoclonal antibodies against HMGA1 protein. The detection method for early screening and auxiliary diagnosis of cancer provided by the present invention detects the HMGA1 content in human serum or urine samples through a kit, has strong specificity, can quickly and high-throughput detect a large number of samples, can effectively judge the risk of cancer in the population to be tested, thereby facilitating early detection and early treatment of relevant patients, improving treatment prognosis, and reducing mortality.

Description

Technical Field

[0001] The present invention relates to the field of in vitro diagnosis, and in particular to a kit for early cancer screening and auxiliary diagnosis and its application. Background Art

[0002] Currently, serum cancer antigen levels are widely used for the initial assessment of cancer. However, because these cancer antigens can also be elevated under other physiological and pathological conditions (such as inflammation), their lack of specificity makes screening and early detection unreliable. Urine also contains a variety of molecules that may serve as biomarkers, including excreted proteins, antibodies, RNA, endogenous metabolites, and organic compounds. Compared with blood, urine has several key advantages as a source of cancer biomarkers. First, urine is easy to obtain, non-invasive, highly acceptable to patients, and available in unlimited quantities. Second, as a product of homeostasis, urine likely reflects changes in chemical composition from multiple body sites. Therefore, there is an urgent need to develop a highly sensitive and specific detection kit using urine as a test sample for early cancer screening and auxiliary diagnosis to benefit more patients.

[0003] The high-mobility group A (HMGA) family of proteins is a group of non-histone chromosomal proteins, including HMGA1a, HMGA1b, HMGA1c, and HMGA2. They bind to AT-rich DNA sequences through their AT-hook domains, inducing DNA bending and affecting gene expression by enhancing promoter binding of transcription factors. HMGA is a key regulatory factor during embryonic development. HMGA genes are significantly expressed during embryonic development but are virtually absent in normal adult tissues. However, recent studies have revealed that HMGA proteins also play a crucial role in the development and progression of both benign and malignant tumors. Data from the TCGA database show that HMGA1 is highly expressed in patients with malignant tumors, and its overexpression naturally leads to misregulation of affected genes, contributing to tumor development and progression. HMGA1 has been reported to play a key role in promoting thyroid cancer by inhibiting p53 and inducing TGF-β1. HMGA1 also promotes the development of non-small cell lung cancer through transcriptional regulation of the Wnt signaling pathway, miR-137, and miR-214. In triple-negative breast cancer, extracellular HMGA1 acts as a ligand for the receptor for advanced glycation end products (RAGE), forming an HMGA1-RAGE autocrine loop and inducing pERK signaling, thereby increasing tumor cell migration and invasion. The recent rise of metabolomics has also demonstrated that HMGA1 can increase glucose uptake, promote aerobic glycolysis, and contribute to the progression of gastric cancer. Cytoplasmic HMGA1 also inhibits p53 mitochondrial apoptosis and is associated with more aggressive tumor histotypes. Therefore, HMGA1 is a potential important tumor biomarker and can serve as an indicator for auxiliary diagnosis of cancer. Furthermore, HMGA1 is a small protein of 10.6-12 kDa and has been reported to be excreted in urine. Kits for detecting urinary HMGA1 concentrations have great potential for early cancer screening and auxiliary diagnosis. Summary of the Invention

[0004] In order to overcome the deficiencies of the prior art, one of the objectives of the present invention is to provide a kit for early screening and auxiliary diagnosis of cancer, which can detect the level of serum cancer antigens in urine samples with high sensitivity and strong specificity.

[0005] A second object of the present invention is to provide a detection method for early screening and auxiliary diagnosis of cancer.

[0006] One of the purposes of the present invention is achieved by the following technical solution:

[0007] A kit for early cancer screening and auxiliary diagnosis, comprising a capture antibody-magnetic bead complex, a fluorescent microsphere detection antibody complex, a magnetic bead cleaning solution, a magnetic bead preservation solution, an antigen diluent, a calibrator, and a quality control product.

[0008] Furthermore, the capture antibody-magnetic bead complex and the fluorescent microsphere detection antibody complex are prepared from monoclonal antibodies against HMGA1 protein.

[0009] Furthermore, the heavy chain amino acid sequence of the monoclonal antibody against HMGA1 protein in the capture antibody magnetic bead complex is shown as SEQ ID NO.1, and the light chain amino acid sequence is shown as SEQ ID NO.2; the heavy chain amino acid sequence of the monoclonal antibody against HMGA1 protein in the fluorescent microsphere detection antibody complex is shown as SEQ ID NO.3, and the light chain amino acid sequence is shown as SEQ ID NO.4.

[0010] Furthermore, the preparation process of the capture antibody magnetic bead complex is as follows:

[0011] 1) Wash the magnetic beads and add activation solution to activate the magnetic beads for 20-40 minutes;

[0012] 2) mixing the HMGA1-1 monoclonal antibody with the magnetic beads prepared in step 1), adding magnetic bead coupling solution and incubating at room temperature for 1-3 hours. After the incubation, magnetic separation was performed and the supernatant was removed;

[0013] 3) washing the precipitate from step 2), adding blocking solution and continuing incubation for 1-3 hours. After the incubation, magnetic separation is performed and the supernatant is removed;

[0014] 4) Washing the precipitate from step 3) and resuspending it in magnetic bead storage solution to obtain a capture antibody-magnetic bead complex.

[0015] Furthermore, the activation solution in step 1) is a 20 mg / mL N-hydroxysuccinimide solution, and the solvent of the solution is 0.1 M MES buffer.

[0016] Furthermore, the blocking solution in step 2) is a 20 mg / mL BSA solution, and the solvent of the solution is 20 mM PBS.

[0017] Furthermore, the preparation process of the fluorescent microsphere detection antibody complex is as follows: after activating the fluorescent microspheres for 20-40 minutes, adding HMGA1-2 monoclonal antibody and incubating for 1-3 hours, centrifuging and discarding the supernatant, adding blocking solution and continuing to incubate for 1-3 hours, centrifuging and discarding the supernatant, resuspending with labeled antibody preservation solution, and storing at 4°C in the dark.

[0018] Furthermore, the labeled antibody storage solution is a 0.9% NaCl and 0.2% FBS solution, and the solvent of the solution is 50mM PBS.

[0019] Furthermore, the magnetic bead cleaning solution is 10mM PBS buffer.

[0020] Furthermore, the magnetic bead storage solution is a 0.09% sodium azide and 0.01% Tween 20 solution, and the solvent of the solution is 20 mM HEPES.

[0021] Furthermore, the antigen diluent is a 1% BSA, 0.01% proclin300 solution, and the solvent of the solution is a 10 mM PBS buffer.

[0022] Furthermore, the calibrator and quality control product are HMGA1 protein antigen.

[0023] The second object of the present invention is achieved by adopting the following technical solution:

[0024] The kit for early cancer screening and auxiliary diagnosis is used to detect the content of HMGA1 protein in serum or urine samples.

[0025] Furthermore, the detection step is:

[0026] (1) Mix the test sample with the capture antibody magnetic bead complex, incubate at 37°C for 10-30 min, magnetically adsorb and discard the supernatant, and wash with magnetic bead washing solution;

[0027] (3) Add fluorescent microspheres to detect the antibody complex, incubate at 37°C for 10-30 min, and discard the supernatant by magnetic adsorption;

[0028] (3) Washing the beads with magnetic cleaning solution, resuspending the beads in magnetic storage solution, and placing them in a single molecule fluorescence immunoassay analyzer to detect the signal value;

[0029] (4) Determine whether HMGA1 is positive in the test sample.

[0030] Compared with the prior art, the present invention has the following beneficial effects:

[0031] The detection method for early cancer screening and auxiliary diagnosis provided by the present invention detects the HMGA1 content in human serum or urine samples through a kit. It has strong specificity and can quickly and high-throughput detect large quantities of samples. It can effectively determine the risk of cancer in the population to be tested, thereby facilitating early detection and treatment of relevant patients, improving treatment prognosis, and reducing mortality. BRIEF DESCRIPTION OF THE DRAWINGS

[0032] Figure 1 It is a standard curve for early cancer screening and auxiliary diagnosis. DETAILED DESCRIPTION

[0033] The present invention will be further described below in conjunction with the accompanying drawings and specific implementation methods. It should be noted that, under the premise of no conflict, the various embodiments or technical features described below can be arbitrarily combined to form new embodiments.

[0034] Example 1

[0035] Acquisition of monoclonal antibodies against HMGA1 protein:

[0036] Five female BALB / c mice were immunized subcutaneously using recombinant human HMGA1 protein (Abcam, Catalog No. ab109927) as an antigen. For the first immunization, 50 μL of recombinant human HMGA1 protein was mixed with an equal volume of Freund's complete adjuvant and emulsified, then injected at multiple points on the back of the mouse. For the second immunization, 50 μL of incomplete Freund's adjuvant mixed with an equal volume of recombinant human HMGA1 protein was injected subcutaneously on the back of the mouse. Booster immunizations were repeated three times every two weeks. One week after the final immunization, 100 μL of recombinant human HMGA1 protein was injected into the tail vein for a pulse immunization. Three days later, blood was collected, and the antibody titer of each mouse's serum was determined by indirect ELISA. Splenocytes from the mouse with the highest serum titer were selected for cell fusion, and serum from healthy female BALB / c mice was used as a negative control.

[0037] Mouse spleens were removed under sterile conditions, and a splenocyte suspension was prepared. The spleen cells were mixed with SP2 / 0 myeloma cells at a ratio of 10:1. 50% polyethylene glycol 1500 was used as a fusion agent, and the cells were fused in a 37°C water bath for 2 minutes. The fused cells were aliquoted into 96-well cell culture plates containing feeder cells and cultured in HAT selection medium (20% fetal bovine serum, 1% double-antibody antibody, 1 / 50 volume of 50× HAT medium, and incomplete 1640 medium) at 37°C in a 5% CO2 incubator. When cell colonies reached 50% of the well area, the cell culture supernatant was collected, positive hybridoma cells were screened by ELISA, and subcloned by limiting dilution. The hybridoma cells to be cloned were gently aspirated from the wells, the number of viable cells was counted using a hemocytometer, and the cells were diluted to a concentration of 5 cells / mL in RPMI-1640 complete medium. Add the cell suspension to a 96-well plate containing prepared feeder cells (spleen cells from unimmunized mice) and add 100 μL of cell diluent per well, ensuring a maximum of one cell per well. Culture in an incubator at 37°C and 5% CO2. Add one drop of medium on day 4 and change the medium on day 7. Cell colonies may form on day 9. Test for antibody activity and transfer the cells to 24-well plates for expansion.

[0038] The two most active monoclonal antibodies were named HMGA1-1 and HMGA1-2, and their heavy and light chain variable region sequences were determined. The heavy chain amino acid sequence of monoclonal antibody HMGA1-1 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 HMGA1-2 is shown in SEQ ID NO. 3, and the light chain amino acid sequence is shown in SEQ ID NO. 4.

[0039] SEQ ID NO.1

[0040] EVQLTQSTPELVRCGASVEMSCKASIYTFTVTNHESRWVRNKMATGIDWILFRDPTTQMIANKVTLTSERSSSTVFCDLSTLTSEEQILYYCTRYGNEQSVETGPWGQGTTLNVSS;

[0041] SEQ ID NO.2

[0042] DIVCNNSPSSLVVTAGNEVNASCFSTHECWYQTRPANTCKLLVYKADGKLVAPGVMEKFLASGSGTEYINTITSAQVDELADYFCMNTTRFKTTNRYAFGLLNKLDAK;

[0043] SEQ ID NO.3

[0044] EVPVVDSAGVLLNPAGSLRISMGASVFTYTTGNSRDMKWVRQSCERRLDAGAGYGPMNIKHYTISRESVRNSLNLSCSTVKSEDNGPFYCVRYNGYDAMEGRWGQANTVTVSS;

[0045] SEQ ID NO.4

[0046] AVITETLASNSPADNLSATCSVSDGWVTEHCDKLFTGILARTSNGDPDGVPVKYSGSLIGERGVLTISVGQTEEDAIYFCYENFYGYACDTFTRITVK.

[0047] When the hybridoma cells in the 24-well plate grow well, take 5×10 5Cells were injected into the peritoneal cavity of mice, and ascites was collected 7 days later. Freshly collected ascites was centrifuged at 2000 rpm for 15 minutes to remove cellular components. The supernatant was filtered through a 0.45µm filter and diluted with an equal volume of equilibration buffer (20 mM Na2HPO4, 0.15 M NaCl, pH 7.0). 0.5 ml of Protein G was loaded onto the chromatography column, returned to room temperature, and the peristaltic pump, recorder, and detector were started. The column was equilibrated with equilibration buffer, followed by sample loading. The flow-through was collected and the column was washed again with equilibration buffer to thoroughly remove contaminants. The elution buffer was replaced with 0.1 M glycine, pH 3.0, and the elution peaks were collected in collection tubes containing an appropriate amount of neutralization buffer (1 M Tris-HCl, pH 8.5). These peaks were designated as eluate 1, eluate 2, etc. Immediately after collection, the eluate was neutralized with Tris-HCl buffer (pH 9.0) to maintain the antibody at a neutral or slightly alkaline pH. The concentration of the purified HMGA1-1 and HMGA1-2 monoclonal antibodies was adjusted to 1 mg / mL and stored at -20°C.

[0048] Example 2

[0049] A kit for early cancer screening and auxiliary diagnosis:

[0050]

[0051] (1) Preparation of capture antibody magnetic bead complex

[0052] The activator N-hydroxysuccinimide (NHS) was prepared in magnetic bead coupling solution (0.1M MES buffer) to a concentration of 20 mg / mL. 200 μL of 3 μm carboxyl magnetic microparticles (beaverbio, cat. no. 70117-5) was transferred to a centrifuge tube and placed on a magnetic stand for 3 minutes, after which the supernatant was removed. 1 mL of activation solution was added and activated at room temperature for 30 minutes. The beads were then placed on a magnetic stand for 3 minutes, after which the supernatant was removed. HMGA1-1 was used as the capture antibody. 100 μL of the capture antibody was added to the activated carboxyl magnetic bead solution, and the magnetic bead coupling solution was added to a total volume of 500 μL. The mixture was mixed thoroughly, incubated at room temperature for 2 hours, and placed on a magnetic stand for 3 minutes, after which the supernatant was removed. The capture antibody-magnetic bead complex was washed three times with 10 mM PBS buffer, mixed thoroughly on a vortex, and then magnetically separated. Add 500 μL of blocking buffer (20 mg / mL BSA dissolved in 20 mM PBS), vortex to disperse the magnetic particles, incubate at room temperature for 1 hour, place on a magnetic stand for 3 minutes, and remove the supernatant. Wash the capture antibody-magnetic bead complex three times with 10 mM PBS buffer, mix thoroughly on a vortexer, and then perform magnetic separation. Add 10 mL of magnetic bead storage solution (0.09% sodium azide, 0.01% Tween 20 dissolved in 20 mM HEPES) and vortex to mix thoroughly to obtain a 0.5 mg / mL capture antibody-magnetic bead complex suspension. Store at 4°C.

[0053] (2) Preparation of fluorescent microspheres to detect antibody complexes

[0054] Take 200 μL of 200 nm carboxyl-modified fluorescent microspheres (Thermo Fisher, Cat. No. F8888) and add 1 mL of label conjugation solution (20 mM HEPES buffer). Centrifuge at 12,000 rpm for 10 minutes, remove the supernatant, and repeat washing three times. Add 1 mL of activation solution and activate at 25°C for 30 minutes. Add 100 μL of HMGA1-2 as the fluorescent detection antibody to the activated fluorescent microsphere solution, mix thoroughly, incubate at room temperature for 2 hours, centrifuge at 12,000 rpm for 10 minutes, and remove the supernatant. Add 500 μL of blocking solution and incubate for 1 hour. Centrifuge at 12,000 rpm for 10 minutes, and remove the supernatant. Add 50 mL of fluorescent microsphere-labeled antibody storage solution (0.9% NaCl and 0.2% FBS dissolved in 50 mM PBS) to a final concentration of 0.1 mg / mL of the fluorescent microsphere-detection antibody complex. Store at 4°C in the dark.

[0055] (3) Preparation of calibrators

[0056] Dissolve bovine serum albumin (BSA) and the preservative proclin 300 in 500 mL of 10 mM PBS buffer to prepare an antigen diluent containing 1% BSA and 0.01% proclin 300. Mix thoroughly and filter through a 0.22 μm microporous filter. Dilute the HMGA1 protein antigen with the antigen diluent to calibrant concentrations of 1 pg / mL, 10 pg / mL, 100 pg / mL, 200 pg / mL, 300 pg / mL, 400 pg / mL, and 500 pg / mL.

[0057] (4) Preparation of quality control products

[0058] The HMGA1 protein antigen was diluted with antigen diluent to 5 pg / mL, 50 pg / mL, and 250 pg / mL, which were respectively referred to as quality control product 1, quality control product 2, and quality control product 3.

[0059] (5) Kit standard curve determination

[0060] Add 100 μL of each calibrant with different concentration gradients to an EP tube. Add 100 μL of the capture antibody-magnetic bead complex, shake to mix, and incubate at 37°C for 20 minutes. Place the EP tube on a magnetic stand for 3 minutes, and completely aspirate the supernatant with a pipette. Add 600 μL of magnetic bead wash buffer, mix thoroughly, and then place the EP tube on a magnetic stand for 3 minutes. Aspirate the supernatant with a pipette, and repeat the wash three times. Add 100 μL of the fluorescent microsphere detection antibody complex, shake to mix, and incubate at 37°C for 20 minutes. Place the EP tube on a magnetic stand for 3 minutes, and completely aspirate the supernatant with a pipette. Add 600 μL of magnetic bead wash buffer, mix thoroughly, and then place the EP tube on a magnetic stand for 3 minutes. Aspirate the supernatant with a pipette, and repeat the wash three times to remove excess fluorescent detection antibody. Resuspend the magnetic beads in magnetic bead storage buffer. Take 10 μL of liquid and add it to the microfluidic chip, place it in the single molecule fluorescence immunoassay analyzer, complete the sample detection and result analysis, repeat each concentration point 10 times, and draw a standard curve based on the results, such as Figure 1 shown.

[0061] Add 100 μL of each of the three quality control products to an EP tube, add 50 μL of the capture antibody-magnetic bead complex, shake to mix, and incubate at 37°C for 20 minutes. Place the EP tube on a magnetic stand for 3 minutes, and completely aspirate the supernatant with a pipette. Add 600 μL of magnetic bead wash buffer, mix thoroughly, and then place the EP tube on a magnetic stand for 3 minutes. Aspirate the supernatant with a pipette, and repeat the wash three times. Add 100 μL of fluorescent microsphere detection antibody complex, shake to mix, and incubate at 37°C for 20 minutes. Place the EP tube on a magnetic stand for 3 minutes, and completely aspirate the supernatant with a pipette. Add 600 μL of magnetic bead wash buffer, mix thoroughly, and then place the EP tube on a magnetic stand for 3 minutes. Aspirate the supernatant with a pipette, and repeat the wash three times to remove excess fluorescent detection antibody. Resuspend the magnetic beads in magnetic bead storage buffer. 10 μL of liquid was added to the microfluidic chip and tested in a single-molecule fluorescence immunoassay instrument. It was found that the concentration of the quality control product was consistent with the prepared concentration ( Figure 1 ).

[0062] Test Example 1

[0063] Blood sample testing:

[0064] Fifteen blood samples from healthy individuals (numbers 1-15) and 15 blood samples from ovarian cancer patients (numbers 16-30) were collected. After blood collection, the samples were allowed to clot at room temperature and then centrifuged at 3000 rpm for 15 minutes. The supernatant was collected and 100 μL of each sample was transferred to an Eppendorf tube. 100 μL of the capture antibody-magnetic bead complex was added, the tubes were shaken to mix, and the tubes were incubated at 37°C for 20 minutes. The tubes were placed on a magnetic stand for 3 minutes, and the supernatant was completely aspirated. 600 μL of magnetic bead wash buffer was added, the tubes were mixed thoroughly, and the tubes were placed on a magnetic stand for 3 minutes. The supernatant was aspirated and the wash cycle was repeated three times. 100 μL of the fluorescent microsphere detection antibody complex was added, the tubes were shaken to mix, and the tubes were incubated at 37°C for 20 minutes. The tubes were placed on a magnetic stand for 3 minutes, and the supernatant was completely aspirated. Add 600 μL of magnetic bead washing buffer, mix thoroughly, and place the EP tube on a magnetic stand for 3 minutes. Aspirate the supernatant with a pipette, repeat the wash three times to remove excess fluorescent detection antibody, and resuspend the beads in magnetic bead storage buffer. Add 10 μL of the solution to the microfluidic chip and place it in a single-molecule fluorescence immunoassay analyzer to measure the signal. Repeat three times and average the results. The results are shown in Table 2.

[0065]

[0066] The results showed that the test kit can accurately distinguish between blood samples from healthy people and blood samples from cancer patients. The blood of healthy people does not contain HMGA1 protein, the signal value is 0, and it is judged as negative. The blood of cancer patients contains HMGA1 protein, the signal value can be detected, and it is judged as positive.

[0067] Test Example 2

[0068] Urine sample testing:

[0069] Fifteen urine samples from healthy individuals (numbered 1-15) and 15 urine samples from ovarian cancer patients (numbered 16-30) were collected. 100 μL of each sample was transferred to an EP tube. 100 μL of the capture antibody-magnetic bead complex was added, the tube was shaken to mix, and the sample was incubated at 37°C for 20 minutes. The tube was placed on a magnetic stand for 3 minutes, and the supernatant was completely aspirated. 600 μL of magnetic bead wash buffer was added, the sample was mixed thoroughly, and the tube was placed on a magnetic stand for 3 minutes. The supernatant was aspirated, and this was repeated three times. 100 μL of the fluorescent microsphere detection antibody complex was added, the sample was shaken to mix, and the sample was incubated at 37°C for 20 minutes. The tube was placed on a magnetic stand for 3 minutes, and the supernatant was aspirated. 600 μL of magnetic bead wash buffer was added, the sample was mixed thoroughly, and the tube was placed on a magnetic stand for 3 minutes. The supernatant was aspirated, and this was repeated three times to remove excess fluorescent detection antibody. The beads were then resuspended in magnetic bead storage buffer. 10 μL of liquid was added to the microfluidic chip, and the chip was placed in a single-molecule fluorescence immunoassay instrument to detect the signal value. The test was repeated three times and the average value was taken. The results are shown in Table 3.

[0070]

[0071] Urine samples from healthy people do not contain HMGA1 protein, the signal value is 0, and it is judged as negative. Urine samples from cancer patients contain HMGA1 protein, the signal value can be detected, and it is judged as positive.

[0072] The above embodiments are only preferred embodiments of the present invention and cannot be used to limit the scope of protection of the present invention. Any non-substantial changes and replacements made by technicians in this field on the basis of the present invention fall within the scope of protection required by the present invention.

Claims

1. A kit for early screening and auxiliary diagnosis of cancer, characterized in that: Including capture antibody magnetic bead complex, fluorescent microsphere detection antibody complex, magnetic bead cleaning solution, magnetic bead storage solution, antigen diluent, calibrator and quality control products; The capture antibody magnetic bead complex and the fluorescent microsphere detection antibody complex are prepared from monoclonal antibodies against HMGA1 protein; the two most active monoclonal antibodies are named HMGA1-1 and HMGA1-2; The preparation process of the capture antibody magnetic bead complex is as follows: A: Wash the magnetic beads and add activation solution to activate the magnetic beads for 20-40 minutes; B: Mix the anti-HMGA1 protein monoclonal antibody HMGA1-1 with the magnetic beads in step A, add magnetic bead coupling solution and incubate at room temperature for 1-3 hours. After the incubation, perform magnetic separation and remove the supernatant; C: Wash the precipitate from step B, add blocking solution and continue incubation for 1-3 hours. After the incubation, perform magnetic separation and remove the supernatant; D: Wash the precipitate from step C and resuspend it in magnetic bead storage solution to obtain a capture antibody magnetic bead complex; The preparation process of the fluorescent microsphere detection antibody complex is as follows: After activating the fluorescent microspheres for 20-40 minutes, add the anti-HMGA1 protein monoclonal antibody HMGA1-2 and incubate for 1-3 hours, centrifuge and discard the supernatant, add the blocking solution and continue incubation for 1-3 hours, centrifuge and discard the supernatant, resuspend with the labeled antibody preservation solution, and store at 4°C in the dark; The heavy chain amino acid sequence of the monoclonal antibody HMGA1-1 against HMGA1 protein in the capture antibody magnetic bead complex 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 HMGA1-2 against HMGA1 protein in the fluorescent microsphere detection antibody complex is shown in SEQ ID NO.3, and the light chain amino acid sequence is shown in SEQ ID NO.4; SEQ ID NO.1 EVQLTQSTPELVRCGASVEMSCKASIYTFTVTNHESRWVRNKMATGIDWILFRDPTTQMIANKVTTLTSERSSSTVFCDLSTLTSEEQILYYCTRYGNEQSVETGPWGQGTTLNVSS; SEQ ID NO.2 DIVCNNSPSSLVVTAGNEVNASCFSTHECWYQTRPANTCKLLVYKADGKLVAPGVMEKFLASGSGTEYINTITSAQVDELADYFCMNTTRFKTTNRYAFGLLNKLDAK; SEQ ID NO.3 EVPVVDSAGVLLNPAGSLRISMGASVFTYTTGNSRDMKWVRQSCERRLDAGAGYGPMNIKHYTISRESVRNSLNLSCSTVKSEDNGPFYCVRYNGYDAMEGRWGQANTVTVSS; SEQ ID NO.4 AVITETLASNSPADNLSATCSVSDGWVTEHCDKLFTGILARTSNGDPDGVPVKYSGSLIGERGVLTISVGQTEEDAIYFCYENFYGYACDTFTRITVK; The magnetic bead cleaning solution is 10mM PBS buffer; the magnetic bead preservation solution is 0.09% sodium azide, 0.01% Tween 20 solution, and the solvent of the magnetic bead preservation solution is 20 mM HEPES; the antigen diluent is 1% BSA, 0.01% proclin300 solution, and the solvent of the antigen diluent is 10mM PBS buffer; the calibrator and quality control product are HMGA1 protein antigen.

2. The kit for early screening and auxiliary diagnosis of cancer according to claim 1, characterized in that: The activation solution described in step A is a 20 mg / mL N-hydroxysuccinimide solution, and the solvent of the activation solution is a 0.1 M MES buffer.

3. The kit for early screening and auxiliary diagnosis of cancer according to claim 1, characterized in that: The blocking solution in step B is a 20 mg / mL BSA solution, and the solvent of the blocking solution is 20 mM PBS.

4. The kit for early screening and auxiliary diagnosis of cancer according to claim 1, characterized in that: The labeled antibody storage solution is a 0.9% NaCl and 0.2% FBS solution, and the solvent of the labeled antibody storage solution is 50mM PBS.

5. Use of the kit for early screening and auxiliary diagnosis of cancer according to any one of claims 1 to 4, characterized in that: Used to detect the level of HMGA1 protein in serum or urine samples.

6. The use of the kit for early screening and auxiliary diagnosis of cancer according to claim 5, characterized in that: The detection steps are: (1) Mix the test sample with the capture antibody magnetic bead complex, react at 37°C for 10-30 min, discard the supernatant by magnetic adsorption, and wash with magnetic bead washing solution; (2) Add fluorescent microspheres to detect the antibody complex, incubate at 37°C for 10-30 min, and discard the supernatant by magnetic adsorption; (3) washing the magnetic beads with a cleaning solution, resuspending the magnetic beads with a storage solution, and placing the beads in a single molecule fluorescence immunoassay analyzer to detect the signal value; (4) Determine whether HMGA1 is positive in the test sample.

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