Enrichment, detection method and application of GPC-3 complex in blood samples

Abstract

The present invention provides enrichment, detection methods and applications of GPC-3 complexes in blood samples. The enrichment method includes A. collecting peripheral blood from liver cancer patients, placing it in an anticoagulant tube and mixing the whole blood; B. adding multiple volumes of red blood cell lysis solution, pipetting and mixing, lysing on ice, centrifuging and discarding the red supernatant. If the cell pellet is still red, repeat this step; C. washing the pellet 1-2 times, adding PBS or normal saline to resuspend the pellet, centrifuging and discarding the supernatant; D. counting white blood cells, adding PBS to resuspend the cell pellet, taking an appropriate amount to fill the pool, letting it stand for 2-3 minutes, and manually counting the white blood cells using the four large squares in the four corners of the low-power microscope. The calculation formula is as follows: WBC / L=N / 4*10 5 *10 4 , and calculate the number of white blood cells per milliliter of the original suspension, WBC / ml original suspension = N / 4*10 4 *Dilution factor; E. Add lysis buffer containing 1% protease inhibitor and 1% phosphatase inhibitor to the obtained cells, refrigerate and let stand for a certain period of time, and then centrifuge to obtain the supernatant as the sample to be tested to achieve GPC-3 enrichment.

Description

Technical Field

[0001] The present invention belongs to the field of biological detection technology, and specifically relates to a method and application of enrichment and detection of GPC-3 in human blood samples, so as to realize rapid and systematic detection of GPC-3 in blood. Background Art

[0002] Glypican 3 (GPC-3) is a member of the glypican family. Human GPC-3 is encoded by the GPC3 gene (Entrez Gene ID: 2719), consisting of 580 amino acids with a molecular weight of approximately 66 kDa. Other aliases for this protein include DGSX, GTR2-2, MXR7, OCI-5, SDYS, SGB, SGBS, and SGBS1. GPC-3 is a complex formed by covalent linkage of protein, sugar, and lipid, anchored primarily to the outer surface of the cell membrane via glycosylphosphatidyl inositol (GPI). Its signal peptide is located at amino acids 1-25 (Filmus J, Capurro MI, Rast J. Glypicans. Genome Biol 2008; 9(5): 224). GPC-3 is abundantly expressed during early development of human embryonic cells and participates in biological processes such as tissue cell morphogenesis and growth. GPC-3 can be detected during fetal liver formation; however, it is usually undetectable in normal adult liver tissue.

[0003] Studies have shown that GPC-3 is specifically highly expressed in hepatocellular carcinoma, with elevated expression in precancerous lesions, atypical hyperplasia, and cancerous tissues, while negative in normal liver tissue or benign liver lesions (Capurro MI et al. Glypican-3: A novel serum and histochemical marker for hepatocellular carcinoma. Gastroenterology 2003; 125(1): 89-97). Its expression level gradually increases during the malignant progression of HCC, with a particularly high detection rate in AFP-negative HCC patients. This has clinical significance for indicating recurrence and metastasis, and for evaluating the efficacy of surgical treatment. Biological function studies have shown that GPC-3 upregulates the Wnt signaling pathway in an autocrine / paracrine manner, regulating the growth, migration, and adhesion of HCC cells, thereby promoting their malignant transformation (HoM, Kim H. Glypican-3: A new target for cancer immunotherapy. Eur J Cancer 2011; 47(3): 333-8). GPC-3 levels in HCC patients are significantly correlated with phenotypes such as HBV infection, TNM stage, periportal tumor thrombus, and extrahepatic metastasis. Downregulating GPC-3 with specific siRNA or anti-GPC-3 antibodies can alter biological behaviors such as cell migration, invasion, and metastasis. Silencing GPC-3 gene transcription can inhibit the growth of xenografts in nude mice (Li Wang et al., Glypican-3 is a biomarker and a therapeutic target of hepatocellular carcinoma, Hepatobiliary Pancreat Dis Int. 2015 Aug; 14(4): 361-6).

[0004] Immunohistochemical testing of liver biopsies and surgical specimens has shown that GPC-3 has a wide range of sensitivity (56.8% to 100%) and specificity (90% to 100%) in the detection of HCC, with higher sensitivity in poorly differentiated carcinomas (Tremosini S et al. Prospective validation of an immunohistochemical panel (glypican 3, heat shock protein 70 and glutamine synthetase) in liver biopsies for diagnosis of very early hepatocellular carcinoma [J]. Gut, 2012, 61(10): 1481-1487.). The diagnostic accuracy of serum GPC-3 combined with alpha-fetoprotein for HCC is as high as 94.3%. In addition, some literature reports have also shown that high expression of GPC-3 can be detected in germ cell tumors, embryonal malignancies, ovarian tumors, and melanoma.

[0005] To enable GPC-3 detection, the applicant has previously conducted a series of studies on the preparation and purification of GPC-3 monoclonal antibodies. They jointly applied for a GPC-3 detection kit with Fuzhou Maixin Biotechnology Development Co., Ltd., and obtained a Class III medical device registration certificate from the China Food and Drug Administration [Registration Number: Guo Shi Yao Jian Xie (Zhun) Zi 2014 No. 3401502]. The kit has been clinically deployed. This kit detects GPC-3 expression in postoperative tissue samples using immunohistochemistry. It can be used for the pathological diagnosis and typing of liver cancer, particularly in difficult cases of liver tumors and for differential diagnosis of benign and malignant tumors.

[0006] However, this test kit can only detect postoperative tissue samples or invasive biopsy tissue samples, and cannot detect body fluids such as blood. Furthermore, the single operation is time-consuming, and its non-invasiveness and timeliness are difficult to guarantee. GPC-3 protein expression is mainly found in the cell cytoplasm, with a very small amount of GPC-3 secreted outside the cell. The existing detection method for blood GPC-3 is ELISA testing of human peripheral blood serum / plasma, which has low expression rate, low detection rate, and low sensitivity, making it unsuitable for clinical application.

[0007] To effectively detect GPC-3 in blood, a Chinese invention patent with patent number CN111593024A uses GPC3 antibodies combined with immunomagnetic beads to capture GPC3-positive CTCs, thereby improving the efficiency of liver cancer sorting. However, this method requires pre-processing the blood sample, which involves removing plasma proteins and plasma nucleic acids, removing red blood cells, centrifuging the sample, and removing white blood cells. Magnetic particles bound to GPC3 antibodies are then slowly added to separate and capture CTCs. The magnetic beads are then washed and detected using an immunofluorescence probe. Although this method achieves liver cancer diagnosis using blood as a detection matrix to a certain extent, the blood pretreatment method is complex and the CTC enrichment effect is poor, and the expected translation is questionable. Summary of the Invention

[0008] The present invention addresses the problem that the GPC-3 content in blood samples is low and difficult to detect using conventional detection methods. It provides a method for enriching and detecting GPC-3 complexes in blood samples, and also provides a kit for detecting the GPC-3 content in blood samples.

[0009] In order to achieve the above object, the technical solution adopted by the present invention is as follows:

[0010] In a first aspect, the present invention provides a method for enriching GPC-3 complexes in a blood sample, comprising the following steps:

[0011] A. Blood sample collection

[0012] Peripheral blood was collected from liver cancer patients and placed in anticoagulant tubes, and then mixed with whole blood;

[0013] B. Red blood cell lysis

[0014] Add multiple volumes of red blood cell lysis buffer, pipette and mix thoroughly, lyse on ice for a certain period of time, centrifuge and discard the red supernatant. If the cell pellet still has red color, repeat this step to remove the red blood cells;

[0015] C. Precipitation and impurity removal

[0016] Wash the precipitate 1-2 times, resuspend it in PBS or saline, centrifuge and discard the supernatant to further remove impurities;

[0017] D. White blood cell count

[0018] Resuspend the cell pellet in PBS, take an appropriate amount to fill the pool, let it stand for 2-3 minutes, and manually count the white blood cells using the four large squares at the four corners of the low-power microscope. The calculation formula is as follows: WBC / L = N / 4*10 5 *10 4 , and calculate the number of white blood cells per milliliter of the original suspension, WBC / ml original suspension = N / 4*10 4 *Dilution multiple, when the number of white blood cells is not less than 10 7When the , proceed to the next step;

[0019] E. Enrichment of GPC-3 complex

[0020] Specific magnetic bead antibodies were used to purify and separate the target CTC cells. The obtained cells were added with a lysis buffer containing 1% protease inhibitors and 1% phosphatase inhibitors. After refrigeration and standing for a certain period of time, the supernatant was centrifuged to obtain the sample to be tested to achieve GPC-3 enrichment.

[0021] Preferably, in step B, the red blood cell lysis solution is an existing product, the main components of which are: ammonium chloride, potassium bicarbonate, EDTA sodium salt, etc.

[0022] The volume ratio of red blood cell lysis buffer to whole blood was 6:1. Mix by pipetting, lyse on ice for 4-6 minutes, centrifuge at 4°C and 400 g for 5 minutes, and discard the red supernatant.

[0023] In step C, when resuspending the precipitate, the volume of PBS or physiological saline should be 4 to 5 times that of the precipitate, centrifuge at 4°C and 400g for 2 to 3 minutes, and discard the supernatant.

[0024] In step E, the protease inhibitors and phosphatase inhibitors are also existing products, and their main components are: sodium pyrophosphate substances, aprotinin, E64 and other polypeptide compounds that inhibit the degradation of protein substances.

[0025] The lysis buffer includes RIPA lysis buffer or NP-40 lysis buffer. The lysis buffer is RIPA lysis buffer, which includes: the main components of the RIPA lysis buffer are Triton X-100 and SDS in a Tris salt buffer system; the main components of the NP-40 lysis buffer are: NP-40 and sodium deoxycholate solution in a Tris salt buffer system.

[0026] The cells were added with lysis buffer, refrigerated and allowed to stand for 30 minutes, and then centrifuged at 4°C and 14,000 g for 10 minutes to obtain the supernatant as the sample to be tested.

[0027] After the GPC-3 complex is enriched by the above method, the specific detection mode is further explored, and the detection can be achieved using existing commonly used clinical detection methods.

[0028] A second aspect of the present invention provides a method for rapidly detecting GPC-3 in a human blood sample, comprising the following steps:

[0029] (1) Enrichment of GPC-3 complex: The above-mentioned enrichment method was used.

[0030] (2) GPC-3 detection: chemiluminescence detection mode or enzyme-linked immunosorbent assay mode.

[0031] Preferably, the steps for establishing the chemiluminescence detection mode are as follows:

[0032] (1) Prepare 10 mL of GPC-3 coating antibody at a concentration of 5 μg / mL, add 100 μL / well to a 96-well white high-absorption chemiluminescent plate, and store at 4°C for 18 h or overnight;

[0033] (2) Remove the reaction plate, discard the remaining liquid, and wash three times with 0.01 mol / L PBST (pH 7.0-7.4), 300 μL / well. After the last wash, pat the reaction plate dry.

[0034] (3) Add 150 μL / well of blocking solution, let stand at room temperature for 2 h, discard the residual liquid, pat dry and air dry the reaction plate, and store in an aluminum foil bag for later use;

[0035] (4) Remove the reaction plate, prepare a gradient concentration solution of the standard, add the prepared sample solution at 100 μL / well, and react at 37°C for 60-90 min;

[0036] (5) Remove the reaction plate, discard the residual liquid, and wash three times with 0.01 mol / L PBST (pH 7.0-7.4), 250 μL / well. After the last wash, pat the reaction plate dry.

[0037] (6) Add 2 μg / mL biotin-labeled anti-GPC-3 antibody working solution to 100 μL / well, react at 37°C for 60-90 min, remove the reaction plate, discard the residual liquid, and wash three times with 0.01 mol / L PBST (pH 7.0-7.4) at 250 μL / well. After the last wash, pat the reaction plate dry.

[0038] (7) Add 100 μL / well of streptavidin-labeled HRP enzyme and react at 37°C for 20-30 min. Remove the reaction plate, discard the residual liquid, and wash three times with PBST (pH 7.0-7.4, concentration 0.01 mol / L) at 250 μL / well. After the last wash, pat the reaction plate dry.

[0039] (8) Add 100 μL / well of HRP luminescent solution and read the value using a chemiluminescence instrument.

[0040] Preferably, the steps of establishing the enzyme-linked immunosorbent assay are as follows:

[0041] (1) Prepare 10 mL of GPC-3 coating antibody at a concentration of 5 μg / mL, add 100 μL / well to a 96-well white high-absorption chemiluminescent plate, and store at 4°C for 18 h or overnight;

[0042] (2) Remove the reaction plate, discard the remaining liquid, and wash three times with 0.01 mol / L PBST (pH 7.0-7.4), 300 μL / well. After the last wash, pat the reaction plate dry.

[0043] (3) Add 150 μL / well of blocking solution, let stand at room temperature for 2 h, discard the residual liquid, pat dry and air dry the reaction plate, and store in an aluminum foil bag for later use;

[0044] (4) Remove the reaction plate, prepare a gradient concentration solution of the standard, add the prepared sample solution at 100 μL / well, and react at 37°C for 60-90 min;

[0045] (5) Remove the reaction plate, discard the residual liquid, and wash three times with 0.01 mol / L PBST (pH 7.0-7.4), 250 μL / well. After the last wash, pat the reaction plate dry.

[0046] (6) Add 2 μg / mL biotin-labeled anti-GPC-3 antibody working solution to 100 μL / well, react at 37°C for 60-90 min, remove the reaction plate, discard the residual liquid, and wash three times with 0.01 mol / L PBST (pH 7.0-7.4) at 250 μL / well. After the last wash, pat the reaction plate dry.

[0047] (7) Add 100 μL / well of streptavidin-labeled HRP enzyme and react at 37°C for 20-30 min. Remove the reaction plate, discard the residual liquid, and wash three times with PBST (pH 7.0-7.4, concentration 0.01 mol / L) at 250 μL / well. After the last wash, pat the reaction plate dry.

[0048] (8) Add 100 μL / well of TMB substrate and protect from light for 7-30 minutes. When the gradient concentration liquid of the standard shows a gradient blue change, add 100 μL / well of stop solution, which is 1 mol / L HCl solution or 2 mol / L H2SO4 solution.

[0049] By comparison with direct detection of the upper plasma / serum after centrifugation, when the chemiluminescence detection mode is used for detection, the CLIA value of the cell lysate treated by the enrichment method of the present invention is several times or even dozens of times that of the plasma / serum CLIA value; when the enzyme-linked immunosorbent assay mode is used for detection, the detection rate of plasma is very low and the sample concentration is basically undetectable, while the detection ability of the cell lysate is significantly higher than that of plasma, and the detection concentration is higher.

[0050] A third aspect of the present invention provides a kit for rapidly detecting GPC-3 content in a blood sample. The chemiluminescent detection kit includes a red blood cell lysate, a cell lysate for enriching GPC-3, a chemiluminescent plate or immunomagnetic beads pre-coated with a GPC-3 antibody, a phosphate or Tris-buffered wash solution, a blocking solution, a biotin-labeled anti-GPC-3 antibody working solution, streptavidin-labeled luminolase, and an enzymatic chemiluminescent solution.

[0051] The enzyme-linked immunosorbent assay kit includes red blood cell lysis buffer, cell lysis buffer for enriching GPC-3, an enzyme-labeled plate pre-coated with GPC-3 antibody, phosphate or Tris buffer washing solution, blocking solution, biotin-labeled anti-GPC-3 antibody working solution, streptavidin-labeled luminol enzyme, TMB substrate, and stop solution.

[0052] When the cell lysate used for enriching GPC-3 is used to enrich the GPC-3 complex, the above step E is followed.

[0053] Functions and effects of the invention

[0054] First, the present invention achieves GPC-3 enrichment through steps such as red blood cell lysis, cell pellet resuspending, and cell lysis to extract the GPC-3 complex. After enrichment, GPC-3 content can be effectively detected using commonly used clinical chemiluminescence detection or enzyme-linked immunosorbent assay (ELISA) methods. This invention solves the problem of the inability to achieve rapid and systematic detection of GPC3 in blood, achieving high expression, detection, and sensitivity. This overcomes the current clinical limitations of limited testing on postoperative tissue or biopsy tissue samples, resulting in a time-consuming, non-invasive, and time-sensitive procedure, and improves patient compliance.

[0055] Secondly, in terms of operation, the enrichment method of the present invention only requires three major procedures: red blood cell lysis, cell sedimentation resuspension, and cell lysis extraction. The pretreatment operation is simple, which greatly improves the operational convenience. In terms of result accuracy, the detection method of the present invention has high expression rate, detection rate, and sensitivity, which helps to improve clinical promotion. BRIEF DESCRIPTION OF THE DRAWINGS

[0056] Figure 1 The standard curve for the blood sample pretreatment sample of Example 1 when the chemiluminescence detection mode is used for detection;

[0057] Figure 2 This is the standard curve when the blood sample pre-treated sample of Comparative Example 1 is detected using the chemiluminescence detection mode;

[0058] Figure 3 This is the standard curve when the blood sample pretreatment sample of Comparative Example 2 is detected using the chemiluminescence detection mode;

[0059] Figure 4 This is the standard curve when the blood sample pretreatment sample in Example 1 is detected using the enzyme-linked immunosorbent assay (ELISA) mode. DETAILED DESCRIPTION

[0060] The present invention will be described in detail below with reference to the examples and accompanying drawings. However, the following examples should not be considered as limiting the scope of the present invention.

[0061] Example GPC-3 enrichment and detection in blood samples

[0062] 1. GPC-3 enrichment in blood samples

[0063] A. Blood sample collection

[0064] Peripheral blood was collected from patients with liver tumors and placed in anticoagulant tubes, and then mixed with whole blood;

[0065] B. Red blood cell lysis

[0066] Add 6 volumes of red blood cell lysis buffer, mix thoroughly by pipetting, lyse on ice for 5 minutes, centrifuge at 400 g for 5 minutes at 4°C, discard the red supernatant, and repeat this step to remove red blood cells if the cell pellet still has red color.

[0067] C. Precipitation and impurity removal

[0068] Wash the pellet 1-2 times, add 5 volumes of PBS or saline to resuspend the pellet, centrifuge at 400g for 2-3 minutes at 4°C, discard the supernatant, and further remove impurities;

[0069] D. White blood cell count

[0070] Add 100 μL PBS to resuspend the cell pellet, take 10 μL to fill the pool, let it stand for 2-3 minutes, and manually count the white blood cells using the four large squares at the four corners of the low-power microscope. The calculation formula is as follows: WBC / L = N / 4*10 5 *10 4 , and calculate the number of white blood cells per milliliter of the original suspension, WBC / ml original suspension = N / 4*10 4 *Dilution factor (10) = N / 4*10 5 ;

[0071] E. Enrichment of GPC-3 complex

[0072] Target CTCs were purified and isolated using specific magnetic beads and antibodies. The cells were then added to RIPA lysis buffer containing 1% protease inhibitors and 1% phosphatase inhibitors. After refrigeration for 30 minutes, the cells were centrifuged at 14,000 g for 10 minutes at 4°C. The supernatant was used as the sample for GPC-3 enrichment. The total protein concentration in the supernatant was measured using a BCA total protein assay kit and used as a later ratio parameter.

[0073] 2. GPC-3 test

[0074] 2.1 Establishment of chemiluminescence detection mode

[0075] (1) Prepare 10 mL of GPC-3 coating antibody at a concentration of 5 μg / mL, add 100 μL / well to a 96-well white high-absorption chemiluminescent plate (reaction plate), and store at 4°C for 18 h or overnight;

[0076] (2) Remove the reaction plate, discard the remaining liquid, and wash three times with PBST (pH 7.0-7.4, concentration 0.01 mol / L), 300 μL / well. After the last wash, pat the reaction plate dry.

[0077] (3) Add blocking solution (mainly containing 1% BSA or casein or salmon sperm, 10% sucrose or 2‰ tween20, etc.) at 150 μL / well and let it stand at room temperature for 2 h;

[0078] (4) Discard the residual liquid, pat the reaction plate dry, turn the reaction plate upside down, dry it, and store it in an aluminum foil bag for later use;

[0079] (5) Remove the reaction plate and prepare a gradient concentration solution of the standard (0, 0.313, 0.625, 1.25, 2.5, 5, 10, 20 ng / ml). Add the prepared sample solution at a rate of 100 μL / well and react at 37°C for 60-90 min.

[0080] (6) Remove the reaction plate, discard the residual liquid, and wash three times with PBST (pH 7.0-7.4, concentration 0.01 mol / L), 250 μL / well. After the last wash, pat the reaction plate dry.

[0081] (7) Add 100 μL / well of biotin-labeled anti-GPC-3 antibody working solution (2 μg / ml), react at 37°C for 60-90 min, and then repeat step 6;

[0082] (8) Add 100 μL / well of streptavidin-labeled HRP enzyme (SA-HRP) and react at 37°C for 20-30 min, then repeat step 6;

[0083] (9) Add 100 μL / well of HRP luminescent solution and read the value using a chemiluminescence instrument.

[0084] 2.2 Establishment of enzyme-linked immunosorbent assay (ELISA) detection mode:

[0085] (1) Prepare 10 mL of GPC-3 coating antibody at a concentration of 5 μg / mL, add 100 μL / well to a 96-well white high-absorption ELISA plate (reaction plate), and store at 4°C for 18 h or overnight;

[0086] (2) Remove the reaction plate, discard the remaining liquid, and wash three times with 0.01 M PBST (pH 7.0-7.4), 300 μL / well. After the last wash, pat the reaction plate dry.

[0087] (3) Add blocking solution (mainly containing 1% BSA or casein or salmon sperm, and may also contain 10% sucrose or 2‰ tween20, etc.) at 150 μL / well and leave at room temperature for 2 h;

[0088] (4) Discard the residual liquid, pat the reaction plate dry, turn the reaction plate upside down, dry it, and store it in an aluminum foil bag for later use

[0089] (5) Remove the reaction plate and prepare a gradient concentration solution of the standard (0, 0.313, 0.625, 1.25, 2.5, 5, 10, 20 ng / ml). Add the prepared sample solution at a rate of 100 μL / well and react at 37°C for 60-90 min.

[0090] (6) Remove the reaction plate, discard the residual liquid, and wash three times with 0.01 M PBST (pH 7.0-7.4), 250 μL / well. After the last wash, pat the reaction plate dry.

[0091] (7) Add 100 μL / well of biotin-labeled anti-GPC-3 antibody working solution (2 μg / ml), react at 37°C for 60-90 min, and then repeat step 6;

[0092] (8) Add 100 μL / well of streptavidin-labeled HRP enzyme (SA-HRP) and react at 37°C for 20-30 min, then repeat step 6;

[0093] (9) Add 100 μL / well of TMB substrate and keep in the dark for 7-30 min (preferably 10 min). When the gradient concentration liquid of the standard shows a gradient blue change, add 100 μL / well of stop solution (1 mol / L HCl solution or 2 mol / L H2SO4 solution).

[0094] In the above two detection modes, the method for preparing a standard solution with a concentration gradient of 0 to 20 ng / mL is as follows: recombinant human GPC-3 full sequence protein (sequence such as SEQ ID NO.1) is selected as the standard, and a gradient dilution method is used to prepare standard solutions with a concentration gradient of 0, 0.313, 0.625, 1.25, 2.5, 5, 10, and 20 ng / ml, respectively.

[0095] The complete protein sequence of recombinant human GPC-3 is as follows:

[0096] MAGTVRTACLVVAMLLSLDFPGQAQPPPPPPDATCHQVRSFFQRLQPGLKWVPETPVPGSDLQVCLPKGPTCCSRKMEEKYQLTARLNMEQLLQSASMELKFLIIQNAAVFQEAFEIVVRHAKNYTNAMFKNNYP SLTPQAFEFVGEFFTDVSLYILGSDINVDDMVNELFDSLFPVIYTQLMNPGLPDSALDINECLRGARRDLKVFGNFPKLIMTQVSKSLQVTRIFLQALNLGIEVINTTDHLKFSKDCGRMLTRMWYCSYCQGLMM VKPCGGYCNVVMQGCMAGVVEIDKYWREYILSLEELVNGMYRIYDMENVLLGLFSTIHDSIQYVQKNAGKLTTTIGKLCAHSQQRQYRSAYYPEDLFIDKKVLKVAHVEHEETLSSRRRELIQKLKSFISFYSAL PGYICSSHSPVAENDTLCWNGQELVERYSQKAARNGMKNQFNLHELKMKGPEPVVSQIIDKLKHINQLLRTMSMPKGRVLDKNLDEEGFESGDCGDDEDECIGGSGDGMIKVKNQLRFLAELAYDLDVDDAPGNSQ QATPKDNEISTFHNLGNVHSPLKLLTSMAISVVCFFFLVH.

[0097] Comparative Example 1: GPC-3 detection using peripheral blood serum / plasma

[0098] EDTA anticoagulated whole blood / heparin Corning whole blood was centrifuged at 3000 rpm for 10 min, and 1 ml of the upper plasma layer was collected into an EP tube for sample detection. The detection method was the same as in Example 1.

[0099] Comparative Example 2: GPC-3 detection of serum / plasma samples after acid-base activation pretreatment

[0100] The upper layer of plasma prepared in Comparative Example 1 was collected and treated with acid and base solutions in a ratio of 5:1:1 (20 μL of 1 M HCl was added to 100 μL of plasma, and after standing for 10 minutes, 20 μL of 1 M NaOH was added). The resulting solution was used as a sample for testing. The testing method was the same as in Example 1.

[0101] In the two comparative examples, the preparation method of the standard solution is the same as that of Example 1.

[0102] Results and Analysis

[0103] 1. Chemiluminescence detection mode

[0104] 1. Standard curve drawing

[0105] The standard products in Example 1 and Comparative Examples 1 and 2 were detected by chemiluminescence detection. The CLIA values ​​of the standard products are shown in Table 1. The standard curves drawn based on the results are shown in Table 1. Figures 1 to 3 shown.

[0106] Table 1 Summary of standard test results

[0107]

[0108] By comparison, it can be seen that at the same standard concentration, the CLIA test values ​​are not much different. One of the standard curves can be selected as a benchmark to determine the CPC-3 content in the actual sample.

[0109] 2. Actual sample testing in the embodiment

[0110] Peripheral blood was collected from eight liver cancer patients in parallel. After pretreatment according to the "1. GPC-3 Enrichment in Blood Samples" section of Example 1, the CLIA values ​​were determined using chemiluminescence detection, and the GPC-3 concentration was calculated based on a GPC-3 recombinant protein standard. The BCA total protein concentration in the lysate was determined using a BCA total protein assay kit, and the GPC-3 / BCA ratio was calculated. The GPC-3 / cell number ratio was also calculated. The results are shown in Table 2:

[0111] Table 2 Summary of the results of GPC-3 content detection by chemiluminescence method:

[0112]

[0113] The enrichment method of the present invention can enrich CPC-3 complexes with a concentration of 0.3 to 1.76 ng / mL and realize corresponding detection, and the sensitivity is higher than that of the immunohistochemical detection method currently used in clinical practice.

[0114] 3. Comparative Example Actual Sample Testing

[0115] Peripheral blood samples from ten liver cancer patients were collected in parallel and processed according to the methods described in Comparative Example 1 and Comparative Example 2, respectively. The samples were then tested using chemiluminescence. The results are shown in Table 3.

[0116] Table 3 Comparison of sample test results of Comparative Example 1 and Comparative Example 2

[0117] Sample No. Comparative Example 1 Detection of CLIA Value Comparative Example 2 Detection of CLIA Value 1 530.33 1988.44 2 1732.94 3806.12 3 513.90 2588.59 4 2058.40 2571.85 5 601.66 2474.91 6 1076.31 2398.96 7 1081.11 2996.96 8 476.27 2398.76 9 771.40 3808.33 10 864.90 3135.19

[0118] According to the results in Table 3, after the upper plasma was pretreated with acid-base activation, the impurities in the blood sample were removed to a certain extent, the GPC-3 enrichment effect was optimized, and the CLIA value was naturally better than that of Comparative Example 1.

[0119] 4. Comparison between Examples and Comparative Examples

[0120] Peripheral blood samples from 15 liver cancer patients were collected in parallel and pretreated according to the methods described in Comparative Example 1 and Example 1, respectively. The samples were then tested using chemiluminescence. The results are shown in Table 4.

[0121] Table 4 Comparison of sample test results of Comparative Example 1 and Example 1

[0122] Sample No. Comparative Example 1 Detection of CLIA Value Example Detection of CLIA Values 1 1732.94 21203.03 2 530.33 6716.11 3 513.9 13481.52 4 2058.4 12336.59 5 601.66 26315.88 6 1076.31 13305.3 7 1081.11 8213.44 8 771.4 3085.81 9 683.97 25326.97 10 1349.87 3903.22 11 498.83 22629.52 12 545.87 2041.52 13 354.92 2516.14 14 536.51 2809.64 15 530.14 6070.25

[0123] There is overlap between samples 1 to 7 and 9 in Table 4 and Table 3, which is equivalent to the eight samples being enriched by the methods of Example, Comparative Examples 1 and 2, respectively. Comparison shows that after the blood samples are enriched and pretreated using the method of the present invention, the GPC-3 enrichment effect is significantly enhanced, with CLIA values ​​reaching several times or even dozens of times that of Comparative Example 1 and 3 to 10 times that of Comparative Example 2, indicating a significant difference in effect.

[0124] 2. Enzyme-linked immunosorbent assay (ELISA) detection mode

[0125] 1. Test results of the embodiment

[0126] 1.1 Standard curve drawing

[0127] The standard products were tested by enzyme-linked immunosorbent assay, and the OD values ​​of the standard products are shown in Table 5:

[0128] Table 5 Test results of standard products in Example 1

[0129] Standard concentration (ng / ml) OD value 0 0.023 0.313 0.049 0.625 0.07 1.25 0.111 2.5 0.199 5 0.372 10 0.774 20 1.423

[0130] The standard curve drawn based on the results is as follows Figure 4 shown.

[0131] 2. Comparison between Examples and Comparative Examples

[0132] Peripheral blood samples from 15 liver cancer patients were collected in parallel and pretreated according to the methods described in Comparative Example 1 and Example, and then tested using enzyme-linked immunosorbent assay. The results are shown in Table 6:

[0133] Table 6 Comparison of test results of comparative example 1 and example samples

[0134]

[0135]

[0136] According to the comparison results in Table 6, the detection rate of plasma after conventional treatment is very low, and the sample concentration is basically undetectable. The detection ability of cell lysate is significantly higher than that of plasma, and the detection concentration is higher.

[0137] The basic principles, main features, and advantages of the present invention are shown and described above. Those skilled in the art should understand that the present invention is not limited to the above embodiments. The above embodiments and descriptions are merely illustrative of the principles of the present invention. Various changes and modifications may be made to the present invention without departing from the spirit and scope of the present invention. Such changes and modifications are intended to fall within the scope of the present invention. The scope of protection claimed in the present invention is defined by the appended claims and their equivalents. Sequence Listing <110> The Third Affiliated Hospital of the Naval Medical University of the Chinese People's Liberation Army <120> Enrichment, detection method and application of GPC-3 complex in blood samples <130> Claims, description <160> 1 <170> SIPOSequenceListing 1.0 <210> 1 <211> 580 <212> PRT <213> Artificial sequence <400> 1 Met Ala Gly Thr Val Arg Thr Ala Cys Leu Val Val Ala Met Leu Leu 1 5 10 15 Ser Leu Asp Phe Pro Gly Gln Ala Gln Pro Pro Pro Pro Pro Pro Asp 20 25 30 Ala Thr Cys His Gln Val Arg Ser Phe Phe Gln Arg Leu Gln Pro Gly 35 40 45 Leu Lys Trp Val Pro Glu Thr Pro Val Pro Gly Ser Asp Leu Gln Val 50 55 60 Cys Leu Pro Lys Gly Pro Thr Cys Cys Ser Arg Lys Met Glu Glu Lys 65 70 75 80 Tyr Gln Leu Thr Ala Arg Leu Asn Met Glu Gln Leu Leu Gln Ser Ala 85 90 95 Ser Met Glu Leu Lys Phe Leu Ile Ile Gln Asn Ala Ala Val Phe Gln 100 105 110 Glu Ala Phe Glu Ile Val Val Arg His Ala Lys Asn Tyr Thr Asn Ala 115 120 125 Met Phe Lys Asn Asn Tyr Pro Ser Leu Thr Pro Gln Ala Phe Glu Phe 130 135 140 Val Gly Glu Phe Phe Thr Asp Val Ser Leu Tyr Ile Leu Gly Ser Asp 145 150 155 160 Ile Asn Val Asp Asp Met Val Asn Glu Leu Phe Asp Ser Leu Phe Pro 165 170 175 Val Ile Tyr Thr Gln Leu Met Asn Pro Gly Leu Pro Asp Ser Ala Leu 180 185 190 Asp Ile Asn Glu Cys Leu Arg Gly Ala Arg Arg Asp Leu Lys Val Phe 195 200 205 Gly Asn Phe Pro Lys Leu Ile Met Thr Gln Val Ser Lys Ser Leu Gln 210 215 220 Val Thr Arg Ile Phe Leu Gln Ala Leu Asn Leu Gly Ile Glu Val Ile 225 230 235 240 Asn Thr Thr Asp His Leu Lys Phe Ser Lys Asp Cys Gly Arg Met Leu 245 250 255 Thr Arg Met Trp Tyr Cys Ser Tyr Cys Gln Gly Leu Met Met Val Lys 260 265 270 Pro Cys Gly Gly Tyr Cys Asn Val Val Met Gln Gly Cys Met Ala Gly 275 280 285 Val Val Glu Ile Asp Lys Tyr Trp Arg Glu Tyr Ile Leu Ser Leu Glu 290 295 300 Glu Leu Val Asn Gly Met Tyr Arg Ile Tyr Asp Met Glu Asn Val Leu 305 310 315 320 Leu Gly Leu Phe Ser Thr Ile His Asp Ser Ile Gln Tyr Val Gln Lys 325 330 335 Asn Ala Gly Lys Leu Thr Thr Thr Ile Gly Lys Leu Cys Ala His Ser 340 345 350 Gln Gln Arg Gln Tyr Arg Ser Ala Tyr Tyr Pro Glu Asp Leu Phe Ile 355 360 365 Asp Lys Lys Val Leu Lys Val Ala His Val Glu His Glu Glu Thr Leu 370 375 380 Ser Ser Arg Arg Arg Glu Leu Ile Gln Lys Leu Lys Ser Phe Ile Ser 385 390 395 400 Phe Tyr Ser Ala Leu Pro Gly Tyr Ile Cys Ser His Ser Pro Val Ala 405 410 415 Glu Asn Asp Thr Leu Cys Trp Asn Gly Gln Glu Leu Val Glu Arg Tyr 420 425 430 Ser Gln Lys Ala Ala Arg Asn Gly Met Lys Asn Gln Phe Asn Leu His 435 440 445 Glu Leu Lys Met Lys Gly Pro Glu Pro Val Val Ser Gln Ile Ile Asp 450 455 460 Lys Leu Lys His Ile Asn Gln Leu Leu Arg Thr Met Ser Met Pro Lys 465 470 475 480 Gly Arg Val Leu Asp Lys Asn Leu Asp Glu Glu Gly Phe Glu Ser Gly 485 490 495 Asp Cys Gly Asp Asp Glu Asp Glu Cys Ile Gly Gly Ser Gly Asp Gly 500 505 510 Met Ile Lys Val Lys Asn Gln Leu Arg Phe Leu Ala Glu Leu Ala Tyr 515 520 525 Asp Leu Asp Val Asp Asp Ala Pro Gly Asn Ser Gln Gln Ala Thr Pro 530 535 540 Lys Asp Asn Glu Ile Ser Thr Phe His Asn Leu Gly Asn Val His Ser 545 550 555 560 Pro Leu Lys Leu Leu Thr Ser Met Ala Ile Ser Val Val Cys Phe Phe 565 570 575 Phe Leu Val His 580

Claims

1. A method for enriching GPC-3 complexes in a blood sample, characterized in that: The following steps are included: A. Blood sample collection Peripheral blood was collected from patients with liver cancer and placed in anticoagulant tubes, and then mixed with whole blood; B. Red blood cell lysis Add multiple volumes of red blood cell lysis buffer, pipette and mix thoroughly, lyse on ice for a certain period of time, centrifuge and discard the red supernatant. If the cell pellet still has red color, repeat the step; C. Precipitation and impurity removal Wash the pellet 1-2 times, add PBS or saline to resuspend the pellet, centrifuge and discard the supernatant; D. White blood cell count Resuspend the cell pellet in PBS, take an appropriate amount to fill the pool, let it stand for 2-3 minutes, and manually count the white blood cells using the four large squares at the four corners of the low-power microscope. The calculation formula is as follows: WBC / L = N / 4*10 5 *10 4 , and calculate the number of white blood cells per ml of the original suspension, WBC / ml original suspension = N / 4*10 4 *Dilution factor, when the number of white blood cells is not less than 10 7 When the , proceed to the next step; E. Enrichment of GPC-3 complex Antibody purification and separation are used to obtain target CTC cells. The obtained cells are counted and the data is recorded. The obtained cells are added to a lysis buffer containing 1% protease inhibitors and 1% phosphatase inhibitors. The lysis buffer includes RIPA lysis buffer or NP-40 lysis buffer. The main components of the RIPA lysis buffer are Triton X-100 and SDS in a Tris salt buffer system; the main components of the NP-40 lysis buffer are NP-40 and sodium deoxycholate solution in a Tris salt buffer system. After refrigeration and standing for a certain period of time, the supernatant is centrifuged to obtain the sample to be tested.

2. The method for enriching GPC-3 complexes in a blood sample according to claim 1, characterized in that: in, In step B, the volume ratio of red blood cell lysis buffer to whole blood is 6:

1. Mix by pipetting, lyse on ice for 4-6 minutes, centrifuge at 400g for 5 minutes at 4°C, and discard the red supernatant.

3. The method for enriching GPC-3 complexes in a blood sample according to claim 1, wherein: in, In step C, when resuspending the precipitate, the volume of PBS or physiological saline should be 4 to 5 times that of the precipitate, centrifuge at 4°C and 400g for 2 to 3 minutes, and discard the supernatant.

4. The method for enriching GPC-3 complexes in a blood sample according to claim 1, wherein: in, In step E, the antibody is a specific magnetic bead antibody pre-coated with a GPC-3 antibody.

5. The method for enriching GPC-3 complexes in a blood sample according to claim 1, wherein: in, In step E, the cells were added with lysis buffer, refrigerated and allowed to stand for 30 minutes, and then centrifuged at 4°C and 14,000 g for 10 minutes to obtain the supernatant as the sample to be tested.

6. The method for enriching GPC-3 complexes in a blood sample according to any one of claims 1 to 5, characterized in that: in, GPC-3 was detected using chemiluminescence detection mode or enzyme-linked immunosorbent assay mode.

7. The method for enriching GPC-3 complexes in a blood sample according to claim 6, wherein: in, The steps to establish the chemiluminescence detection mode are as follows: (1) Prepare 10 mL of GPC-3 coating antibody at a concentration of 5 μg / mL, add 100 μL / well to a 96-well white high-absorption chemiluminescent plate, and store at 4°C for 18 h or overnight; (2) Remove the reaction plate, discard the remaining liquid, and wash three times with 0.01 mol / L PBST (pH 7.0-7.4), 300 μL / well. After the last wash, pat the reaction plate dry. (3) Add 150 μL / well of blocking solution, let stand at room temperature for 2 h, discard the residual liquid, pat dry and air dry the reaction plate, and store in an aluminum foil bag for later use; (4) Remove the reaction plate, prepare a gradient concentration solution of the standard, add the prepared sample solution at 100 μL / well, and react at 37°C for 60-90 min; (5) Remove the reaction plate, discard the residual liquid, and wash three times with 0.01 mol / L PBST (pH 7.0-7.4), 250 μL / well. After the last wash, pat the reaction plate dry. (6) Add 2 μg / mL biotin-labeled anti-GPC-3 antibody working solution to 100 μL / well, react at 37°C for 60-90 min, remove the reaction plate, discard the residual liquid, and wash three times with 0.01 mol / L PBST (pH 7.0-7.4) at 250 μL / well. After the last wash, pat the reaction plate dry. (7) Add 100 μL / well of streptavidin-labeled HRP enzyme and react at 37°C for 20-30 min. Remove the reaction plate, discard the residual liquid, and wash three times with PBST (pH 7.0-7.4, concentration 0.01 mol / L) at 250 μL / well. After the last wash, pat the reaction plate dry. (8) Add 100 μL / well of HRP luminescent solution and read the value using a chemiluminescence analyzer; (9) The above steps were used to detect the CLIA value of GPC-3 complex in the peripheral blood of liver cancer patients, and the GPC-3 concentration was calculated based on the standard.

8. The method for enriching GPC-3 complexes in a blood sample according to claim 6, Its characteristics are: The steps for establishing the enzyme-linked immunosorbent assay are as follows: (1) Prepare 10 mL of GPC-3 coating antibody at a concentration of 5 μg / mL, add 100 μL / well to a 96-well white high-absorption chemiluminescent plate, and store at 4°C for 18 h or overnight; (2) Remove the reaction plate, discard the remaining liquid, and wash three times with 0.01 mol / L PBST (pH 7.0-7.4), 300 μL / well. After the last wash, pat the reaction plate dry. (3) Add 150 μL / well of blocking solution, let stand at room temperature for 2 h, discard the residual liquid, pat dry and air dry the reaction plate, and store in an aluminum foil bag for later use; (4) Remove the reaction plate, prepare a gradient concentration solution of the standard, add the prepared sample solution at 100 μL / well, and react at 37°C for 60-90 min; (5) Remove the reaction plate, discard the residual liquid, and wash three times with 0.01 mol / L PBST (pH 7.0-7.4), 250 μL / well. After the last wash, pat the reaction plate dry. (6) Add 2 μg / mL biotin-labeled anti-GPC-3 antibody working solution to 100 μL / well, react at 37°C for 60-90 min, remove the reaction plate, discard the residual liquid, and wash three times with 0.01 mol / L PBST (pH 7.0-7.4) at 250 μL / well. After the last wash, pat the reaction plate dry. (7) Add 100 μL / well of streptavidin-labeled HRP enzyme and react at 37°C for 20-30 min. Remove the reaction plate, discard the residual liquid, and wash three times with PBST (pH 7.0-7.4, concentration 0.01 mol / L) at 250 μL / well. After the last wash, pat the reaction plate dry. (8) Add 100 μL / well of TMB substrate and protect from light for 7-30 min. When the gradient concentration liquid of the standard shows a gradient blue change, add 100 μL / well of stop solution; (9) The above steps were used to detect the OD value of the GPC-3 complex in the peripheral blood of liver cancer patients, and the GPC-3 concentration was calculated based on the standard.

9. The method for enriching GPC-3 complexes in a blood sample according to claim 8, wherein: in, The stop solution is 1 mol / L HCl solution or 2 mol / L H2SO4 solution.

10. A kit for rapidly detecting the content of GPC-3 in a blood sample, characterized in that: Including red blood cell lysate, cell lysate for enriching GPC-3, chemiluminescent plate or immunomagnetic beads pre-coated with GPC-3 antibody, phosphate or Tris buffer washing solution, blocking solution, biotin-labeled anti-GPC-3 antibody working solution, streptavidin-labeled luminol enzyme, enzymatic chemiluminescent solution, The cell lysate for enriching GPC-3 contains 1% protease inhibitors and 1% phosphatase inhibitors, including RIPA lysate or NP-40 lysate. The main components of the RIPA lysate are Triton X-100 and SDS in a Tris salt buffer system; the main components of the NP-40 lysate are NP-40 and sodium deoxycholate solution in a Tris salt buffer system.

11. A kit for rapidly detecting the content of GPC-3 in a blood sample, characterized in that: The kit includes red blood cell lysis buffer, cell lysis buffer for enriching GPC-3, enzyme-labeled plate pre-coated with GPC-3 antibody, phosphate or Tris buffer washing solution, blocking solution, biotin-labeled anti-GPC-3 antibody working solution, streptavidin-labeled luminol enzyme, TMB substrate and stop solution. The cell lysate for enriching GPC-3 contains 1% protease inhibitors and 1% phosphatase inhibitors, including RIPA lysate or NP-40 lysate. The main components of the RIPA lysate are Triton X-100 and SDS in a Tris salt buffer system; the main components of the NP-40 lysate are NP-40 and sodium deoxycholate solution in a Tris salt buffer system.

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