WO2017157194A1 - 一种用于蛋白聚糖高灵敏度检测的试剂盒及其制备方法 - Google Patents

一种用于蛋白聚糖高灵敏度检测的试剂盒及其制备方法 Download PDF

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WO2017157194A1
WO2017157194A1 PCT/CN2017/075758 CN2017075758W WO2017157194A1 WO 2017157194 A1 WO2017157194 A1 WO 2017157194A1 CN 2017075758 W CN2017075758 W CN 2017075758W WO 2017157194 A1 WO2017157194 A1 WO 2017157194A1
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antibody
graphene
monoclonal antibody
buffer
proteoglycan
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French (fr)
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李福川
韩乃寒
王文爽
蔡晓娟
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Shandong University
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Shandong University
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    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/68—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/577—Immunoassay; Biospecific binding assay; Materials therefor involving monoclonal antibodies binding reaction mechanisms characterised by the use of monoclonal antibodies

Definitions

  • the invention relates to a kit for high-sensitivity detection of proteoglycans and a preparation method thereof, and belongs to the technical field of biological analysis and medical detection.
  • Proteoglycan is a complex biological macromolecule in which one or more glycosaminoglycan (GAG) chains are covalently coupled to the core protein.
  • GAG is a linear polysaccharide consisting of repeating disaccharide units, which are mainly divided into four categories: hyaluronic acid (HA), chondroitin sulfate (CS)/dermatan sulfate (DS), keratan sulfate (KS) and heparin ( Hep) / heparan sulfate (HS).
  • HA hyaluronic acid
  • CS chondroitin sulfate
  • DS dermatan sulfate
  • KS keratan sulfate
  • Hep heparin
  • HS heparan sulfate
  • hyaluronic acid does not combine with core protein to form proteoglycan.
  • proteoglycans are coupled to some N- or O-
  • PG is widely distributed in the extracellular matrix and is also present on the cell surface as well as secretory granules within the cell.
  • PG participates in many physiological processes, such as the existence of the original components of the tissue structure, regulating the proliferation, adhesion, migration and mutation of cells, and also participating in the binding of growth factors and cytokines, signal transduction of cells, and nerves. The process of growth and morphogenesis of the element.
  • the expression of PG is significantly changed, so a part of proteoglycan is used as a specific marker molecule for detecting certain diseases, such as Glypican-3 (GPC3), a member of the proteoglycan Glypican family, which is currently widely used. It is considered to be a specific marker molecule for hepatocellular carcinoma (HCC).
  • GPC3 Glypican-3
  • HCC hepatocellular carcinoma
  • GPC3 consists of a single-stranded core protein, a Heparin Sulfate (HS) chain, and a glycosylphosphatidylinositol (GPI), which is anchored to the cell membrane by GPI.
  • GPC3 is significantly expressed in 75% of HCC, but it is not expressed in normal liver tissues and benign lesions, so it is a new HCC marker molecule that is more specific and sensitive than alpha-fetoprotein.
  • the proteoglycan Due to the molecular weight of the proteoglycan core protein and its structure, and the number, type, length, degree of sulfation and coupling site of the covalently coupled GAG chain, the proteoglycan has complex composition and various structures, thus giving Its detection has brought many difficulties.
  • the most commonly used detection method is sandwich ELISA, but its detection sensitivity is low, only about 50% of HCC patients can detect the presence of significant GPC3 in serum, and because the first antibody is on the ELISA plate.
  • the adsorption coating and the binding of the second antibody to the antigen need to be incubated overnight, and it takes three days to complete an experiment, which takes a long time, which greatly limits the application of the method and brings difficulties to the detection of GPC3.
  • the present invention is directed to the deficiencies of the prior art, and provides a kit for high sensitivity detection of proteoglycans and a preparation method thereof.
  • the kit utilizes the binding of graphene coupled to the GPC3 core protein to the high-sensitivity rapid detection of the HCC marker GPC3 in serum/plasma by fluorescence quenching of ScGFP bound to the GPC3HS side chain.
  • a kit for high sensitivity detection of proteoglycans comprising:
  • the carboxylated graphene coupled to the monoclonal antibody comprises: a carboxylated graphene coupled to the monoclonal antibody 1, the monoclonal antibody 1
  • the carboxy terminal amino acid sequence of the core protein of proteoglycan is prepared as an antibody prepared by antigen immunization; carboxygraphene coupled to monoclonal antibody 2, which is the amino terminal amino acid of the core protein of proteoglycan
  • the sequence is prepared as an antibody prepared by antigen immunization;
  • the carboxylated graphene coupled to the polyclonal antibody comprises: a carboxylated graphene coupled with polyclonal antibody 3, which is a carboxyl terminal amino acid of a core protein of proteoglycan
  • the sequence is prepared as an antibody prepared by antigen immunization; a carboxylated graphene conjugated to poly
  • Buffer A solution containing 5 to 50 mM Tris-HCl and a concentration of 50 to 200 mM NaCl.
  • the proteoglycans are GPC3, GPC1, GPC2, GPC4, GPC5, GPC6, Syndecan-1, Syndecan-2, Syndecan-3, Syndecan-4, Versican, Aggrecan, Brevican, Decorin, Lumican, Perleacan, Agrin, CD44 or NG2; further preferably, the proteoglycan is GPC3 or Proteoglycan Syndecan-2; optimally, when the proteoglycan is GPC3, the amino acid sequence length of the carboxy terminal is 50-90 The amino acid amino acid sequence has an amino terminal sequence of 490 to 530 amino acids.
  • the concentration of the carboxylated graphene coupled to the monoclonal antibody 1 is 5 to 20 ⁇ g/mL.
  • the concentration of the carboxylated graphene coupled to the monoclonal antibody 2 is 5 to 20 ⁇ g/mL.
  • the concentration of the carboxylated graphene coupled to the polyclonal antibody 3 is 5 to 20 ⁇ g/mL.
  • the concentration of the carboxylated graphene coupled to the polyclonal antibody 4 is 5 to 20 ⁇ g/mL.
  • the concentration of the highly positively charged fluorescent protein is 0.001 to 0.01 ⁇ g/ ⁇ L; further preferably, the highly positively charged fluorescent protein is a highly positive green fluorescent protein (ScGFP).
  • Preferred according to the invention also includes a microplate, tin foil paper necessary for assembling the kit.
  • the preparation method of the above kit for high-sensitivity detection of proteoglycans is as follows:
  • the surfactant is ethylenediaminetetraacetic acid (EDTA) or sodium dodecyl sulfate (SDS), adding 1 ⁇ 5 ⁇ L of surfactant per ml of the reaction system;
  • the blocking agent is bovine serum albumin (BSA), and a blocking agent is added to the reaction system to a mass concentration of 0.5% to 2%.
  • the reaction mass concentration of the carboxyl graphene is 0.8 to 1.2 mg/mL, and the reaction mass concentration ratio of the carboxyl graphene to the monoclonal antibody 1 is 10:1, and the carboxyl group is
  • the reaction mass concentration ratio of graphene to monoclonal antibody 2 is 10:1
  • the reaction mass concentration ratio of carboxyl graphene to polyclonal antibody 3 is 10:1
  • the reaction mass concentration ratio of carboxyl graphene to polyclonal antibody 4 is 10 :1.
  • the preparation method of the monoclonal antibody 1 is as follows:
  • step (ii) immunizing the BALB/C mice with the antigen prepared in the step (i) three times, the antigen immunization amount is 50-100 ⁇ g/time, and then performing spleen cells and myeloma cells with a titer of ⁇ 2000 by the PEG method. Fusion and screening for positive clones;
  • step (iii) Injecting Freund's incomplete adjuvant (purchased from SIGMA) into the peritoneal cavity of BALB/C mice, and injecting sufficient amount of ascites into the peritoneal cavity of the mouse, inoculation in the abdominal cavity of the mouse was positive in step (ii). After cloning, ascites was taken two weeks later, and the supernatant was centrifuged, and after purification, monoclonal antibody 1 was obtained.
  • Freund's incomplete adjuvant purchased from SIGMA
  • step (i) is carried out using an NI-NTA affinity chromatography column.
  • the purification of the step (iii) is as follows:
  • the supernatant was diluted 2.5-fold with PBS buffer, and a protein A/G affinity chromatography column (GE) was used, respectively, with a buffer of TRIS-HCl pH 8.0 containing 0.5 M NaCl and TRIS-HCl pH 9.0 containing 0.5 M NaCl.
  • the buffer was fully eluted and decontaminated.
  • the antibody was recovered by elution with 100 mM triethylamine. The first three volumes of eluate were collected and immediately neutralized with 1 M NaH 2 PO 4 , and finally with ultrafiltration with a molecular weight of 10 KD. The tube is desalted and the antibody is replaced in PBS.
  • the preparation method of the monoclonal antibody 2 is as follows:
  • step (b) immunizing the BALB/C mice with the antigen prepared in the step (a) three times, the antigen immunization amount is 50-100 ⁇ g/time, and then performing the spleen cell and the myeloma cells of the mouse having a titer of ⁇ 2000 by the PEG method. Fusion and screening for positive clones;
  • step (c) Injecting Freund's incomplete adjuvant into the peritoneal cavity of BALB/C mice. After a sufficient amount of ascites was produced in the peritoneal cavity of the mouse, the positive clone prepared in step (b) was inoculated into the peritoneal cavity of the mouse, and extracted two weeks later. In the ascites, the supernatant was centrifuged, and after purification, monoclonal antibody 2 was obtained.
  • step (a) is carried out using an NI-NTA affinity chromatography column.
  • the purification of the step (c) is as follows:
  • the supernatant was diluted 2.5-fold with PBS buffer, and a protein A/G affinity chromatography column (GE) was used, respectively, with a buffer of TRIS-HCl pH 8.0 containing 0.5 M NaCl and TRIS-HCl pH 9.0 containing 0.5 M NaCl.
  • the buffer was fully eluted and decontaminated.
  • the antibody was recovered by elution with 100 mM triethylamine. The first three volumes of eluate were collected and immediately neutralized with 1 M NaH 2 PO 4 , and finally with ultrafiltration with a molecular weight of 10 KD. The tube is desalted and the antibody is replaced in PBS.
  • the preparation method of the polyclonal antibody 3 is as follows:
  • the expression of the carboxy terminal amino acid sequence of the core protein of proteoglycan is inserted into the PTRACER eukaryotic expression vector, transfected into HEK293 cells, purified from the cell culture supernatant, and diluted to 1.0-2.0 mg/mL in PBS to prepare an antigen;
  • step b the antigen obtained in step a is immunized female New Zealand white rabbit 3 times, the antigen immunization amount is 200-400 ⁇ g / time, then the blood is taken, the supernatant is centrifuged, and purified, the polyclonal antibody 3 is obtained;
  • step b The purification of step b is as follows:
  • the supernatant was diluted 2.5-fold with PBS buffer, and a protein A/G affinity chromatography column (GE) was used, respectively, with a buffer of TRIS-HCl pH 8.0 containing 0.5 M NaCl and TRIS-HCl pH 9.0 containing 0.5 M NaCl.
  • the buffer was fully eluted and decontaminated.
  • the antibody was recovered by elution with 100 mM triethylamine. The first three volumes of eluate were collected and immediately neutralized with 1 M NaH 2 PO 4 , and finally with ultrafiltration with a molecular weight of 10 KD.
  • the tube is desalted and the antibody is replaced in PBS buffer.
  • the preparation method of the polyclonal antibody 4 is as follows:
  • the expression of the amino terminal amino acid sequence of the core protein of proteoglycan is inserted into the PTRACER eukaryotic expression vector, transfected into HEK293 cells, purified from the cell culture supernatant, and diluted to 1.0-2.0 mg/mL in PBS to prepare an antigen;
  • the antigen prepared in step a immunized female New Zealand white rabbit 3 times the antigen immunization amount is 200 ⁇ 400 ⁇ g / time, then take blood, centrifuge to take the supernatant, after purification, to obtain polyclonal antibody 4;
  • step b The purification of step b is as follows:
  • the supernatant was diluted 2.5-fold with PBS buffer, and a protein A/G affinity chromatography column (GE) was used, respectively, with a buffer of TRIS-HCl pH 8.0 containing 0.5 M NaCl and TRIS-HCl pH 9.0 containing 0.5 M NaCl.
  • the buffer was fully eluted and decontaminated.
  • the antibody was recovered by elution with 100 mM triethylamine. The first three volumes of eluate were collected and immediately neutralized with 1 M NaH 2 PO 4 , and finally with ultrafiltration with a molecular weight of 10 KD.
  • the tube is desalted and the antibody is replaced in PBS buffer.
  • the high positively charged fluorescent protein is a high positive green fluorescent protein (ScGFP).
  • ScGFP high positive green fluorescent protein
  • the preparation of a highly positively charged fluorescent protein is a conventional technique in the art, such as the construction expression method described in Lawrence, MS; Phillips, KJ; Liu, DRJAm. Chem. Soc. 2007, 129, 10110-10112. .
  • the kit for high-sensitivity detection of proteoglycan according to the present invention can realize the proteoglycan in the homogeneous system without the cumbersome steps of adsorbing and coating the enzyme labeling plate and without washing after antigen and antibody binding. Rapid detection and analysis, high sensitivity;
  • the kit for detecting high sensitivity of proteoglycan according to the present invention which is capable of specifically binding to the core protein of the proteoglycan by the antibody coupled to the carboxylated graphene in a homogeneous system, and at the same time, the target protein is aggregated.
  • the sugar side chain on the sugar can be The green fluorescent protein (ScGFP) with high positive charge is connected to each other by charge, and finally added to the carboxylated graphene coupled with another antibody to form a sandwich structure, which can be used for the proteoglycan side chain in the middle of the interlayer.
  • ScGFP green fluorescent protein
  • the above-mentioned ScGFP performs better fluorescence quenching, further improves the sensitivity of detection, and thus solves the shortcomings of low sensitivity and long reaction time of ELISA, and can solve the rapid proteoglycan in the serum of patients without expensive equipment. Sensitive detection problem;
  • the preparation method for the high-sensitivity detection of proteoglycan according to the invention is simple, easy to operate, low in cost, and convenient for large-scale popularization and application.
  • Figure 1 Bar graph of the effect of the addition sequence of ScGFP, conjugated monoclonal antibody ( ⁇ GCN, ⁇ GCC), and carboxygraphene and GPC3 on fluorescence intensity;
  • A carboxygraphene coupled with ⁇ GCN and GPC3, ScGFP are mixed, and then add carboxygraphene coupled with ⁇ GCC;
  • B two carboxygraphenes coupled with ⁇ GCN and ⁇ GCC, respectively, mixed with GPC3 and then added to ScGFP;
  • C two The carboxylated graphene coupled with ⁇ GCN and ⁇ GCC was mixed with ScGFP and added to GPC3;
  • D the carboxylated graphene coupled with ⁇ GCC and GPC3 and ScGFP were mixed, and then the carboxylated graphene coupled with ⁇ GCN was added.
  • Figure 2 Bar graph of the detection results of GPC3 in buffer
  • Figure 3 Histogram of the detection results of GPC3 in FBS
  • Figure 4 A histogram comparing the results of GPC3 and GPC3 ⁇ GAG;
  • Figure 6 Bar graph of the detection results of Syndecan-2 in buffer
  • Figure 7 Schematic diagram of the preparation process of carboxylated graphene coupled with antibody 1;
  • Figure 8 is a schematic view showing the preparation process of carboxylated graphene coupled with antibody 2;
  • Figure 9 Schematic diagram of the process of proteoglycan GPC3 detection process
  • Example 9 is a histogram of detection results of GPC3 using a kit for detecting high sensitivity of proteoglycan GPC3;
  • Figure 11 A histogram of the detection results of GPC3 detected by the kit for detecting high sensitivity of proteoglycan GPC3 in Example 11;
  • a kit for high sensitivity detection of proteoglycan GPC3, comprising:
  • the carboxygraphene of the monoclonal antibody 1 is conjugated to the carboxy terminal 70 amino acid sequence fragment of the core protein of the proteoglycan as an antibody prepared by antigen immunization; the conjugated monoclonal antibody 1
  • the concentration of carboxyl graphene is 10 ⁇ g / mL;
  • the carboxylated graphene of the monoclonal antibody 2 is conjugated to the amino terminal 510 amino acid sequence fragment of the core protein of the proteoglycan as an antibody prepared by antigen immunization; the coupled monoclonal antibody 2
  • the concentration of carboxyl graphene is 10 ⁇ g / mL;
  • Green fluorescent protein with high positive charge (ScGFP); concentration of green fluorescent protein with high positive charge is 0.005 ⁇ g / ⁇ L;
  • Buffer a solution containing a concentration of 10 mM Tris-HCl and a concentration of 100 mM NaCl;
  • microplate and tin foil necessary for assembling the kit.
  • the preparation method of the above kit for high-sensitivity detection of proteoglycans is as follows:
  • Monoclonal antibody 2 was added to the carboxylated graphene solution, mixed, allowed to stand for 1 min, then surfactant EDTA was added, then the blocking agent was added, mixed uniformly, and allowed to stand for 3 min to obtain the carboxyl group coupled with monoclonal antibody 2.
  • Graphene (the schematic diagram of the preparation process is shown in Figure 8);
  • the surfactant is ethylenediaminetetraacetic acid (EDTA), 5 ⁇ L of a surfactant is added per ml of the reaction system;
  • the blocking agent is bovine serum albumin (BSA), and a blocking agent is added to the reaction system to the quality.
  • the concentration is 1.5%.
  • the reaction mass concentration of the carboxyl graphene is 1.0 mg/mL, and the reaction mass concentration ratio of the carboxyl graphene to the monoclonal antibody 1 is 10:1, and the carboxylated graphene and the monoclonal antibody 2 are The reaction mass concentration ratio was 10:1.
  • the preparation method of the monoclonal antibody 1 is as follows:
  • step (iii) Injecting Freund's incomplete adjuvant into the peritoneal cavity of BALB/C mice. After a sufficient amount of ascites was produced in the peritoneal cavity of the mouse, the positive clone prepared in step (ii) was inoculated into the peritoneal cavity of the mouse, and extracted two weeks later. In the ascites, the supernatant was centrifuged, and after purification, monoclonal antibody 1 was obtained.
  • the supernatant was diluted 2.5-fold with PBS on an A/G affinity chromatography column (GE) using a buffer of TRIS-HCl pH 8.0 containing 0.5 M NaCl and a buffer of TRIS-HCl pH 9.0 containing 0.5 M NaCl, respectively. Fully elute and remove impurities. Finally, the antibody was recovered by elution with 100 mM triethylamine. The first three column volumes of eluate were collected and immediately neutralized with 1 M NaH 2 PO 4 , and finally desalted with an ultrafiltration tube with a molecular weight cutoff of 10 KD. And the antibody was replaced in PBS.
  • GE A/G affinity chromatography column
  • the preparation method of the monoclonal antibody 2 is as follows:
  • step (b) The antigen prepared in the step (a) was immunized to the BALB/C mice three times, the antigen immunization amount was 80 ⁇ g/time, and then the mouse spleen cells and the myeloma cells having a titer of 2000 were fused by the PEG method. And screen out positive clones;
  • step (c) Injecting Freund's incomplete adjuvant into the peritoneal cavity of BALB/C mice. After a sufficient amount of ascites was produced in the peritoneal cavity of the mouse, the positive clone prepared in step (b) was inoculated into the peritoneal cavity of the mouse, and extracted two weeks later. In the ascites, the supernatant was centrifuged, and after purification, monoclonal antibody 2 was obtained.
  • step (c) The purification of the step (c) is as follows:
  • the supernatant was diluted 2.5-fold with PBS on an A/G affinity chromatography column (GE) using a buffer of TRIS-HCl pH 8.0 containing 0.5 M NaCl and a buffer of TRIS-HCl pH 9.0 containing 0.5 M NaCl, respectively. Fully elute and remove impurities. Finally, the antibody was recovered by elution with 100 mM triethylamine. The first three column volumes of eluate were collected and immediately neutralized with 1 M NaH 2 PO 4 , and finally desalted with an ultrafiltration tube with a molecular weight cutoff of 10 KD. And the antibody was replaced in PBS.
  • GE A/G affinity chromatography column
  • step (2) the preparation of a highly positively charged fluorescent protein is a conventional technique in the art, as described in Lawrence, MS; Phillips, KJ; Liu, DRJAm. Chem. Soc. 2007, 129, 10110- The constructive expression method described in 10112.
  • group A is a mixture of ⁇ GCN-linked carboxyl graphene and GPC3, ScGFP, and then coupled with ⁇ GCC-conjugated carboxyl graphene
  • group B is two kinds of carboxyl graphene coupled with ⁇ GCN and ⁇ GCC, respectively, mixed with GPC3 and added to ScGFP
  • the two groups were carboxy graphene coupled with ⁇ GCN and ⁇ GCC, respectively, mixed with ScGFP and then added to GPC3
  • group D was mixed with ⁇ GCC-linked carboxyl graphene and GPC3, ScGFP, and then coupled with ⁇ GCN-conjugated carboxyl graphene.
  • the carboxylated graphene and GPC3 coupled with ⁇ GCN were added and left for 5-10 min, then ScGFP was added, and the mixture was allowed to stand at room temperature for 10-30 min.
  • the carboxylated graphene coupled with ⁇ GCC was added for 5-20 min as the reaction time.
  • the results showed that with the increase of NaCl concentration, the fluorescence quenching ability of ScGFP increased first and then decreased. According to this, the NaCl concentration was 50-200 mM as the concentration of salt added in the reaction system.
  • the carboxylated graphene was prepared by coupling different amounts of antibody.
  • a 10 mM Tris-HCl, 100 mM NaCl solution 0.05-0.2 ⁇ g of carboxylated graphene coupled with different ⁇ GCN, GPC3 0.001-0.005 ng, mixed, and allowed to stand at room temperature for 5 min; Add 0.01-0.5 ⁇ g of ScGFP, mix and store at room temperature for 15min; avoid adding the corresponding coupling
  • the amount of carboxylated graphene of ⁇ GCC was 0.05 to 0.2 ⁇ g, and the mixture was allowed to stand at room temperature for 10 minutes in the dark. After the incubation, the fluorescence intensity (Ex: 470 nm, Em: 509 nm) was measured.
  • GPC3 was dissolved in fetal bovine serum (FBS) as a stock solution.
  • FBS fetal bovine serum
  • the GPC3 stock solution was then diluted to a different concentration with FBS at a mass concentration of 20% for testing.
  • FBS fetal bovine serum
  • In 40 ⁇ L of 10 mM Tris-HCl and 100 mM NaCl solution add 0.05-0.2 ⁇ g of carboxygraphene coupled with ⁇ GCN, GPC3 0-0.04 ng, mix and leave at room temperature for 5 min; then add 0.01-0.5 ⁇ g of ScGFP, mix, room temperature The light was allowed to stand for 15 min; finally, 0.05-0.2 ⁇ g of the carboxylated graphene coupled with ⁇ GCC was added, mixed, and allowed to stand at room temperature for 10 min in the dark. After the incubation, the fluorescence intensity (Ex: 470 nm, Em: 509 nm) was measured.
  • a kit for high sensitivity detection of proteoglycan Syndecan-2 comprising:
  • the Syndecan-2 monoclonal antibody 1 is Human Syndecan-2 MAb (Clone 305507), Rat IgG2A (purchased from R&D Systems, Inc.); the coupled anti-Syndecan -2 monoclonal antibody 1 has a concentration of carboxyl graphene of 10 ⁇ g / mL;
  • Coupling anti-Syndecan-2 monoclonal antibody 2 to carboxygraphene said Syndecan-2 monoclonal antibody 2 is Human Syndecan-2 MAb (Clone 305515), Rat IgG2B (purchased from R&D Systems, Inc.); the coupled anti-Syndecan -2 monoclonal antibody 2 has a concentration of carboxyl graphene of 10 ⁇ g / mL;
  • Green fluorescent protein with high positive charge (ScGFP); concentration of green fluorescent protein with high positive charge is 0.005 ⁇ g / ⁇ L;
  • Buffer a solution containing a concentration of 10 mM Tris-HCl and a concentration of 100 mM NaCl;
  • microplate and tin foil necessary for assembling the kit.
  • the above kit for the high sensitivity detection of proteoglycan Syndecan-2 was prepared in the same manner as in Example 1.
  • a kit for high sensitivity detection of proteoglycan GPC3, comprising:
  • the carboxygraphene of the polyclonal antibody 3 is conjugated to the carboxy terminal 290 amino acid sequence fragment of the core protein of the proteoglycan as an antibody prepared by antigen immunization; the conjugated polyclonal antibody 3
  • the concentration of carboxyl graphene is 10 ⁇ g / mL;
  • the carboxygraphene of the polyclonal antibody 4 is conjugated to the amino terminal 290 amino acid sequence fragment of the core protein of the proteoglycan as an antibody prepared by antigen immunization; the conjugated polyclonal antibody 4
  • the concentration of carboxyl graphene is 10 ⁇ g / mL;
  • Green fluorescent protein with high positive charge (ScGFP); concentration of green fluorescent protein with high positive charge is 0.005 ⁇ g / ⁇ L;
  • Buffer a solution containing a concentration of 10 mM Tris-HCl and a concentration of 100 mM NaCl;
  • microplate and tin foil necessary for assembling the kit.
  • the preparation method of the above kit for high-sensitivity detection of proteoglycans is as follows:
  • Polyclonal antibody 4 was added to the carboxylated graphene solution, mixed, allowed to stand for 1 min, then the surfactant EDTA was added, then the blocking agent was added, mixed uniformly, and allowed to stand for 3 min to obtain the carboxyl group coupled with polyclonal antibody 4.
  • the surfactant is ethylenediaminetetraacetic acid (EDTA), 5 ⁇ L of a surfactant is added per ml of the reaction system;
  • the blocking agent is bovine serum albumin (BSA), and a blocking agent is added to the reaction system to the quality.
  • the concentration is 1.5%.
  • the reaction mass concentration of the carboxyl graphene is 1.0 mg/mL, and the reaction mass concentration ratio of the carboxyl graphene to the polyclonal antibody 3 is 10:1, and the carboxylated graphene and the polyclonal antibody 4 are The reaction mass concentration ratio was 10:1.
  • the preparation method of the polyclonal antibody 3 is as follows:
  • step (ii) immunizing female New Zealand white rabbits with the antigen prepared in step (i) three times, the antigen immunization amount is 300 ⁇ g / time, then taking blood, centrifuging the supernatant, and purifying, and obtaining polyclonal antibody 3;
  • the purification of the step (ii) is as follows:
  • the supernatant was diluted 2.5-fold with PBS buffer, and a protein A/G affinity chromatography column (GE) was used, respectively, with a buffer of TRIS-HCl pH 8.0 containing 0.5 M NaCl and TRIS-HCl pH 9.0 containing 0.5 M NaCl.
  • the buffer was fully eluted and decontaminated.
  • the antibody was recovered by elution with 100 mM triethylamine. The first three volumes of eluate were collected and immediately neutralized with 1 M NaH 2 PO 4 , and finally with ultrafiltration with a molecular weight of 10 KD.
  • the tube is desalted and the antibody is replaced in PBS buffer.
  • the preparation method of the polyclonal antibody 4 is as follows:
  • step (b) immunizing female New Zealand white rabbits with the antigen prepared in step (a) three times, the antigen immunization amount is 300 ⁇ g / time, then taking blood, centrifuging to take the supernatant, and purifying, to obtain polyclonal antibody 4;
  • the purification of the step (b) is as follows:
  • the supernatant was diluted 2.5-fold with PBS buffer, and a protein A/G affinity chromatography column (GE) was used, respectively, with a buffer of TRIS-HCl pH 8.0 containing 0.5 M NaCl and TRIS-HCl pH 9.0 containing 0.5 M NaCl.
  • the buffer was fully eluted and decontaminated.
  • the antibody was recovered by elution with 100 mM triethylamine. The first three volumes of eluate were collected and immediately neutralized with 1 M NaH 2 PO 4 , and finally with ultrafiltration with a molecular weight of 10 KD.
  • the tube is desalted and the antibody is replaced in PBS buffer.
  • step (2) the preparation of a highly positively charged fluorescent protein is a conventional technique in the art, as described in Lawrence, MS; Phillips, KJ; Liu, DRJAm. Chem. Soc. 2007, 129, 10110- The constructive expression method described in 10112.
  • polyclonal antibodies can also be used for the detection of GPC3, and as the concentration of GPC3 increases, the intensity of fluorescence quenching is also gradually increased.
  • a kit for high sensitivity detection of proteoglycan GPC3, comprising:
  • the carboxygraphene of the monoclonal antibody 1 is conjugated to the carboxy terminal 70 amino acid sequence fragment of the core protein of the proteoglycan as an antibody prepared by antigen immunization; the conjugated monoclonal antibody 1
  • the concentration of carboxyl graphene is 10 ⁇ g / mL;
  • the carboxygraphene of the polyclonal antibody 4 is conjugated to the amino terminal sequence fragment of the core protein of the proteoglycan as an antibody prepared by antigen immunization; the coupled polyclonal antibody 4 carboxylated graphite
  • concentration of the alkene is 10 ⁇ g / mL;
  • the carboxylated graphene of the monoclonal antibody 2 is conjugated to the amino terminal 510 amino acid sequence fragment of the core protein of the proteoglycan as an antibody prepared by antigen immunization; the coupled monoclonal antibody 2
  • the concentration of carboxyl graphene is 10 ⁇ g / mL;
  • the carboxygraphene of the polyclonal antibody 3 is conjugated to the carboxy terminal amino acid sequence fragment of the core protein of the proteoglycan as an antibody prepared by antigen immunization; the conjugated polyclonal antibody 3 carboxylated graphite
  • concentration of the alkene is 10 ⁇ g / mL;
  • Green fluorescent protein with high positive charge (ScGFP); concentration of green fluorescent protein with high positive charge is 0.005 ⁇ g / ⁇ L;
  • Buffer a solution containing a concentration of 10 mM Tris-HCl and a concentration of 100 mM NaCl;
  • microplate and tin foil necessary for assembling the kit.
  • the preparation method of the above kit for high-sensitivity detection of proteoglycans is as follows:
  • the polyclonal antibody was added to the carboxylated graphene solution, mixed, allowed to stand for 1 min, then the surfactant EDTA was added, and then the blocking agent was added, mixed uniformly, and allowed to stand for 3 min to obtain a carboxylated graphene conjugated with the polyclonal antibody. ;
  • the surfactant is ethylenediaminetetraacetic acid (EDTA), 5 ⁇ L of a surfactant is added per ml of the reaction system;
  • the blocking agent is bovine serum albumin (BSA), and a blocking agent is added to the reaction system to the quality.
  • the concentration is 1.5%.
  • the reaction mass concentration of the carboxyl graphene is 1.0 mg/mL, and the reaction mass concentration ratio of the carboxyl graphene to the monoclonal antibody is 10:1, and the reaction quality of the carboxyl graphene and the polyclonal antibody The concentration ratio is 10:1.

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Abstract

一种用于蛋白聚糖高灵敏度检测的试剂盒及其制备方法。试剂盒包括：偶联单克隆抗体的羧基石墨烯和/或偶联多克隆抗体的羧基石墨烯；带高正电荷的荧光蛋白；缓冲液。所述用于蛋白聚糖高灵敏度检测的试剂盒，无需抗体吸附包被酶标板，且无抗原抗体结合后的洗涤等繁琐步骤，可以实现均相体系中目的蛋白聚糖的快速检测分析，灵敏度高。

Description

一种用于蛋白聚糖高灵敏度检测的试剂盒及其制备方法 技术领域
本发明涉及一种用于蛋白聚糖高灵敏度检测的试剂盒及其制备方法，属于生物分析和医学检测技术领域。
背景技术
蛋白聚糖(proteoglycan，PG)是一类复杂的生物大分子，在核心蛋白上共价偶联着一条或多条糖胺聚糖(glycosaminoglycan,GAG)链。GAG是由重复的二糖单位组成的直链多糖，主要分为四类：透明质酸(HA)、硫酸软骨素(CS)/硫酸皮肤素(DS)、硫酸角质素(KS)及肝素(Hep)/硫酸乙酰肝素(HS)。其中透明质酸不与核心蛋白结合组成蛋白聚糖。蛋白聚糖除含糖胺聚糖链外，还偶联一些N-或O-链接的寡糖链。
PG在细胞外基质中广泛分布，也存在于细胞表面以及细胞内的分泌颗粒中。在机体中PG参与了众多生理学过程，如作为组织结构的构成原件存在，调控细胞的增殖、粘附、迁移和变异等过程，还参与生长因子、细胞因子的结合，细胞的信号转导，神经元的生长和形态发生等过程。在癌变微环境中，PG的表达显著改变，因此一部分蛋白聚糖被用作检测某些疾病的特异性标志分子，如蛋白聚糖Glypican的家族成员之一Glypican-3(GPC3)，目前被普遍认为是肝细胞癌(hepatocellular carcinoma,HCC)的特异性标志分子。
GPC3由单链核心蛋白、硫酸乙酰肝素(Heparin Sulfate，HS)链和糖基化磷脂酰肌醇(glycosylphosphatidylinositol，GPI)组成，通过GPI锚定于细胞膜上。GPC3在75％的HCC中显著表达，但在肝脏正常组织和良性病变中并不表达，因此是比甲胎球蛋白更为特异和灵敏的新型HCC标志分子。
由于蛋白聚糖核心蛋白分子量的大小及其结构不同，且共价偶联的GAG链的数量、种类、长度、硫酸化程度及偶联部位不同，使得蛋白聚糖组成复杂，结构多样，因而给其检测带来了诸多困难。在GPC3的检测中，目前最常用的检测方法为夹心ELISA法，但其检测灵敏度偏低，只能检测到50％左右HCC病人血清中有明显GPC3存在；且由于第一抗体在酶标板上的吸附包被及第二抗体与抗原的结合需要孵育过夜，完成一次实验需要三天时间，耗时较长，这极大的限制了此方法的应用，给GPC3的检测带来了困难。
发明内容
本发明针对现有技术的不足，提供一种用于蛋白聚糖高灵敏度检测的试剂盒及其制备方法。该试剂盒可运用耦联抗体的石墨烯与GPC3核心蛋白的结合，对结合在GPC3HS侧链上的ScGFP的荧光淬灭作用达到对血清/血浆中HCC的标志物GPC3的高灵敏度快速检测。
一种用于蛋白聚糖高灵敏度检测的试剂盒，包括：
偶联单克隆抗体的羧基石墨烯和/或偶联多克隆抗体的羧基石墨烯；偶联单克隆抗体的羧基石墨烯包括：偶联单克隆抗体1的羧基石墨烯，所述单克隆抗体1为蛋白聚糖的核心蛋白的羧基端氨基酸序列做为抗原免疫后制得的抗体；偶联单克隆抗体2的羧基石墨烯，所述单克隆抗体2为蛋白聚糖的核心蛋白的氨基端氨基酸序列做为抗原免疫后制得的抗体；偶联多克隆抗体的羧基石墨烯包括：偶联多克隆抗体3的羧基石墨烯，所述多克隆抗体3为蛋白聚糖的核心蛋白的羧基端氨基酸序列做为抗原免疫后制得的抗体；偶联多克隆抗体4的羧基石墨烯，所述多克隆抗体4为蛋白聚糖的核心蛋白的氨基端氨基酸序列做为抗原免疫后制得的抗体；
带高正电荷的荧光蛋白；
缓冲液：含有浓度5～50mM Tris-HCl和浓度50～200mM NaCl的溶液。
根据本发明优选的，所述蛋白聚糖为GPC3、GPC1、GPC2、GPC4、GPC5、GPC6、Syndecan-1、Syndecan-2、Syndecan-3、Syndecan-4、Versican、Aggrecan、Brevican、Decorin、Lumican、Perleacan、Agrin、CD44或NG2；进一步优选的，所述的蛋白聚糖为GPC3或蛋白聚糖Syndecan-2；最优的，所述蛋白聚糖为GPC3时，羧基端氨基酸序列长度为50～90个氨基酸，氨基端氨基酸序列长度为490～530个氨基酸。
根据本发明优选的，所述偶联单克隆抗体1的羧基石墨烯的浓度为5～20μg/mL。
根据本发明优选的，所述偶联单克隆抗体2的羧基石墨烯的浓度为5～20μg/mL。
根据本发明优选的，所述偶联多克隆抗体3的羧基石墨烯的浓度为5～20μg/mL。
根据本发明优选的，所述偶联多克隆抗体4的羧基石墨烯的浓度为5～20μg/mL。
根据本发明优选的，所述带高正电荷的荧光蛋白的浓度为0.001～0.01μg/μL；进一步优选的，所述的带高正电荷荧光蛋白为高正电荷绿色荧光蛋白(ScGFP)。
根据本发明优选的，还包括装配试剂盒必须的微孔板、锡箔纸。
上述用于蛋白聚糖高灵敏度检测的试剂盒的制备方法，步骤如下：
(1)将单克隆抗体1加入到羧基石墨烯溶液中，混匀，静置1min，然后加入表面活性剂，再加入封闭剂，混合均匀，静置封闭1～3min，制得偶联单克隆抗体1的羧基石墨烯；
将单克隆抗体2加入到羧基石墨烯溶液中，混匀，静置1min，然后加入表面活性剂，再加入封闭剂，混合均匀，静置封闭1～3min，制得偶联单克隆抗体2的羧基石墨烯；
将多克隆抗体3加入到羧基石墨烯溶液中，混匀，静置1min，然后加入表面活性剂，再加入封闭剂，混合均匀，静置封闭1～3min，制得偶联多克隆抗体3的羧基石墨烯；
和/或，
将多克隆抗体4加入到羧基石墨烯溶液中，混匀，静置1min，然后加入表面活性剂，再加入封闭剂，混合均匀，静置封闭1～3min，制得偶联多克隆抗体4的羧基石墨烯；
(2)制备带高正电荷荧光蛋白，配制带高正电荷荧光蛋白溶液；
(3)配制含有浓度5～50mM Tris-HCl和浓度50～200mM NaCl的溶液，制得缓冲液；
(4)分别将步骤(1)制得的偶联单克隆抗体1的羧基石墨烯、步骤(1)制得的偶联单克隆抗体2的羧基石墨烯、步骤(2)制得的带高正电荷荧光蛋白溶液和步骤(3)制得的缓冲液经装配后，制得用于蛋白聚糖高灵敏度检测的试剂盒。
根据本发明优选的，所述步骤(1)中，表面活性剂为乙二胺四乙酸(EDTA)或十二烷基磺酸钠(SDS)，每毫升反应体系加入表面活性剂1～5μL；封闭剂为牛血清白蛋白(BSA)，反应体系中加入封闭剂至质量浓度为0.5％～2％。
根据本发明优选的，所述步骤(1)中，所述羧基石墨烯的反应质量浓度为0.8～1.2mg/mL，羧基石墨烯与单克隆抗体1的反应质量浓度比为10：1，羧基石墨烯与单克隆抗体2的反应质量浓度比为10：1，羧基石墨烯与多克隆抗体3的反应质量浓度比为10：1，羧基石墨烯与多克隆抗体4的反应质量浓度比为10：1。
根据本发明优选的，所述步骤(1)中，单克隆抗体1的制备方法如下：
(i)蛋白聚糖的核心蛋白的羧基端氨基酸序列的表达基因插入PTRACER真核表达载体(购自INVITROGEN)，转染HEK293细胞(购自上海细胞所)，从细胞培养上清中纯化，PBS稀释至1.0～2.0mg/mL，制得抗原；
(ii)将步骤(i)制得的抗原免疫BALB/C小鼠3次，抗原免疫量为50～100μg/次，然后用PEG法将效价≥2000的小鼠脾细胞和骨髓瘤细胞进行融合，并筛选出阳性克隆；
(iii)向BALB/C小鼠腹腔中注射弗氏不完全佐剂(购自SIGMA公司)，待小鼠腹腔中产生足量腹水后，于小鼠腹腔中接种步骤(ii)制得的阳性克隆，两周后抽取腹水，离心取上清，纯化后，制得单克隆抗体1。
根据本发明进一步优选的，所述步骤(i)的纯化，为用NI-NTA亲和层析柱进行纯化。
根据本发明进一步优选的，所述步骤(iii)的纯化，步骤如下：
将上清用PBS缓冲液稀释2.5倍，上蛋白A/G亲和层析柱(GE)，分别用含0.5M NaCl的TRIS-HClpH 8.0的缓冲液和含0.5M NaCl的TRIS-HClpH 9.0的缓冲液充分洗脱除杂，最后用100mM三乙胺洗脱回收抗体，收集前三柱体积的洗脱液，并立即用1M的NaH2PO4中和，最后用截流分子量为10KD的超滤管脱盐，并将抗体置换到PBS中，即得。
根据本发明优选的，所述步骤(1)中，单克隆抗体2的制备方法如下：
(a)蛋白聚糖的核心蛋白的氨基端氨基酸序列的表达基因插入PTRACER真核表达载体，转染HEK293细胞，从细胞培养上清中纯化，PBS稀释至1.0～2.0mg/mL，制得抗原；
(b)将步骤(a)制得的抗原免疫BALB/C小鼠3次，抗原免疫量为50～100μg/次，然后用PEG法将效价≥2000的小鼠脾细胞和骨髓瘤细胞进行融合，并筛选出阳性克隆；
(c)向BALB/C小鼠腹腔中注射弗氏不完全佐剂，待小鼠腹腔中产生足量腹水后，于小鼠腹腔中接种步骤(b)制得的阳性克隆，两周后抽取腹水，离心取上清，纯化后，制得单克隆抗体2。
根据本发明进一步优选的，所述步骤(a)的纯化，为用NI-NTA亲和层析柱进行纯化。
根据本发明进一步优选的，所述步骤(c)的纯化，步骤如下：
将上清用PBS缓冲液稀释2.5倍，上蛋白A/G亲和层析柱(GE)，分别用含0.5M NaCl的TRIS-HClpH 8.0的缓冲液和含0.5M NaCl的TRIS-HClpH 9.0的缓冲液充分洗脱除杂，最后用100mM三乙胺洗脱回收抗体，收集前三柱体积的洗脱液，并立即用1M的NaH2PO4中和，最后用截流分子量为10KD的超滤管脱盐，并将抗体置换到PBS中，即得。
根据本发明优选的，所述步骤(1)中，多克隆抗体3的制备方法如下：
a、蛋白聚糖的核心蛋白羧基端氨基酸序列的表达基因插入PTRACER真核表达载体，转染HEK293细胞，从细胞培养上清中纯化，PBS稀释至1.0～2.0mg/mL，制得抗原；
b、将步骤a制得的抗原免疫雌性新西兰大白兔3次，抗原免疫量为200～400μg/次，然后取血，离心取上清，纯化后，制得多克隆抗体3；
所述步骤b的纯化，步骤如下：
将上清用PBS缓冲液稀释2.5倍，上蛋白A/G亲和层析柱(GE)，分别用含0.5M NaCl的TRIS-HClpH 8.0的缓冲液和含0.5M NaCl的TRIS-HClpH 9.0的缓冲液充分洗脱除杂，最后用100mM三乙胺洗脱回收抗体，收集前三柱体积的洗脱液，并立即用1M的NaH2PO4中和，最后用截流分子量为10KD的超滤管脱盐，并将抗体置换到PBS缓冲液中，即得。
根据本发明优选的，所述步骤(1)中，多克隆抗体4的制备方法如下：
a、蛋白聚糖的核心蛋白氨基端氨基酸序列的表达基因插入PTRACER真核表达载体，转染HEK293细胞，从细胞培养上清中纯化，PBS稀释至1.0～2.0mg/mL，制得抗原；
b、将步骤a制得的抗原免疫雌性新西兰大白兔3次，抗原免疫量为200～400μg/次，然后取血，离心取上清，纯化后，制得多克隆抗体4；
所述步骤b的纯化，步骤如下：
将上清用PBS缓冲液稀释2.5倍，上蛋白A/G亲和层析柱(GE)，分别用含0.5M NaCl的TRIS-HClpH 8.0的缓冲液和含0.5M NaCl的TRIS-HClpH 9.0的缓冲液充分洗脱除杂，最后用100mM三乙胺洗脱回收抗体，收集前三柱体积的洗脱液，并立即用1M的NaH2PO4中和，最后用截流分子量为10KD的超滤管脱盐，并将抗体置换到PBS缓冲液中，即得。
根据本发明优选的，所述步骤(2)中，带高正电荷荧光蛋白为高正电荷绿色荧光蛋白(ScGFP)。带高正电荷荧光蛋白的制备为本领域的常规技术，如可参考文献Lawrence,M.S.；Phillips,K.J.；Liu,D.R.J.Am.Chem.Soc.2007,129,10110-10112.中记载的构建表达方法。
上述工艺步骤如无特别说明，均可采用本领域的常规操作。
有益效果
1、本发明所述用于蛋白聚糖高灵敏度检测的试剂盒，无需抗体吸附包被酶标板，且无抗原抗体结合后的洗涤等繁琐步骤，可以实现均相体系中目的蛋白聚糖的快速检测分析，灵敏度高；
2、本发明所述用于蛋白聚糖高灵敏度检测的试剂盒，在均相体系中通过偶联在羧基石墨烯上的抗体，特异性的结合目的蛋白聚糖的核心蛋白，同时目的蛋白聚糖上的糖侧链又可 与带高正电荷的绿色荧光蛋白(ScGFP)通过电荷作用相互连接，待最后加入偶联另一种抗体的羧基石墨烯后，形成一种夹心结构，可对处于夹层中间目的蛋白聚糖侧链上的ScGFP进行更好的荧光淬灭作用，进一步提高检测的灵敏度，从而解决ELISA法检测灵敏度低，反应时间过长等缺点，可解决在无昂贵设备条件下病人血清中目的蛋白聚糖的快速灵敏检测问题；
3、本发明所述用于蛋白聚糖高灵敏度检测的试剂盒的制备方法简单，易操作，成本低廉，便于大规模推广应用。
附图说明
图1：ScGFP、偶联单克隆抗体(αGCN、αGCC)的羧基石墨烯和GPC3加入顺序对荧光强度的影响柱状图；
A：偶联αGCN的羧基石墨烯和GPC3、ScGFP混合后加入偶联αGCC的羧基石墨烯；B：两种分别偶联αGCN和αGCC的羧基石墨烯与GPC3混合后加入ScGFP；C：两种分别偶联αGCN和αGCC的羧基石墨烯与ScGFP混合后加入GPC3；D：偶联αGCC的羧基石墨烯和GPC3、ScGFP混合后加入偶联αGCN的羧基石墨烯。
图2：缓冲液中GPC3的检测结果柱状图；
图3：FBS中GPC3的检测结果柱状图；
图4：GPC3与GPC3ΔGAG的检测结果比较柱状图；
图5：临床血清中GPC3的检测结果。
图6：缓冲液中Syndecan-2的检测结果柱状图；
图7：偶联抗体1的羧基石墨烯的制备过程示意图；
图8：偶联抗体2的羧基石墨烯的制备过程示意图；
图9：蛋白聚糖GPC3检测过程的过程示意图；
图10：实施例9采用蛋白聚糖GPC3高灵敏度检测的试剂盒检测GPC3的检测结果柱状图；
图11：实施例11采用蛋白聚糖GPC3高灵敏度检测的试剂盒检测GPC3的检测结果柱状图；
具体实施方式
以下实施例的阐述，是为了全面公开本发明如何实施的一些常用技术，而不是为了限制本发明的应用范围。发明人已经尽最大努力确保实施例中各参数的准确性(例如质量，温度等)，但是一些实验误差和偏差也应该予以考虑。
实施例1、
一种用于蛋白聚糖GPC3高灵敏度检测的试剂盒，包括：
偶联单克隆抗体1的羧基石墨烯，所述单克隆抗体1为蛋白聚糖的核心蛋白的羧基端70个氨基酸序列片段做为抗原免疫后制得的抗体；所述偶联单克隆抗体1的羧基石墨烯的浓度为10μg/mL；
偶联单克隆抗体2的羧基石墨烯，所述单克隆抗体2为蛋白聚糖的核心蛋白的氨基端510个氨基酸序列片段做为抗原免疫后制得的抗体；所述偶联单克隆抗体2羧基石墨烯的浓度为10μg/mL；
带高正电荷的绿色荧光蛋白(ScGFP)；带高正电荷的绿色荧光蛋白的浓度为0.005μg/μL；
缓冲液：含有浓度10mM Tris-HCl和浓度100mM NaCl的溶液；
以及，装配试剂盒必须的微孔板、锡箔纸。
上述用于蛋白聚糖高灵敏度检测的试剂盒的制备方法，步骤如下：
(1)将单克隆抗体1加入到羧基石墨烯溶液中，混匀，静置1min，然后加入表面活性剂EDTA，再加入封闭剂，混合均匀，静置封闭3min，制得偶联单克隆抗体1的羧基石墨烯(制备过程示意图如图7所示)；
将单克隆抗体2加入到羧基石墨烯溶液中，混匀，静置1min，然后加入表面活性剂EDTA，再加入封闭剂，混合均匀，静置封闭3min，制得偶联单克隆抗体2的羧基石墨烯(制备过程示意图如图8所示)；
(2)制备带高正电荷荧光蛋白，配制带高正电荷荧光蛋白溶液；
(3)配制含有浓度10mM Tris-HCl和浓度100mM NaCl的溶液，制得缓冲液；
(4)分别将步骤(1)制得的偶联单克隆抗体1的羧基石墨烯、步骤(1)制得的偶联单克隆抗体2的羧基石墨烯、步骤(2)制得的带高正电荷荧光蛋白溶液和步骤(3)制得的缓冲液经装配后，制得用于蛋白聚糖高灵敏度检测的试剂盒。
所述步骤(1)中，表面活性剂为乙二胺四乙酸(EDTA)，每毫升反应体系加入表面活性剂5μL；封闭剂为牛血清白蛋白(BSA)，反应体系中加入封闭剂至质量浓度为1.5％。
所述步骤(1)中，所述羧基石墨烯的反应质量浓度为1.0mg/mL，羧基石墨烯与单克隆抗体1的反应质量浓度比为10：1，羧基石墨烯与单克隆抗体2的反应质量浓度比为10：1。
所述步骤(1)中，单克隆抗体1的制备方法如下：
(i)用GPC3全长蛋白基因核心蛋白的羧基端70个氨基酸序列片段(αGCC，SEQ ID NO.1)的表达基因插入PTRACER真核表达载体，转染HEK293细胞，用NI-NTA亲和层析柱从细胞培养上清中纯化，PBS稀释至2.0mg/mL，制得抗原；
(ii)将步骤(i)制得的抗原免疫BALB/C小鼠3次，抗原免疫量为80μg/次，然后用PEG法将效价达到2000的小鼠脾细胞和骨髓瘤细胞进行融合，并筛选出阳性克隆；
(iii)向BALB/C小鼠腹腔中注射弗氏不完全佐剂，待小鼠腹腔中产生足量腹水后，于小鼠腹腔中接种步骤(ii)制得的阳性克隆，两周后抽取腹水，离心取上清，纯化后，制得单克隆抗体1。
所述步骤(iii)的纯化，步骤如下：
将上清用PBS稀释2.5倍，上蛋白A/G亲和层析柱(GE)，分别用含0.5M NaCl的TRIS-HClpH 8.0的缓冲液和含0.5M NaCl的TRIS-HClpH 9.0的缓冲液充分洗脱除杂，最后用100mM三乙胺洗脱回收抗体，收集前三柱体积的洗脱液，并立即用1M的NaH2PO4中和，最后用截流分子量为10KD的超滤管脱盐，并将抗体置换到PBS中，即得。
所述步骤(1)中，单克隆抗体2的制备方法如下：
(a)用GPC3全长蛋白基因除羧基端的70个氨基酸序列外的其他氨基酸序列片段(αGCN，SEQ ID NO.2)的表达基因插入PTRACER真核表达载体，转染HEK293细胞，用NI-NTA亲和层析柱从细胞培养上清中纯化，PBS稀释至2.0mg/mL，制得抗原；
(b)将步骤(a)制得的抗原免疫BALB/C小鼠3次，抗原免疫量为80μg/次，然后用PEG法将效价达到2000的小鼠脾细胞和骨髓瘤细胞进行融合，并筛选出阳性克隆；
(c)向BALB/C小鼠腹腔中注射弗氏不完全佐剂，待小鼠腹腔中产生足量腹水后，于小鼠腹腔中接种步骤(b)制得的阳性克隆，两周后抽取腹水，离心取上清，纯化后，制得单克隆抗体2。
所述步骤(c)的纯化，步骤如下：
将上清用PBS稀释2.5倍，上蛋白A/G亲和层析柱(GE)，分别用含0.5M NaCl的TRIS-HClpH 8.0的缓冲液和含0.5M NaCl的TRIS-HClpH 9.0的缓冲液充分洗脱除杂，最后用100mM三乙胺洗脱回收抗体，收集前三柱体积的洗脱液，并立即用1M的NaH2PO4中和，最后用截流分子量为10KD的超滤管脱盐，并将抗体置换到PBS中，即得。
所述步骤(2)中，带高正电荷荧光蛋白的制备为本领域的常规技术，如可参考文献Lawrence,M.S.；Phillips,K.J.；Liu,D.R.J.Am.Chem.Soc.2007,129,10110-10112.中记载的构建表达方法。
实施例2、反应条件的优化
试剂添加顺序对GPC3检测的的影响
在微孔板40μL反应体系中依照不同顺序加入偶联αGCN的羧基石墨烯0.05～0.2μg、偶联αGCC的羧基石墨烯0.05～0.2μg、ScGFP 0.01～0.5μg、GPC3 0.001～0.005ng，混匀、室温避光放置15min，孵育结束后，测定荧光强度(Ex：470nm，Em：509nm)。
其中：A组为偶联αGCN的羧基石墨烯和GPC3、ScGFP混合后加入偶联αGCC的羧基石墨烯；B组为两种分别偶联αGCN和αGCC的羧基石墨烯与GPC3混合后加入ScGFP；C组为两种分别偶联αGCN和αGCC的羧基石墨烯与ScGFP混合后加入GPC3；D组为偶联αGCC的羧基石墨烯和GPC3、ScGFP混合后加入偶联αGCN的羧基石墨烯。
结果显示，先加入偶联一种抗体的羧基石墨烯和GPC3，随后加入ScGFP，最后加入偶联另一种抗体的石墨烯的顺序对荧光的淬灭作用最强，因此选用此顺序进行后续实验(如图1)。
加入试剂孵育时间对GPC3检测的影响
在微孔板40μL反应体系中加入偶联αGCN的羧基石墨烯0.05～0.2μg、GPC3 0.001～0.005ng，混匀、室温放置不同的时间；然后统一加入ScGFP 0.01～0.5μg，混匀、室温避光放置5min；最后加入偶联αGCC的羧基石墨烯0.05～0.2μg，混匀、室温避光放置10min，孵育结束后，测定荧光强度(Ex：470nm，Em：509nm)。
结果显示，加入前两者后放置5min即可基本达到最佳荧光淬灭效果。
在微孔板40μL PBS(pH7.4)溶液中加入偶联αGCN的羧基石墨烯0.05～0.2μg，GPC30.001～0.005ng后混匀、室温放置5min；随后加入ScGFP 0.01～0.5μg，混匀、室温避光放置不同时间，最后加入偶联αGCC的羧基石墨烯0.05～0.2μg，混匀、室温避光放置10min，孵育结束后，测定荧光强度(Ex：470nm，Em：509nm)。
结果显示，加入混合物后放置15min后基本达到荧光淬灭平衡状态。
在微孔板40μL PBS(pH7.4)溶液中加入αGCN的羧基石墨烯0.05～0.2μg、GPC30.001～0.005ng，混匀、室温反应5min；随后加入ScGFP 0.01～0.5μg，混匀、室温避光放置15min；最后加入偶联αGCC的羧基石墨烯0.05～0.2μg，混匀、室温避光放置不同的时间，孵育结束后，测定荧光强度(Ex：470nm，Em：509nm)。
结果显示，加入羧基石墨烯后10min即可基本达到荧光淬灭平衡。
因此最后选择加入偶联αGCN的羧基石墨烯和GPC3后放置5～10min，随后加入ScGFP，室温避光放置10～30min，最后加入偶联αGCC的羧基石墨烯避光放置5～20min作为反应时间。
不同pH的不同缓冲溶液对GPC3检测的影响
在不同pH值的10mM HAc-NaAc、NaH2PO4-Na2HPO4、Tris-HCl缓冲液中加入偶联αGCN的羧基石墨烯0.05～0.2μg，GPC3 0.001～0.005ng，混匀、室温放置5min；随后加入ScGFP 0.01～0.5μg，混匀、室温避光放置15min；最后加入偶联αGCC的羧基石墨烯0.05～0.2μg，混匀、室温避光放置10min，孵育结束后，测定荧光强度(Ex：470nm，Em：509nm)。
结果显示在Tris-HCl(pH 7～10.0)缓冲液中，对ScGFP的荧光淬灭能力最强。
不同NaCl浓度对GPC3检测的影响
在含不同浓度NaCl的10mM Tris-HCl缓冲液中加入偶联αGCN的羧基石墨烯0.05～0.2μg，GPC3 0.001～0.005ng，混匀、室温放置5min；随后加入ScGFP 0.01～0.5μg，混匀、室温避光放置15min；最后加入偶联αGCC的羧基石墨烯，混匀、室温避光放置10min，孵育结束后，测定荧光强度(Ex：470nm，Em：509nm)。
结果显示随着NaCl浓度的增加，对ScGFP的荧光淬灭能力呈现先增强后减弱的情况，据此选择NaCl浓度为50～200mM作为反应体系中加入盐的浓度。
羧基石墨烯偶联抗体量对GPC3检测的影响
制备偶联不同含量抗体的羧基石墨烯，在10mM Tris-HCl，100mM NaCl溶液中加入偶联不同含量αGCN的羧基石墨烯0.05～0.2μg，GPC3 0.001～0.005ng，混匀、室温放置5min；随后加入ScGFP 0.01～0.5μg，混匀、室温避光放置15min；最后加入相对应的偶联不同含 量αGCC的羧基石墨烯0.05～0.2μg，混匀、室温避光放置10min，孵育结束后，测定荧光强度(Ex：470nm，Em：509nm)。
结果显示，偶联0.05～0.2mg/mL抗体的羧基石墨烯具有最好的淬灭效果。
实施例3、GPC3在缓冲液中的检测
在微孔板40μL 10mM Tris-HCl、100mM NaCl溶液中，加入偶联αGCN的羧基石墨烯0.05～0.2μg，GPC3 0～0.04ng，混匀、室温放置5min；随后加入ScGFP 0.01～0.5μg，混匀，室温避光放置15min；最后加入偶联αGCC的羧基石墨烯0.05～0.2μg，混匀、室温避光放置10min，孵育结束后，测定荧光强度(Ex：470nm，Em：509nm)。蛋白聚糖GPC3检测过程的过程示意图如图9所示。
结果显示，随着GPC3浓度的升高，其介导的荧光淬灭强度也逐渐增高，检测灵敏度可以达到10pg/mL以下(如图2)，远高于夹心ELISA法。
实施例4、GPC3在血清中的检测
将GPC3溶于胎牛血清(FBS)作为贮备液。然后用质量浓度为20％的FBS稀释GPC3贮藏液成不同浓度，待检。在40μL 10mM Tris-HCl、100mM NaCl溶液中，加入偶联αGCN的羧基石墨烯0.05～0.2μg，GPC3 0～0.04ng，混匀、室温放置5min；随后加入ScGFP 0.01～0.5μg，混匀、室温避光放置15min；最后加入偶联αGCC的羧基石墨烯0.05～0.2μg，混匀、室温避光放置10min，孵育结束后，测定荧光强度(Ex：470nm，Em：509nm)。
结果显示，血清中GPC3含量与荧光淬灭强度呈显著的剂量依赖性，灵敏度与在缓冲液中相似，达到皮克级(如图3)。
实施例5、GPC3和GPC3ΔGAG检测的比较
在40μL 10mM Tris-HCl(pH 10.0)，100mM NaCl溶液中加入偶联αGCN的羧基石墨烯0.05～0.2μg，并分别加入不同浓度的GPC3和其非糖基化突变GPC3ΔGAG，混匀、室温放置5min；随后加入ScGFP 0.01～0.5μg，混匀、室温避光放置15min；最后加入偶联αGCC的羧基石墨烯0.05～0.2μg，混匀、室温避光放置10min，孵育结束后，测定荧光强度(Ex：470nm，Em：509nm)。
结果显示，GPC3的非糖基化突变GPC3ΔGAG几乎不能介导羧基石墨烯对ScGFP的荧光淬灭(如图4)，表明GPC3的糖胺聚糖侧链HS对于ScGFP的结合至关重要，同时也表明上述由偶联单克隆抗体1的羧基石墨烯和偶联单克隆抗体2的羧基石墨烯装配的试剂盒对GPC3检测的专一性。
实施例6、临床血清中GPC3的检测和比较分析
在32μL 10mM Tris-HCl，100mM NaCl溶液中加入偶联αGCN的羧基石墨烯0.05～0.2μg，8μL五倍稀释的血清(正常人、良性肝病病人或HCC病人)，混匀，室温放置5min；随后加入ScGFP 0.01～0.5μg，混匀、室温避光放置15min；最后加入加入偶联αGCC的羧基石墨烯0.05～0.2μg，混匀、室温避光放置10min，孵育结束后，测定荧光强度(Ex：470nm，Em：509nm)。
结果显示，正常人和良性肝病病人血清样本的荧光强度分布范围基本相似，无显著差异。但HCC病人血清的荧光强度显著下降，意味着GPC3的存在介导了石墨烯对ScGFP的荧光淬灭，与正常组和良性肝病组相比存在极为显著的差异。其中有近14例HCC病人的血清的荧光强度显著低于正常和良性肝病组，占HCC病人总数的60％(如图5)，高于以前报道的45～50％。本发明所建立的GPC3均相检测技术，灵敏度远高于常用的ELISA法，这可能是其检出率较高的原因。
实施例7、Syndecan-2在缓冲液中的检测
一种用于蛋白聚糖Syndecan-2高灵敏度检测的试剂盒，包括：
偶联抗Syndecan-2单克隆抗体1的羧基石墨烯，所述Syndecan-2单克隆抗体1为Human Syndecan-2MAb(Clone 305507),Rat IgG2A(购自R&D Systems公司)；所述偶联抗Syndecan-2单克隆抗体1的羧基石墨烯的浓度为10μg/mL；
偶联抗Syndecan-2单克隆抗体2的羧基石墨烯，所述Syndecan-2单克隆抗体2为Human Syndecan-2MAb(Clone 305515),Rat IgG2B(购自R&D Systems公司)；所述偶联抗Syndecan-2单克隆抗体2的羧基石墨烯的浓度为10μg/mL；
带高正电荷的绿色荧光蛋白(ScGFP)；带高正电荷的绿色荧光蛋白的浓度为0.005μg/μL；
缓冲液：含有浓度10mM Tris-HCl和浓度100mM NaCl的溶液；
以及，装配试剂盒必须的微孔板、锡箔纸。
上述用于蛋白聚糖Syndecan-2高灵敏度检测的试剂盒的制备方法同实施例1。
在微孔板40μL 10mM Tris-HCl、100mM NaCl溶液中，加入偶联抗Syndecan-2单克隆抗体1(Human Syndecan-2MAb(Clone 305507),Rat IgG2A)的羧基石墨烯0.05～0.2μg，Syndecan-2 0～0.05ng，混匀、室温放置5min；随后加入ScGFP 0.01～0.5μg，混匀，室温避光放置15min；最后加入偶联抗Syndecan-2单克隆抗体2(Human Syndecan-2MAb(Clone 305515),Rat IgG2B)的羧基石墨烯0.05～0.2μg，混匀、室温避光放置10min，孵育结束后，测定荧光强度(Ex：470nm，Em：509nm)。结果显示，随着Syndecan-2浓度的升高，其辅助荧光淬灭强度也逐渐增高(如图6)。
实施例8、
一种用于蛋白聚糖GPC3高灵敏度检测的试剂盒，包括：
偶联多克隆抗体3的羧基石墨烯，所述多克隆抗体3为蛋白聚糖的核心蛋白的羧基端290个氨基酸序列片段做为抗原免疫后制得的抗体；所述偶联多克隆抗体3的羧基石墨烯的浓度为10μg/mL；
偶联多克隆抗体4的羧基石墨烯，所述多克隆抗体4为蛋白聚糖的核心蛋白的氨基端290个氨基酸序列片段做为抗原免疫后制得的抗体；所述偶联多克隆抗体4羧基石墨烯的浓度为10μg/mL；
带高正电荷的绿色荧光蛋白(ScGFP)；带高正电荷的绿色荧光蛋白的浓度为0.005μg/μL；
缓冲液：含有浓度10mM Tris-HCl和浓度100mM NaCl的溶液；
以及，装配试剂盒必须的微孔板、锡箔纸。
上述用于蛋白聚糖高灵敏度检测的试剂盒的制备方法，步骤如下：
(1)将多克隆抗体3加入到羧基石墨烯溶液中，混匀，静置1min，然后加入表面活性剂EDTA，再加入封闭剂，混合均匀，静置封闭3min，制得偶联多克隆抗体3的羧基石墨烯；
将多克隆抗体4加入到羧基石墨烯溶液中，混匀，静置1min，然后加入表面活性剂EDTA，再加入封闭剂，混合均匀，静置封闭3min，制得偶联多克隆抗体4的羧基石墨烯；
(2)制备带高正电荷荧光蛋白，配制带高正电荷荧光蛋白溶液；
(3)配制含有浓度10mM Tris-HCl和浓度100mM NaCl的溶液，制得缓冲液；
(4)分别将步骤(1)制得的偶联多克隆抗体3的羧基石墨烯、步骤(1)制得的偶联多克隆抗体4的羧基石墨烯、步骤(2)制得的带高正电荷荧光蛋白溶液和步骤(3)制得的缓冲液经装配后，制得用于蛋白聚糖高灵敏度检测的试剂盒。
所述步骤(1)中，表面活性剂为乙二胺四乙酸(EDTA)，每毫升反应体系加入表面活性剂5μL；封闭剂为牛血清白蛋白(BSA)，反应体系中加入封闭剂至质量浓度为1.5％。
所述步骤(1)中，所述羧基石墨烯的反应质量浓度为1.0mg/mL，羧基石墨烯与多克隆抗体3的反应质量浓度比为10：1，羧基石墨烯与多克隆抗体4的反应质量浓度比为10：1。
所述步骤(1)中，多克隆抗体3的制备方法如下：
(i)用GPC3全长蛋白基因核心蛋白的羧基端290个氨基酸序列片段(SEQ ID NO.3)的表达基因插入PTRACER真核表达载体，转染HEK293细胞，用NI-NTA亲和层析柱从细胞培养上清中纯化，PBS稀释至2.0mg/mL，制得抗原；
(ii)将步骤(i)制得的抗原免疫雌性新西兰大白兔3次，抗原免疫量为300μg/次，然后取血，离心取上清，纯化后，制得多克隆抗体3；
所述步骤(ii)的纯化，步骤如下：
将上清用PBS缓冲液稀释2.5倍，上蛋白A/G亲和层析柱(GE)，分别用含0.5M NaCl的TRIS-HClpH 8.0的缓冲液和含0.5M NaCl的TRIS-HClpH 9.0的缓冲液充分洗脱除杂，最后用100mM三乙胺洗脱回收抗体，收集前三柱体积的洗脱液，并立即用1M的NaH2PO4中和，最后用截流分子量为10KD的超滤管脱盐，并将抗体置换到PBS缓冲液中，即得。
所述步骤(1)中，多克隆抗体4的制备方法如下：
(a)用GPC3全长蛋白基因核心蛋白的氨基端290个氨基酸序列片段(SEQ ID NO.4)的表达基因插入PTRACER真核表达载体，转染HEK293细胞，用NI-NTA亲和层析柱从细胞培养上清中纯化，PBS稀释至2.0mg/mL，制得抗原；
(b)将步骤(a)制得的抗原免疫雌性新西兰大白兔3次，抗原免疫量为300μg/次，然后取血，离心取上清，纯化后，制得多克隆抗体4；
所述步骤(b)的纯化，步骤如下：
将上清用PBS缓冲液稀释2.5倍，上蛋白A/G亲和层析柱(GE)，分别用含0.5M NaCl的TRIS-HClpH 8.0的缓冲液和含0.5M NaCl的TRIS-HClpH 9.0的缓冲液充分洗脱除杂，最后用100mM三乙胺洗脱回收抗体，收集前三柱体积的洗脱液，并立即用1M的NaH2PO4中和，最后用截流分子量为10KD的超滤管脱盐，并将抗体置换到PBS缓冲液中，即得。
所述步骤(2)中，带高正电荷荧光蛋白的制备为本领域的常规技术，如可参考文献Lawrence,M.S.；Phillips,K.J.；Liu,D.R.J.Am.Chem.Soc.2007,129,10110-10112.中记载的构建表达方法。
实施例9、多克隆抗体用于GPC3的检测
在微孔板40μL 10mM Tris-HCl、100mM NaCl溶液中，加入实施例8制得的偶联多克隆抗体3的羧基石墨烯0.05～0.2μg，GPC3 0～0.04ng，混匀、室温放置5min；随后加入ScGFP 0.01～0.5μg，混匀，室温避光放置15min；最后再加入实施例8制得的偶联GPC3多克隆抗体4的羧基石墨烯0.05～0.2μg，混匀、室温避光放置10min，孵育结束后，测定荧光强度(Ex：470nm，Em：509nm)，结果如图10所示；
结果显示，同单克隆抗体一样，多克隆抗体也可用于GPC3的检测，且随着GPC3浓度的升高，其介导的荧光淬灭强度也逐渐增高。
实施例10、
一种用于蛋白聚糖GPC3高灵敏度检测的试剂盒，包括：
偶联单克隆抗体1的羧基石墨烯，所述单克隆抗体1为蛋白聚糖的核心蛋白的羧基端70个氨基酸序列片段做为抗原免疫后制得的抗体；所述偶联单克隆抗体1的羧基石墨烯的浓度为10μg/mL；
偶联多克隆抗体4的羧基石墨烯，所述多克隆抗体4为蛋白聚糖的核心蛋白的氨基端氨基酸序列片段做为抗原免疫后制得的抗体；所述偶联多克隆抗体4羧基石墨烯的浓度为10μg/mL；
或
偶联单克隆抗体2的羧基石墨烯，所述单克隆抗体2为蛋白聚糖的核心蛋白的氨基端510个氨基酸序列片段做为抗原免疫后制得的抗体；所述偶联单克隆抗体2的羧基石墨烯的浓度为10μg/mL；
偶联多克隆抗体3的羧基石墨烯，所述多克隆抗体3为蛋白聚糖的核心蛋白的羧基端氨基酸序列片段做为抗原免疫后制得的抗体；所述偶联多克隆抗体3羧基石墨烯的浓度为10μg/mL；
带高正电荷的绿色荧光蛋白(ScGFP)；带高正电荷的绿色荧光蛋白的浓度为0.005μg/μL；
缓冲液：含有浓度10mM Tris-HCl和浓度100mM NaCl的溶液；
以及，装配试剂盒必须的微孔板、锡箔纸。
上述用于蛋白聚糖高灵敏度检测的试剂盒的制备方法，步骤如下：
(1)将单克隆抗体加入到羧基石墨烯溶液中，混匀，静置1min，然后加入表面活性剂EDTA，再加入封闭剂，混合均匀，静置封闭3min，制得偶联单克隆抗体的羧基石墨烯；
将多克隆抗体加入到羧基石墨烯溶液中，混匀，静置1min，然后加入表面活性剂EDTA，再加入封闭剂，混合均匀，静置封闭3min，制得偶联多克隆抗体的羧基石墨烯；
(2)制备带高正电荷荧光蛋白，配制带高正电荷荧光蛋白溶液；
(3)配制含有浓度10mM Tris-HCl和浓度100mM NaCl的溶液，制得缓冲液；
(4)分别将步骤(1)制得的偶联单克隆抗体的羧基石墨烯、步骤(1)制得的偶联多克隆抗体的羧基石墨烯、步骤(2)制得的带高正电荷荧光蛋白溶液和步骤(3)制得的缓冲液经装配后，制得用于蛋白聚糖高灵敏度检测的试剂盒。
所述步骤(1)中，表面活性剂为乙二胺四乙酸(EDTA)，每毫升反应体系加入表面活性剂5μL；封闭剂为牛血清白蛋白(BSA)，反应体系中加入封闭剂至质量浓度为1.5％。
所述步骤(1)中，所述羧基石墨烯的反应质量浓度为1.0mg/mL，羧基石墨烯与单克隆抗体的反应质量浓度比为10：1，羧基石墨烯与多克隆抗体的反应质量浓度比为10：1。
实施例11、单、多克隆抗体混合用于GPC3的检测
在微孔板40μL 10mM Tris-HCl、100mM NaCl溶液中，加入制得的偶联单克隆抗体1的羧基石墨烯0.05～0.2μg，GPC3 0～0.04ng，混匀、室温放置5min；随后加入ScGFP 0.01～0.5μg，混匀，室温避光放置15min；最后再加入偶联GPC3多克隆抗体4的羧基石墨烯0.05～0.2μg，混匀、室温避光放置10min，孵育结束后，测定荧光强度(Ex：470nm，Em：509nm)，结果如图11所示；
结果显示，同单克隆抗体一样，单、多克隆抗体混合也可用于GPC3的检测，随着GPC3浓度的升高，其介导的荧光淬灭强度也逐渐增高。
说明书中涉及的参考文献：
1.Llovet JM,Burroughs A,Bruix J.Hepatocellular carcinoma.Lancet 2003,362:1907-17.
2.Filmus J,Selleck SB.Glypicans:proteoglycans with a surprise.J.Clin.Invest.2001,108:497–501.
3.Z-W Zhu,H Friess,L Wang,M Abou-Shady,A Zimmermann,et al.Enhanced glypican-3expression differentiates the majority of hepatocellular carcinomas from benign hepatic disorders.Gut 2001,48:558–564.
4.Chunyang Lei,Yan Huang,Zhou Nie,et al.A Supercharged Fluorescent Protein as a Versatile Probe for Homogenous DNA Detection and Methylation Analysis.Angewandte Chemie 2014,53:8358-8362.
5.Michael S,Lawrence,Kevin J.Phillips,et al.Supercharging proteins can impart unusual  resilience. J. Am. Chem. Soc. 2007, 129: 10110–10112.
6.Capurro M, Wanless IR, Sherman M, et al. Glypican-3: a novel serum and histochemical marker for hepatocellular carcinoma. Gastroenterology 2003, 125: 81–90.
7.Achilleas D. Theocharis, Spyridon S. Skandalis, George N. Tzanakakis, et al. Proteoglycans in health and disease: novel roles for proteoglycans in malignancy and their pharmacological targeting. FEBS J. 2010, 277: 3904-3923.
8.Mariana I Capurro, Ping Xu, Wen Shi, et al. Glypican-3 Inhibits HedgehogSignaling During Development by Competing with Patched for HedgehogBinding. Dev. Cell. 2008,14(5) : 700-11.

Claims (10)

  1. 一种用于蛋白聚糖高灵敏度检测的试剂盒，包括：
    偶联单克隆抗体的羧基石墨烯和/或偶联多克隆抗体的羧基石墨烯；偶联单克隆抗体的羧基石墨烯包括：偶联单克隆抗体1的羧基石墨烯，所述单克隆抗体1为蛋白聚糖的核心蛋白的羧基端氨基酸序列做为抗原免疫后制得的抗体；偶联单克隆抗体2的羧基石墨烯，所述单克隆抗体2为蛋白聚糖的核心蛋白的氨基端氨基酸序列做为抗原免疫后制得的抗体；偶联多克隆抗体的羧基石墨烯包括：偶联多克隆抗体3的羧基石墨烯，所述多克隆抗体3为蛋白聚糖的核心蛋白的羧基端氨基酸序列做为抗原免疫后制得的抗体；偶联多克隆抗体4的羧基石墨烯，所述多克隆抗体4为蛋白聚糖的核心蛋白的氨基端氨基酸序列做为抗原免疫后制得的抗体；
    带高正电荷的荧光蛋白；
    缓冲液：含有浓度5～50mM Tris-HCl和浓度50～200mM NaCl的溶液。
  2. 如权利要求1所述的试剂盒，其特征在于，所述蛋白聚糖为GPC3、GPC1、GPC2、GPC4、GPC5、GPC6、Syndecan-1、Syndecan-2、Syndecan-3、Syndecan-4、Versican、Aggrecan、Brevican、Decorin、Lumican、Perleacan、Agrin、CD44或NG2；进一步优选的，所述的蛋白聚糖为GPC3或蛋白聚糖Syndecan-2；最优的，所述蛋白聚糖为GPC3时，羧基端氨基酸序列长度为50～90个氨基酸，氨基端氨基酸序列长度为490～530个氨基酸。
  3. 如权利要求1所述的试剂盒，其特征在于，所述偶联单克隆抗体1的羧基石墨烯的浓度为5～20μg/mL；
    优选的，所述偶联单克隆抗体2的羧基石墨烯的浓度为5～20μg/mL；
    优选的，所述偶联多克隆抗体3的羧基石墨烯的浓度为5～20μg/mL；
    优选的，所述偶联多克隆抗体4的羧基石墨烯的浓度为5～20μg/mL；
    优选的，所述带高正电荷的荧光蛋白的浓度为0.001～0.01μg/μL；进一步优选的，所述的带高正电荷荧光蛋白为高正电荷绿色荧光蛋白(ScGFP)；
    优选的，还包括装配试剂盒必须的微孔板、锡箔纸。
  4. 权利要求1所述用于蛋白聚糖高灵敏度检测的试剂盒的制备方法，其特征在于，步骤如下：
    (1)将单克隆抗体1加入到羧基石墨烯溶液中，混匀，静置1min，然后加入表面活性剂，再加入封闭剂，混合均匀，静置封闭1～3min，制得偶联单克隆抗体1的羧基石墨烯；
    将单克隆抗体2加入到羧基石墨烯溶液中，混匀，静置1min，然后加入表面活性剂，再加入封闭剂，混合均匀，静置封闭1～3min，制得偶联单克隆抗体2的羧基石墨烯；
    将多克隆抗体3加入到羧基石墨烯溶液中，混匀，静置1min，然后加入表面活性剂，再加入封闭剂，混合均匀，静置封闭1～3min，制得偶联多克隆抗体3的羧基石墨烯；
    和/或，
    将多克隆抗体4加入到羧基石墨烯溶液中，混匀，静置1min，然后加入表面活性剂，再加入封闭剂，混合均匀，静置封闭1～3min，制得偶联多克隆抗体4的羧基石墨烯；
    (2)制备带高正电荷荧光蛋白，配制带高正电荷荧光蛋白溶液；
    (3)配制含有浓度5～50mM Tris-HCl和浓度50～200mM NaCl的溶液，制得缓冲液；
    (4)分别将步骤(1)制得的偶联单克隆抗体1的羧基石墨烯、步骤(1)制得的偶联单克隆抗体2的羧基石墨烯、步骤(2)制得的带高正电荷荧光蛋白溶液和步骤(3)制得的缓冲液经装配后，制得用于蛋白聚糖高灵敏度检测的试剂盒。
  5. 如权利要求4所述的制备方法，其特征在于，所述步骤(1)中，表面活性剂为乙二胺四乙酸(EDTA)或十二烷基磺酸钠(SDS)，每毫升反应体系加入表面活性剂1～5μL；封闭剂为牛血清白蛋白(BSA)，反应体系中加入封闭剂至质量浓度为0.5％～2％。
  6. 如权利要求4所述的制备方法，其特征在于，所述步骤(1)中，所述羧基石墨烯的反应质量浓度为0.8～1.2mg/mL，羧基石墨烯与单克隆抗体1的反应质量浓度比为10：1，羧基石墨烯与单克隆抗体2的反应质量浓度比为10：1，羧基石墨烯与多克隆抗体3的反应质量浓度比为10:1，羧基石墨烯与多克隆抗4的反应质量浓度比为10:1。
  7. 如权利要求4所述的制备方法，其特征在于，所述步骤(1)中，单克隆抗体1的制备方法如下：
    (i)蛋白聚糖的核心蛋白的羧基端50～90个氨基酸序列的表达基因插入PTRACER真核表达载体，转染HEK293细胞，从细胞培养上清中纯化，PBS稀释至1.5～2.5mg/mL，制得抗原；
    (ii)将步骤(i)制得的抗原免疫BALB/C小鼠3次，抗原免疫量为50～100mg/次，然后用PEG法将效价≥2000的小鼠脾细胞和骨髓瘤细胞进行融合，并筛选出阳性克隆；
    (iii)向BALB/C小鼠腹腔中注射弗氏不完全佐剂，待小鼠腹腔中产生足量腹水后，于小鼠腹腔中接种步骤(ii)制得的阳性克隆，两周后抽取腹水，离心取上清，纯化后，制得单克隆抗体1。
  8. 如权利要求7所述的制备方法，其特征在于，所述步骤(i)的纯化，为用NI-NTA亲和层析柱进行纯化；
    优选的，所述步骤(iii)的纯化，步骤如下：
    将上清用PBS缓冲液稀释2.5倍，上蛋白A/G亲和层析柱(GE)，分别用含0.5M NaCl的TRIS-HClpH 8.0的缓冲液和含0.5M NaCl的TRIS-HClpH 9.0的缓冲液充分洗脱除杂，最后用100mM三乙胺洗脱回收抗体，收集前三柱体积的洗脱液，并立即用1M的NaH2PO4中和，最后用截流分子量为10KD的超滤管脱盐，并将抗体置换到PBS缓冲液中，即得。
  9. 如权利要求4所述的制备方法，其特征在于，所述步骤(1)中，单克隆抗体2的制备方法如下：
    (a)蛋白聚糖的核心蛋白的氨基端490～530个氨基酸序列的表达基因插入PTRACER真核表达载体，转染HEK293细胞，从细胞培养上清中纯化，PBS缓冲液稀释至1.5～2.5mg/mL，制得抗原；
    (b)将步骤(a)制得的抗原免疫BALB/C小鼠3次，抗原免疫量为50～100mg/次，然后用PEG法将效价≥2000的小鼠脾细胞和骨髓瘤细胞进行融合，并筛选出阳性克隆；
    (c)向BALB/C小鼠腹腔中注射弗氏不完全佐剂，待小鼠腹腔中产生足量腹水后，于小鼠腹腔中接种步骤(b)制得的阳性克隆，两周后抽取腹水，离心取上清，纯化后，制得单克隆抗体2；
    所述步骤(a)的纯化，为用NI-NTA亲和层析柱进行纯化；
    进一步优选的，所述步骤(c)的纯化，步骤如下：
    将上清用PBS缓冲液稀释2.5倍，上蛋白A/G亲和层析柱(GE)，分别用含0.5M NaCl的TRIS-HClpH 8.0的缓冲液和含0.5M NaCl的TRIS-HClpH 9.0的缓冲液充分洗脱除杂，最后用100mM三乙胺洗脱回收抗体，收集前三柱体积的洗脱液，并立即用1M的NaH2PO4中和，最后用截流分子量为10KD的超滤管脱盐，并将抗体置换到PBS中，即得。
  10. 如权利要求4所述的制备方法，其特征在于，所述步骤(1)中，多克隆抗体3的制备方法如下：
    a、蛋白聚糖的核心蛋白的羧基端290个氨基酸序列表达基因插入PTRACER真核表达载体，转染HEK293细胞，从细胞培养上清中纯化，PBS缓冲液稀释至1.0～2.0mg/mL，制得抗原；
    b、将步骤a制得的抗原免疫雌性新西兰大白兔3次，抗原免疫量为200～400μg/次，然后取血，离心取上清，纯化后，制得多克隆抗体3；
    所述步骤b的纯化，步骤如下：
    将上清用PBS缓冲液稀释2.5倍，上蛋白A/G亲和层析柱(GE)，分别用含0.5M NaCl的TRIS-HClpH 8.0的缓冲液和含0.5M NaCl的TRIS-HClpH 9.0的缓冲液充分洗脱除杂，最后用100mM三乙胺洗脱回收抗体，收集前三柱体积的洗脱液，并立即用1M的NaH2PO4中和，最后用截流分子量为10KD的超滤管脱盐，并将抗体置换到PBS缓冲液中，即得。
    优选的，所述步骤(1)中，多克隆抗体4的制备方法如下：
    a、蛋白聚糖的核心蛋白的氨基端290个氨基酸序列表达基因插入PTRACER真核表达载体，转染HEK293细胞，从细胞培养上清中纯化，PBS缓冲液稀释至1.0～2.0mg/mL，制得抗原；
    b、将步骤a制得的抗原免疫雌性新西兰大白兔3次，抗原免疫量为200～400μg/次，然后取血，离心取上清，纯化后，制得多克隆抗体4；
    所述步骤b的纯化，步骤如下：
    将上清用PBS缓冲液稀释2.5倍，上蛋白A/G亲和层析柱(GE)，分别用含0.5M NaCl的TRIS-HClpH 8.0的缓冲液和含0.5M NaCl的TRIS-HClpH 9.0的缓冲液充分洗脱除杂，最后用100mM三乙胺洗脱回收抗体，收集前三柱体积的洗脱液，并立即用1M的NaH2PO4中和，最后用截流分子量为10KD的超滤管脱盐，并将抗体置换到PBS缓冲液中，即得。
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Cited By (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN110987591A (zh) * 2019-12-16 2020-04-10 苏州市纤维检验院 一种微波消解同时测定纺织品中的砷和汞的方法
CN113884676A (zh) * 2021-09-30 2022-01-04 杭州联科生物技术股份有限公司 一种通过将荧光微球偶联抗体检测目的蛋白的方法
CN116713044A (zh) * 2023-04-18 2023-09-08 深圳市龙岗中心医院 一种基于垂直石墨烯和纳米金的荧光检测芯片、其制备方法及在ad标志物检测中的应用
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Families Citing this family (11)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN105606827B (zh) * 2016-03-14 2017-12-12 山东大学 一种用于蛋白聚糖高灵敏度检测的试剂盒及其制备方法
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CN110095602A (zh) * 2019-05-24 2019-08-06 山东师范大学 一种基于氧化石墨烯的细胞检测试剂盒、方法及应用
CN112946253B (zh) * 2019-12-11 2022-11-08 广东菲鹏生物有限公司 用于免疫诊断试剂中固相载体的封闭阶段处理剂及应用和产品
CN111735965B (zh) * 2020-07-02 2023-07-25 北京美联泰科生物技术有限公司 心肌肌钙蛋白i检测试剂和制备方法以及心肌肌钙蛋白i检测试剂盒
CN113391070B (zh) * 2021-06-03 2024-12-03 山东大学 一种改进的磷酯酰肌醇蛋白聚糖3定量检测方法
CN113652391B (zh) * 2021-07-14 2024-07-09 大连民族大学 基于硼酸定向偶联免疫亲和的外泌体捕获分析一体化方法
CN114217078A (zh) * 2021-12-23 2022-03-22 西安依朵生物科技有限公司 一种检测聚集蛋白聚糖的试纸条、试剂盒及检测方法
CN119125571A (zh) * 2024-08-19 2024-12-13 安徽医科大学 用于类风湿关节炎诊断的生物标志物及其应用

Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN101186650A (zh) * 2002-09-04 2008-05-28 中外制药株式会社 抗存在于血液中的gpc3的分泌性n-端肽或c-端肽的抗体
CN104678092A (zh) * 2015-01-28 2015-06-03 山东大学 高正电荷荧光蛋白在糖胺聚糖及其类似物分析检测中的应用
CN105606827A (zh) * 2016-03-14 2016-05-25 山东大学 一种用于蛋白聚糖高灵敏度检测的试剂盒及其制备方法

Family Cites Families (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN102603896B (zh) * 2012-02-13 2015-03-04 中国科学院苏州纳米技术与纳米仿生研究所 一种纳米氧化石墨烯蛋白质复合物及其制备方法与应用
CN102872458A (zh) * 2012-10-24 2013-01-16 佛山市第一人民医院 一种治疗性疫苗佐剂
CN104345082B (zh) * 2013-08-06 2017-02-15 中国科学院苏州纳米技术与纳米仿生研究所 生物传感器、生物传感器的制作方法及其检测方法
CN104142393B (zh) * 2013-10-30 2016-03-30 郑州轻工业学院 一种生物传感器敏感膜及其在检测盐酸克伦特罗方面的应用

Patent Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN101186650A (zh) * 2002-09-04 2008-05-28 中外制药株式会社 抗存在于血液中的gpc3的分泌性n-端肽或c-端肽的抗体
CN104678092A (zh) * 2015-01-28 2015-06-03 山东大学 高正电荷荧光蛋白在糖胺聚糖及其类似物分析检测中的应用
CN105606827A (zh) * 2016-03-14 2016-05-25 山东大学 一种用于蛋白聚糖高灵敏度检测的试剂盒及其制备方法

Non-Patent Citations (4)

* Cited by examiner, † Cited by third party
Title
LEE, J.S. ET AL.: "Graphene-Based Chemiluminescence Resonance Energy Transfer for Homogeneous Immunoassay", vol. 6, no. 4, 14 March 2012 (2012-03-14), pages 2978 - 2983, XP055422278 *
LEI, JIUQIAN ET AL.: "A simple and Sensitive Immunoassay for the Determination of Human Chorionic Gonadotropin by Graphene-based Chemiluminescence Resonance Energy Transfer", BIOSENSORS AND BIOELECTRONICS, vol. 54, 31 October 2013 (2013-10-31), pages 72 - 77, XP055422266 *
LIU, MENG ET AL.: "Distance-independent Quenching of Quantum Dots by Nanoscale-graphene in Self-assembled Sandwich Immunoassay", CHEM. COMMUN, vol. 46, 21 September 2010 (2010-09-21), pages 7909 - 7911, XP055422272 *
WANG, WENSHUANG ET AL.: "Supercharged Fluorescent Protein as a Versatile Probe for the Detection of Glycosaminoglycans in Vitro and - in Vivo", ANALYTICAL CHEMISTRY, XP055422246 *

Cited By (6)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN110987591A (zh) * 2019-12-16 2020-04-10 苏州市纤维检验院 一种微波消解同时测定纺织品中的砷和汞的方法
CN110987591B (zh) * 2019-12-16 2022-04-05 苏州市纤维检验院 一种微波消解同时测定纺织品中的砷和汞的方法
CN113884676A (zh) * 2021-09-30 2022-01-04 杭州联科生物技术股份有限公司 一种通过将荧光微球偶联抗体检测目的蛋白的方法
CN116713044A (zh) * 2023-04-18 2023-09-08 深圳市龙岗中心医院 一种基于垂直石墨烯和纳米金的荧光检测芯片、其制备方法及在ad标志物检测中的应用
CN116713044B (zh) * 2023-04-18 2024-05-14 深圳市龙岗中心医院 一种基于垂直石墨烯和纳米金的荧光检测芯片、其制备方法及在ad标志物检测中的应用
CN119125572A (zh) * 2024-08-19 2024-12-13 安徽医科大学 用于膝骨关节炎诊断的生物标志物及其应用

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