CN113933507A - Hyaluronidase quantitative determination method based on biotin-streptavidin system - Google Patents
Hyaluronidase quantitative determination method based on biotin-streptavidin system Download PDFInfo
- Publication number
- CN113933507A CN113933507A CN202111370793.2A CN202111370793A CN113933507A CN 113933507 A CN113933507 A CN 113933507A CN 202111370793 A CN202111370793 A CN 202111370793A CN 113933507 A CN113933507 A CN 113933507A
- Authority
- CN
- China
- Prior art keywords
- hyaluronidase
- magnetic beads
- working reagent
- sample
- biotin
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Pending
Links
- 108010003272 Hyaluronate lyase Proteins 0.000 title claims abstract description 72
- 229960002773 hyaluronidase Drugs 0.000 title claims abstract description 72
- 102000001974 Hyaluronidases Human genes 0.000 title claims abstract 22
- 238000004445 quantitative analysis Methods 0.000 title abstract description 4
- 239000011324 bead Substances 0.000 claims abstract description 96
- YBJHBAHKTGYVGT-ZKWXMUAHSA-N (+)-Biotin Chemical compound N1C(=O)N[C@@H]2[C@H](CCCCC(=O)O)SC[C@@H]21 YBJHBAHKTGYVGT-ZKWXMUAHSA-N 0.000 claims abstract description 42
- 238000000034 method Methods 0.000 claims abstract description 29
- 108010090804 Streptavidin Proteins 0.000 claims abstract description 22
- 229960002685 biotin Drugs 0.000 claims abstract description 21
- 235000020958 biotin Nutrition 0.000 claims abstract description 21
- 239000011616 biotin Substances 0.000 claims abstract description 21
- 238000001514 detection method Methods 0.000 claims abstract description 8
- 239000003153 chemical reaction reagent Substances 0.000 claims description 133
- 238000011534 incubation Methods 0.000 claims description 41
- 239000007788 liquid Substances 0.000 claims description 26
- 125000002088 tosyl group Chemical group [H]C1=C([H])C(=C([H])C([H])=C1C([H])([H])[H])S(*)(=O)=O 0.000 claims description 20
- 239000000872 buffer Substances 0.000 claims description 14
- 239000002245 particle Substances 0.000 claims description 14
- 102000004169 proteins and genes Human genes 0.000 claims description 11
- 108090000623 proteins and genes Proteins 0.000 claims description 11
- 150000003839 salts Chemical class 0.000 claims description 11
- 239000007850 fluorescent dye Substances 0.000 claims description 8
- 230000008878 coupling Effects 0.000 claims description 6
- 238000010168 coupling process Methods 0.000 claims description 6
- 238000005859 coupling reaction Methods 0.000 claims description 6
- MHMNJMPURVTYEJ-UHFFFAOYSA-N fluorescein-5-isothiocyanate Chemical compound O1C(=O)C2=CC(N=C=S)=CC=C2C21C1=CC=C(O)C=C1OC1=CC(O)=CC=C21 MHMNJMPURVTYEJ-UHFFFAOYSA-N 0.000 claims description 6
- 230000005284 excitation Effects 0.000 claims description 4
- 239000004593 Epoxy Substances 0.000 claims description 3
- 125000003178 carboxy group Chemical group [H]OC(*)=O 0.000 claims description 3
- 125000002887 hydroxy group Chemical group [H]O* 0.000 claims description 3
- 125000002924 primary amino group Chemical group [H]N([H])* 0.000 claims description 3
- 238000007781 pre-processing Methods 0.000 claims description 2
- 238000003556 assay Methods 0.000 claims 1
- 238000003745 diagnosis Methods 0.000 abstract description 3
- 238000000338 in vitro Methods 0.000 abstract description 2
- 102000009066 Hyaluronoglucosaminidase Human genes 0.000 description 50
- 239000003795 chemical substances by application Substances 0.000 description 21
- 235000002639 sodium chloride Nutrition 0.000 description 13
- 239000000243 solution Substances 0.000 description 11
- 206010061902 Pancreatic neoplasm Diseases 0.000 description 10
- 201000002528 pancreatic cancer Diseases 0.000 description 10
- 206010028980 Neoplasm Diseases 0.000 description 9
- HEMHJVSKTPXQMS-UHFFFAOYSA-M Sodium hydroxide Chemical compound [OH-].[Na+] HEMHJVSKTPXQMS-UHFFFAOYSA-M 0.000 description 9
- 201000011510 cancer Diseases 0.000 description 9
- 208000015486 malignant pancreatic neoplasm Diseases 0.000 description 8
- 208000008443 pancreatic carcinoma Diseases 0.000 description 8
- 239000000047 product Substances 0.000 description 8
- 239000004094 surface-active agent Substances 0.000 description 8
- KIUKXJAPPMFGSW-DNGZLQJQSA-N (2S,3S,4S,5R,6R)-6-[(2S,3R,4R,5S,6R)-3-Acetamido-2-[(2S,3S,4R,5R,6R)-6-[(2R,3R,4R,5S,6R)-3-acetamido-2,5-dihydroxy-6-(hydroxymethyl)oxan-4-yl]oxy-2-carboxy-4,5-dihydroxyoxan-3-yl]oxy-5-hydroxy-6-(hydroxymethyl)oxan-4-yl]oxy-3,4,5-trihydroxyoxane-2-carboxylic acid Chemical compound CC(=O)N[C@H]1[C@H](O)O[C@H](CO)[C@@H](O)[C@@H]1O[C@H]1[C@H](O)[C@@H](O)[C@H](O[C@H]2[C@@H]([C@@H](O[C@H]3[C@@H]([C@@H](O)[C@H](O)[C@H](O3)C(O)=O)O)[C@H](O)[C@@H](CO)O2)NC(C)=O)[C@@H](C(O)=O)O1 KIUKXJAPPMFGSW-DNGZLQJQSA-N 0.000 description 7
- TWRXJAOTZQYOKJ-UHFFFAOYSA-L Magnesium chloride Chemical compound [Mg+2].[Cl-].[Cl-] TWRXJAOTZQYOKJ-UHFFFAOYSA-L 0.000 description 6
- WCUXLLCKKVVCTQ-UHFFFAOYSA-M Potassium chloride Chemical compound [Cl-].[K+] WCUXLLCKKVVCTQ-UHFFFAOYSA-M 0.000 description 6
- FAPWRFPIFSIZLT-UHFFFAOYSA-M Sodium chloride Chemical compound [Na+].[Cl-] FAPWRFPIFSIZLT-UHFFFAOYSA-M 0.000 description 6
- 229920002674 hyaluronan Polymers 0.000 description 6
- 229960003160 hyaluronic acid Drugs 0.000 description 6
- JIAARYAFYJHUJI-UHFFFAOYSA-L zinc dichloride Chemical compound [Cl-].[Cl-].[Zn+2] JIAARYAFYJHUJI-UHFFFAOYSA-L 0.000 description 6
- 239000003223 protective agent Substances 0.000 description 5
- LENZDBCJOHFCAS-UHFFFAOYSA-N tris Chemical compound OCC(N)(CO)CO LENZDBCJOHFCAS-UHFFFAOYSA-N 0.000 description 5
- 108091003079 Bovine Serum Albumin Proteins 0.000 description 4
- 229940098773 bovine serum albumin Drugs 0.000 description 4
- 239000006228 supernatant Substances 0.000 description 4
- GPRLSGONYQIRFK-MNYXATJNSA-N triton Chemical compound [3H+] GPRLSGONYQIRFK-MNYXATJNSA-N 0.000 description 4
- 108091001451 PEGPH20 Proteins 0.000 description 3
- 229920001213 Polysorbate 20 Polymers 0.000 description 3
- 239000007983 Tris buffer Substances 0.000 description 3
- 229910001629 magnesium chloride Inorganic materials 0.000 description 3
- 235000010486 polyoxyethylene sorbitan monolaurate Nutrition 0.000 description 3
- 239000000256 polyoxyethylene sorbitan monolaurate Substances 0.000 description 3
- 239000001103 potassium chloride Substances 0.000 description 3
- 235000011164 potassium chloride Nutrition 0.000 description 3
- 239000011780 sodium chloride Substances 0.000 description 3
- 239000000725 suspension Substances 0.000 description 3
- 239000011592 zinc chloride Substances 0.000 description 3
- 235000005074 zinc chloride Nutrition 0.000 description 3
- 101001065501 Escherichia phage MS2 Lysis protein Proteins 0.000 description 2
- 239000007836 KH2PO4 Substances 0.000 description 2
- 229920001214 Polysorbate 60 Polymers 0.000 description 2
- 210000004204 blood vessel Anatomy 0.000 description 2
- 238000002512 chemotherapy Methods 0.000 description 2
- 230000000052 comparative effect Effects 0.000 description 2
- ZPWVASYFFYYZEW-UHFFFAOYSA-L dipotassium hydrogen phosphate Chemical compound [K+].[K+].OP([O-])([O-])=O ZPWVASYFFYYZEW-UHFFFAOYSA-L 0.000 description 2
- 229910000396 dipotassium phosphate Inorganic materials 0.000 description 2
- 201000010099 disease Diseases 0.000 description 2
- 208000037265 diseases, disorders, signs and symptoms Diseases 0.000 description 2
- 229910000397 disodium phosphate Inorganic materials 0.000 description 2
- DBLXOVFQHHSKRC-UHFFFAOYSA-N ethanesulfonic acid;2-piperazin-1-ylethanol Chemical group CCS(O)(=O)=O.OCCN1CCNCC1 DBLXOVFQHHSKRC-UHFFFAOYSA-N 0.000 description 2
- 238000002474 experimental method Methods 0.000 description 2
- 210000001035 gastrointestinal tract Anatomy 0.000 description 2
- SDUQYLNIPVEERB-QPPQHZFASA-N gemcitabine Chemical compound O=C1N=C(N)C=CN1[C@H]1C(F)(F)[C@H](O)[C@@H](CO)O1 SDUQYLNIPVEERB-QPPQHZFASA-N 0.000 description 2
- 229960005277 gemcitabine Drugs 0.000 description 2
- 210000002865 immune cell Anatomy 0.000 description 2
- 239000012535 impurity Substances 0.000 description 2
- 235000011147 magnesium chloride Nutrition 0.000 description 2
- 239000000203 mixture Substances 0.000 description 2
- 230000004048 modification Effects 0.000 description 2
- 238000012986 modification Methods 0.000 description 2
- 229910000402 monopotassium phosphate Inorganic materials 0.000 description 2
- NETZHAKZCGBWSS-CEDHKZHLSA-N nalbuphine Chemical compound C([C@]12[C@H]3OC=4C(O)=CC=C(C2=4)C[C@@H]2[C@]1(O)CC[C@@H]3O)CN2CC1CCC1 NETZHAKZCGBWSS-CEDHKZHLSA-N 0.000 description 2
- 229960000805 nalbuphine Drugs 0.000 description 2
- 235000010482 polyoxyethylene sorbitan monooleate Nutrition 0.000 description 2
- 229920000136 polysorbate Polymers 0.000 description 2
- 229920000053 polysorbate 80 Polymers 0.000 description 2
- 239000003755 preservative agent Substances 0.000 description 2
- 230000002335 preservative effect Effects 0.000 description 2
- PYWVYCXTNDRMGF-UHFFFAOYSA-N rhodamine B Chemical compound [Cl-].C=12C=CC(=[N+](CC)CC)C=C2OC2=CC(N(CC)CC)=CC=C2C=1C1=CC=CC=C1C(O)=O PYWVYCXTNDRMGF-UHFFFAOYSA-N 0.000 description 2
- AJPJDKMHJJGVTQ-UHFFFAOYSA-M sodium dihydrogen phosphate Chemical compound [Na+].OP(O)([O-])=O AJPJDKMHJJGVTQ-UHFFFAOYSA-M 0.000 description 2
- 229910000162 sodium phosphate Inorganic materials 0.000 description 2
- 230000004083 survival effect Effects 0.000 description 2
- 238000011282 treatment Methods 0.000 description 2
- 210000004881 tumor cell Anatomy 0.000 description 2
- 238000005406 washing Methods 0.000 description 2
- 102000004190 Enzymes Human genes 0.000 description 1
- 108090000790 Enzymes Proteins 0.000 description 1
- 229920002683 Glycosaminoglycan Polymers 0.000 description 1
- 230000002159 abnormal effect Effects 0.000 description 1
- 230000009471 action Effects 0.000 description 1
- 230000032683 aging Effects 0.000 description 1
- 238000013459 approach Methods 0.000 description 1
- 239000007864 aqueous solution Substances 0.000 description 1
- QVGXLLKOCUKJST-UHFFFAOYSA-N atomic oxygen Chemical compound [O] QVGXLLKOCUKJST-UHFFFAOYSA-N 0.000 description 1
- 230000004888 barrier function Effects 0.000 description 1
- 230000008901 benefit Effects 0.000 description 1
- 238000011088 calibration curve Methods 0.000 description 1
- 210000004027 cell Anatomy 0.000 description 1
- 230000010261 cell growth Effects 0.000 description 1
- 229940044683 chemotherapy drug Drugs 0.000 description 1
- 238000013399 early diagnosis Methods 0.000 description 1
- 229940088598 enzyme Drugs 0.000 description 1
- 210000000981 epithelium Anatomy 0.000 description 1
- 210000004907 gland Anatomy 0.000 description 1
- 230000036541 health Effects 0.000 description 1
- 238000002868 homogeneous time resolved fluorescence Methods 0.000 description 1
- 238000009169 immunotherapy Methods 0.000 description 1
- 230000006872 improvement Effects 0.000 description 1
- 201000010985 invasive ductal carcinoma Diseases 0.000 description 1
- 230000036210 malignancy Effects 0.000 description 1
- 230000009245 menopause Effects 0.000 description 1
- 230000001394 metastastic effect Effects 0.000 description 1
- 206010061289 metastatic neoplasm Diseases 0.000 description 1
- 230000009871 nonspecific binding Effects 0.000 description 1
- 229910052760 oxygen Inorganic materials 0.000 description 1
- 239000001301 oxygen Substances 0.000 description 1
- 239000002244 precipitate Substances 0.000 description 1
- 238000002360 preparation method Methods 0.000 description 1
- 230000008569 process Effects 0.000 description 1
- 229940120723 recombinant human hyaluronidase Drugs 0.000 description 1
- 230000033764 rhythmic process Effects 0.000 description 1
- 239000013049 sediment Substances 0.000 description 1
- 238000011255 standard chemotherapy Methods 0.000 description 1
- 230000008685 targeting Effects 0.000 description 1
- 238000002560 therapeutic procedure Methods 0.000 description 1
- 210000001519 tissue Anatomy 0.000 description 1
- 238000011269 treatment regimen Methods 0.000 description 1
- 239000012224 working solution Substances 0.000 description 1
Images
Classifications
-
- 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
- G01N33/573—Immunoassay; Biospecific binding assay; Materials therefor for enzymes or isoenzymes
-
- 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
- G01N33/531—Production of immunochemical test materials
- G01N33/532—Production of labelled immunochemicals
- G01N33/533—Production of labelled immunochemicals with fluorescent label
-
- 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
- G01N33/543—Immunoassay; Biospecific binding assay; Materials therefor with an insoluble carrier for immobilising immunochemicals
- G01N33/54313—Immunoassay; Biospecific binding assay; Materials therefor with an insoluble carrier for immobilising immunochemicals the carrier being characterised by its particulate form
- G01N33/54326—Magnetic particles
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N2333/00—Assays involving biological materials from specific organisms or of a specific nature
- G01N2333/90—Enzymes; Proenzymes
- G01N2333/914—Hydrolases (3)
- G01N2333/924—Hydrolases (3) acting on glycosyl compounds (3.2)
- G01N2333/926—Hydrolases (3) acting on glycosyl compounds (3.2) acting on alpha -1, 4-glucosidic bonds, e.g. hyaluronidase, invertase, amylase
- G01N2333/928—Hydrolases (3) acting on glycosyl compounds (3.2) acting on alpha -1, 4-glucosidic bonds, e.g. hyaluronidase, invertase, amylase acting on alpha -1, 4-glucosidic bonds, e.g. hyaluronidase, invertase, amylase
Landscapes
- Health & Medical Sciences (AREA)
- Immunology (AREA)
- Life Sciences & Earth Sciences (AREA)
- Engineering & Computer Science (AREA)
- Molecular Biology (AREA)
- Biomedical Technology (AREA)
- Chemical & Material Sciences (AREA)
- Hematology (AREA)
- Urology & Nephrology (AREA)
- Biotechnology (AREA)
- Microbiology (AREA)
- Cell Biology (AREA)
- Food Science & Technology (AREA)
- Medicinal Chemistry (AREA)
- Physics & Mathematics (AREA)
- Analytical Chemistry (AREA)
- Biochemistry (AREA)
- General Health & Medical Sciences (AREA)
- General Physics & Mathematics (AREA)
- Pathology (AREA)
- Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)
Abstract
The invention relates to a quantitative determination method of hyaluronidase based on a biotin-streptavidin system, belonging to the technical field of in vitro diagnosis. The invention provides a method for quantitatively determining hyaluronidase in a sample, which comprises the steps of capturing hyaluronidase in the sample by using magnetic beads 1 coupled with hyaluronidase antibodies, then combining the captured hyaluronidase by using biotin coupled with hyaluronidase antibodies, then marking the biotin by using streptavidin coupled with fluorescence marks, and finally obtaining the concentration of the hyaluronidase in the sample according to a fluorescence intensity value; the method is based on a biotin-streptavidin system, and the biotin-streptavidin system can amplify a hyaluronidase signal in a sample so as to facilitate detection, so that the method effectively improves the accuracy and detection limit of quantitative determination of the hyaluronidase.
Description
Technical Field
The invention relates to a quantitative determination method of hyaluronidase based on a biotin-streptavidin system, belonging to the technical field of in vitro diagnosis.
Background
Tumors are diseases caused by abnormal growth of cells in organisms due to various factors, and are classified into malignant tumors and benign tumors according to their characteristics and damage to the organisms. Malignant tumors, also known as cancers, are metastatic and invasive. The malignant tumor seriously threatens human health, and under the combined action of factors such as aging population, tense life rhythm, bad living habits, atmospheric pollution and the like, the incidence rate of the malignant tumor is higher and higher, and the incidence age is younger and younger.
Pancreatic cancer is one of the common malignant tumors in the digestive tract, and is called the king of cancer in the field of tumors. According to the records of the J.Lancet, the five-year survival rate of pancreatic cancer after diagnosis is about 10%, which is one of the worst malignant tumors. Pancreatic cancer is clinically insidious and atypical, and is a difficult malignancy of the digestive tract to diagnose and treat, with about 90% ductal adenocarcinomas originating from the epithelium of the gland duct. Its morbidity and mortality has increased dramatically in recent years. The early diagnosis rate of pancreatic cancer is low, the operative mortality rate is high, and the cure rate is low. The incidence rate of the disease is higher for men than for women, the ratio of men to women is 1.5-2: 1, male patients are far more common than women before menopause, and the incidence rate of postmenopausal women is similar to that of men.
Pancreatic cancer is surrounded by very dense fibrous tissue, of which about 10% of the cells to the right are tumor cells, which are surrounded by hyaluronic acid. Hyaluronic acid is a glycosaminoglycan, which accumulates on the surface of tumor cells and in the surrounding space. During the progression of immune cells to the tumor, hyaluronic acid forms a barrier and compresses blood vessels, impeding the progress of chemotherapy. The application of hyaluronidase targeting hyaluronic acid to pancreatic cancer treatment is a promising approach for pancreatic cancer treatment.
It is hyaluronic acid that PEGPH20 targets, which is based on a recombinant human hyaluronidase, rHuPH20 enzyme. The innovative therapy can degrade hyaluronic acid accumulated around pancreatic tumor, reduce tumor pressure, and make blood vessel easier to deliver oxygen, chemotherapy drugs and immune cells in tumor region. With the help of PEGPH20, the ability of chemotherapy or immunotherapy agents to penetrate pancreatic tumors is greatly improved, and the potential for malignant growth is also suppressed. In a phase II multicenter, randomized clinical study, named "HALO 202", PEGPH20 combined with nalbuphine and gemcitabine treatment regimen showed a statistically significant improvement in Progression Free Survival (PFS) compared to High-level hyaluronic acid (HA-High) pancreatic cancer patients receiving standard chemotherapy (nalbuphine and gemcitabine). On the basis, finding a reliable method for quantitatively determining hyaluronidase in a sample is extremely important for the reasonable clinical application of PEGPH 20.
Disclosure of Invention
In order to solve the above problems, the present invention provides a method for quantitatively determining hyaluronidase in a sample, the method comprising the steps of:
a capturing step: mixing a sample with a working reagent 1 containing magnetic beads 1, and then incubating to obtain an incubation liquid 1; a hyaluronidase antibody is coupled on the magnetic beads 1;
combining steps: mixing the incubation liquid 1 with a working reagent 2 containing biotin and then incubating to obtain an incubation liquid 2; the biotin is conjugated to a hyaluronidase antibody;
a marking step: mixing the incubation liquid 2 with a working reagent 3 containing streptavidin, and then incubating to obtain an incubation liquid 3; the streptavidin is coupled with a fluorescent label;
a resuspension step: after the magnetic beads 1 in the incubation liquid 3 are collected and washed, the magnetic beads 1 are resuspended in the working reagent 4 to obtain a resuspension liquid;
a detection step: and measuring the fluorescence intensity value of the resuspension, and calculating the concentration of the hyaluronidase in the sample according to the relation between the fluorescence intensity value and the concentration of the hyaluronidase.
In one embodiment of the present invention, before the capturing step, a pre-processing step is further included; the pretreatment steps are as follows: mixing a sample with a working reagent 5 containing magnetic beads 2, incubating, and removing the magnetic beads 2 from the sample after the incubation is finished to obtain a pretreated sample; streptavidin is coupled on the magnetic beads 2.
In one embodiment of the invention, the fluorescent label is at least one of FITC, rhodamine, ICG, CY3, CY3.5, CY5, CY5.5, or CY 7.
In one embodiment of the invention, the fluorescent label is FITC; the fluorescence intensity values of the resuspension were determined as follows: and (3) measuring the fluorescence intensity value of the resuspension at the excitation wavelength of 485nm and the emission wavelength of 528nm by using a microplate reader.
In one embodiment of the invention, the calculation is: bringing the fluorescence intensity value into a fitting equation obtained by fitting according to the relation between the fluorescence intensity value and the hyaluronidase concentration; the fitting equation is as follows:
in the fitting equation, x is the concentration of hyaluronidase in the sample in ng/mL, and y is the fluorescence intensity value of the resuspension.
In one embodiment of the present invention, the magnetic beads 1 are at least one of tosyl magnetic beads, carboxyl magnetic beads, amino magnetic beads, hydroxyl magnetic beads, or epoxy magnetic beads.
In one embodiment of the present invention, the magnetic beads 1 are tosyl magnetic beads.
In one embodiment of the present invention, the particle size of the tosyl magnetic bead is 0.5 to 2 μm.
In one embodiment of the present invention, the particle size of the tosyl magnetic bead is 1 μm.
In one embodiment of the present invention, the coupling ratio of the tosyl magnetic bead and the hyaluronidase antibody is 1 mg: 5-30 μ g.
In one embodiment of the present invention, the coupling ratio of the tosyl magnetic bead and the hyaluronidase antibody is 1 mg: 20 μ g.
In one embodiment of the invention, the components of the working reagent 1, the working reagent 2, the working reagent 3 and/or the working reagent 4 comprise a buffer, a salt and a protein protectant.
In one embodiment of the invention, the buffer is 4-hydroxyethylpiperazine ethanesulfonic acid (HEPES), Tris (hydroxymethyl) aminomethane (Tris), NaH2PO4&Na2HPO4And K2HPO4&KH2PO4At least one of; the salt is at least one of potassium chloride, magnesium chloride, sodium chloride and zinc chloride; the protein protective agent is bovine serum albumin.
In one embodiment of the invention, the working reagent 1, the working reagent 2, the working reagent 3 and/or the working reagent 4 comprises 5-20 g/L of buffer, 2-5 g/L of salt and 0.5-2 g/L of protein protective agent.
In one embodiment of the invention, the working reagent 1, working reagent 2, working reagent 3, working reagent 4 and/or working reagent 5 comprises 15g/L buffer, 3g/L salt and 1.5g/L protein protectant.
In one embodiment of the present invention, the components of the working agent 1, the working agent 2, the working agent 3, the working agent 4 and/or the working agent 5 further comprise a surfactant.
In one embodiment of the present invention, the working reagent 1, the working reagent 2, the working reagent 3, the working reagent 4 and/or the working reagent 5 further comprises 0.1 to 1ml/L of a surfactant.
In one embodiment of the present invention, the composition of the working reagent 1, the working reagent 2, the working reagent 3, the working reagent 4 and/or the working reagent 5 further comprises 0.5ml/L of a surfactant.
In one embodiment of the invention, the surfactant is at least one of tween 20, tween 40, tween 60, tween 80, triton 100 and triton 114.
In one embodiment of the present invention, the components of the working agent 1, the working agent 2, the working agent 3, the working agent 4 and/or the working agent 5 further comprise a preservative.
In one embodiment of the present invention, the pH of the working reagent 1, the working reagent 2, the working reagent 3, the working reagent 4, and/or the working reagent 5 is 6.0 to 8.0.
In one embodiment of the present invention, the pH of the working reagent 1, the working reagent 2, the working reagent 3, the working reagent 4 and/or the working reagent 5 is 7.4.
In one embodiment of the present invention, the pH of the working reagent 1, the working reagent 2, the working reagent 3, the working reagent 4, and/or the working reagent 5 is adjusted using an aqueous sodium hydroxide solution having a mass concentration of 8%.
In one embodiment of the invention, the incubation is: incubating for 5-30 min at 25-43 ℃.
In one embodiment of the invention, the incubation is: incubate at 37 ℃ for 10 min.
The invention also provides a kit for quantitatively determining hyaluronidase in a sample, which comprises a working reagent 1 containing magnetic beads 1, a working reagent 2 containing biotin and a working reagent 3 containing streptavidin; a hyaluronidase antibody is coupled on the magnetic beads 1; the biotin is conjugated to a hyaluronidase antibody; the streptavidin is coupled with a fluorescent label.
In one embodiment of the invention, the kit further comprises a working reagent 4.
In one embodiment of the present invention, the kit further comprises a working reagent 5 comprising magnetic beads 2; streptavidin is coupled on the magnetic beads 2.
In one embodiment of the invention, the fluorescent label is at least one of FITC, rhodamine, ICG, CY3, CY3.5, CY5, CY5.5, or CY 7.
In one embodiment of the present invention, the magnetic beads 1 are at least one of tosyl magnetic beads, carboxyl magnetic beads, amino magnetic beads, hydroxyl magnetic beads, or epoxy magnetic beads.
In one embodiment of the present invention, the magnetic beads 1 are tosyl magnetic beads.
In one embodiment of the present invention, the particle size of the tosyl magnetic bead is 0.5 to 2 μm.
In one embodiment of the present invention, the particle size of the tosyl magnetic bead is 1 μm.
In one embodiment of the present invention, the coupling ratio of the tosyl magnetic bead and the hyaluronidase antibody is 1 mg: 5-30 μ g.
In one embodiment of the present invention, the coupling ratio of the tosyl magnetic bead and the hyaluronidase antibody is 1 mg: 20 μ g.
In one embodiment of the present invention, the magnetic beads 2 have a particle size of 1 to 5 μm.
In one embodiment of the present invention, the magnetic beads 2 have a particle size of 3 μm.
In one embodiment of the present invention, the components of the working reagent 1, the working reagent 2, the working reagent 3, the working reagent 4 and/or the working reagent 5 comprise a buffer, a salt and a protein protectant.
In one embodiment of the invention, the buffer is 4-hydroxyethylpiperazine ethanesulfonic acid (HEPES), Tris (hydroxymethyl) aminomethane (Tris), NaH2PO4&Na2HPO4And K2HPO4&KH2PO4At least one of; the salt is at least one of potassium chloride, magnesium chloride, sodium chloride and zinc chloride; the protein protective agent is bovine serum albumin.
In one embodiment of the invention, the working reagent 1, the working reagent 2, the working reagent 3, the working reagent 4 and/or the working reagent 5 comprises 5-20 g/L of buffer, 2-5 g/L of salt and 0.5-2 g/L of protein protective agent.
In one embodiment of the invention, the working reagent 1, working reagent 2, working reagent 3, working reagent 4 and/or working reagent 5 comprises 15g/L buffer, 3g/L salt and 1.5g/L protein protectant.
In one embodiment of the present invention, the components of the working agent 1, the working agent 2, the working agent 3, the working agent 4 and/or the working agent 5 further comprise a surfactant.
In one embodiment of the present invention, the working reagent 1, the working reagent 2, the working reagent 3, the working reagent 4 and/or the working reagent 5 further comprises 0.1 to 1ml/L of a surfactant.
In one embodiment of the present invention, the composition of the working reagent 1, the working reagent 2, the working reagent 3, the working reagent 4 and/or the working reagent 5 further comprises 0.5ml/L of a surfactant.
In one embodiment of the invention, the surfactant is at least one of tween 20, tween 40, tween 60, tween 80, triton 100 and triton 114.
In one embodiment of the present invention, the components of the working agent 1, the working agent 2, the working agent 3, the working agent 4 and/or the working agent 5 further comprise a preservative.
In one embodiment of the present invention, the pH of the working reagent 1, the working reagent 2, the working reagent 3, the working reagent 4, and/or the working reagent 5 is 6.0 to 8.0.
In one embodiment of the present invention, the pH of the working reagent 1, the working reagent 2, the working reagent 3, the working reagent 4 and/or the working reagent 5 is 7.4.
In one embodiment of the present invention, the pH of the working reagent 1, the working reagent 2, the working reagent 3, the working reagent 4, and/or the working reagent 5 is adjusted using an aqueous sodium hydroxide solution having a mass concentration of 8%.
The invention also provides the application of the method or the kit in the hyaluronidase quantitative determination.
The technical scheme of the invention has the following advantages:
the invention provides a method for quantitatively determining hyaluronidase in a sample, which comprises the steps of capturing hyaluronidase in the sample by using magnetic beads 1 coupled with hyaluronidase antibodies, then combining the captured hyaluronidase by using biotin coupled with hyaluronidase antibodies, then marking the biotin by using streptavidin coupled with fluorescence marks, and finally obtaining the concentration of the hyaluronidase in the sample according to a fluorescence intensity value; the method is based on a biotin-streptavidin system, and the biotin-streptavidin system can amplify a hyaluronidase signal in a sample so as to facilitate detection, so that the method effectively improves the accuracy and detection limit of quantitative determination of the hyaluronidase.
Furthermore, before the capturing step, the method also comprises a pretreatment step, wherein in the pretreatment step, the streptavidin-coupled magnetic beads 2 are used for capturing biotin in the sample, so that the interference of the presence of the biotin in the sample on the quantitative determination of the hyaluronidase is effectively avoided, and the quantitative determination result is more accurate.
Furthermore, the magnetic bead 1 used in the method is a tosyl magnetic bead with the particle size of 1 μm, and the magnetic bead can well capture hyaluronidase in a sample, avoid adsorbing excessive protein impurities, effectively avoid the interference of the presence of the protein impurities in the sample on the quantitative determination of the hyaluronidase, and enable the quantitative determination result to be more accurate.
Furthermore, the magnetic beads 2 used in the method are streptavidin magnetic beads with the particle size of 3 μm, and the magnetic beads are not easy to influence hyaluronidase in the sample while capturing biotin in the sample, so that the accuracy of the quantitative determination result of the hyaluronidase is further improved.
Further, the working reagents used in the method comprise a buffer, a salt, and a protein protectant; the buffer and the salt can effectively maintain the ionic property and the pH value stability, meanwhile, the fluorescent signal is improved, the protein protective agent can seal the unbound magnetic beads 1, biotin and streptavidin, the nonspecific signal generated by nonspecific binding is reduced, and the accuracy of the quantitative determination result of the hyaluronidase is further improved.
Drawings
FIG. 1: and fitting and drawing a standard curve by taking the concentration of the hyaluronidase calibrator solution as an x axis and the fluorescence intensity value as a y axis.
Detailed Description
The following examples are provided to further understand the present invention, not to limit the scope of the present invention, but to provide the best mode, not to limit the content and the protection scope of the present invention, and any product similar or similar to the present invention, which is obtained by combining the present invention with other prior art features, falls within the protection scope of the present invention.
The following examples do not show specific experimental procedures or conditions, and can be performed according to the procedures or conditions of the conventional experimental procedures described in the literature in the field. The reagents or instruments used are not indicated by manufacturers, and are all conventional reagent products which can be obtained commercially.
Example 1: kit for quantitatively determining hyaluronidase in sample
The present embodiment provides a kit for quantitatively determining hyaluronidase in a sample, the kit comprising:
magnetic bead 1 was a 5mg/mL working reagent 1, the magnetic bead 1 was a tosyl magnetic bead (available from JSR corporation) having a particle size of 1 μm, and the magnetic bead 1 was coupled with a hyaluronidase antibody (available from Bioss corporation) at a coupling ratio of 1 mg: 20 mu g of the extract;
working reagent 2 with biotin concentration of 12.5nM, biotin conjugated to hyaluronidase antibody (biotin labeled antibody purchased from Abcam corporation);
working reagent 3 with a streptavidin concentration of 2.5nM, to which FITC has been coupled (FITC-labeled streptavidin from Solarbio);
a working reagent 4;
a working reagent 5 having a concentration of 2mg/mL of magnetic beads 2, the magnetic beads 2 being streptavidin magnetic beads (purchased from Dynal) having a particle size of 3 μm;
the working reagent 1, the working reagent 2, the working reagent 3, the working reagent 4 and the working reagent 5 are composed of 15g/L Tris, 1g/L potassium chloride, 1g/L magnesium chloride, 0.5g/L sodium chloride, 0.5g/L zinc chloride, 1.5g/L bovine serum albumin and 0.5ml/L Tween 20, and the pH of the working solution is adjusted to 7.4 by using a sodium hydroxide aqueous solution with the mass concentration of 8%.
The preparation process of the working reagent 1 with the concentration of the magnetic beads 1 being 5mg/mL is as follows:
putting 1mg of tosyl magnetic beads into a 1.5mL centrifuge tube, putting the centrifuge tube on a magnetic rack, and discarding the supernatant to obtain magnetic bead bodies; after washing the magnetic bead bodies with 1mL of PBS buffer (PH 7.4, 0.01M) for 2 times, resuspending the magnetic bead bodies with 1mL of PBS buffer (PH 7.4, 0.01M) to obtain a magnetic bead suspension; continuously adding 20 mu g of hyaluronidase antibody into the magnetic bead suspension to obtain an incubation system; incubating the incubation system for 20h at 25 ℃ to obtain an incubation solution, and reversing the incubation system and uniformly mixing the incubation system once every 5h during incubation; placing the incubation liquid on a magnetic frame, and discarding the supernatant to obtain magnetic beads 1; after washing the magnetic beads 1 with 1mL of PBS buffer (PH 7.4, 0.01M) 2 times, the magnetic beads were resuspended at a concentration of 5mg/mL using the working reagent 1, and the working reagent 1 having a concentration of 5mg/mL of the magnetic beads 1 was obtained.
Example 2: method for quantitatively determining hyaluronidase in sample
The present example provides a method for quantitatively determining hyaluronidase in a sample, using the kit described in example 1, comprising the following specific steps:
a pretreatment step: mixing 10 mu L of sample with 5 mu L of working reagent 5 containing magnetic beads 2, and incubating at 37 ℃ for 10min to obtain an incubation product; the incubation product is reversed and mixed evenly and then placed on a magnetic frame, and the sediment is discarded to obtain a pretreated sample;
a capturing step: mixing the pretreated sample with 10 mu L of working reagent 1 containing magnetic beads 1, and incubating at 37 ℃ for 10min to obtain an incubation liquid 1;
combining steps: mixing the incubation liquid 1 with 10 mu L of the working reagent 2 containing biotin, and incubating for 10min at 37 ℃ to obtain an incubation liquid 2;
a marking step: mixing the incubation liquid 2 with 10 mu L of a working reagent 3 containing streptavidin, and incubating for 10min at 37 ℃ to obtain an incubation liquid 3;
a resuspension step: the incubation liquid 3 is placed on a magnetic frame after being inverted and mixed evenly, and the supernatant is discarded to obtain magnetic beads 1; resuspending the magnetic beads 1 in 10 mu L of working reagent 4 to obtain a resuspension solution;
a detection step: measuring the fluorescence intensity value of the resuspension at the excitation wavelength of 485nm and the emission wavelength of 528nm by using a microplate reader; bringing the fluorescence intensity value into a fitting equation obtained by fitting according to the relation between the fluorescence intensity value and the hyaluronidase concentration; the fitting equation is as follows:
in the fitting equation, x is the concentration of hyaluronidase in the sample, the unit ng/mL, y is the fluorescence intensity value of the resuspension, R2＝0.996。
The obtaining process of the fitting equation comprises the following steps:
configuration of the calibration product: a total of 7 hyaluronidase calibrator solutions were prepared using working reagent 1 at hyaluronidase (available from Gibco) concentrations of 800.0ng/mL, 400.0ng/mL, 200.0ng/mL, 100.0ng/mL, 50.0ng/mL, 25.0ng/mL, and 12.5ng/mL, respectively;
a pretreatment step: mixing 10 mu L of hyaluronidase calibrator solution with each concentration with 5 mu L of working reagent 5 containing magnetic beads 2, and incubating at 37 ℃ for 10min to obtain incubation products; respectively reversing and uniformly mixing all incubation products, then placing the incubation products on a magnetic frame, and removing precipitates to obtain a pretreated sample;
a capturing step: mixing each pretreated sample with 10 mu L of working reagent 1 containing magnetic beads 1, and incubating at 37 ℃ for 10min to obtain incubation liquid 1;
combining steps: mixing each incubation solution 1 with 10 mu L of biotin-containing working reagent 2, and incubating for 10min at 37 ℃ to obtain incubation solution 2;
a marking step: mixing each incubation liquid 2 with 10 mu L of a working reagent 3 containing streptavidin, and incubating for 10min at 37 ℃ to obtain incubation liquid 3;
a resuspension step: respectively reversing and uniformly mixing the incubation liquids 3, then placing the incubation liquids on a magnetic frame, and removing the supernatant to obtain magnetic beads 1; resuspending each magnetic bead 1 in 10 μ L of working reagent 4 to obtain a resuspension solution;
a detection step: measuring the fluorescence intensity value of each heavy suspension at the excitation wavelength of 485nm and the emission wavelength of 528nm by using a microplate reader; and (3) taking the concentration of the hyaluronidase calibrator solution as an x axis and the fluorescence intensity value as a y axis, drawing a calibration curve, and obtaining a fitting equation obtained by fitting according to the relation between the fluorescence intensity value and the hyaluronidase concentration.
Comparative example 1: method for quantitatively determining hyaluronidase in sample
This comparative example provides a method for the quantitative determination of hyaluronidase in a sample based on homogeneous time-resolved fluorescence, which differs from example 2 in that:
the first scheme is as follows: there is no pretreatment step.
Scheme II: the magnetic beads 1 were replaced with tosyl magnetic beads having a particle size of 3 μm.
The third scheme is as follows: the magnetic beads 1 were replaced with tosyl magnetic beads having a particle size of 500 nm.
And the scheme is as follows: the magnetic beads 2 were replaced with streptavidin magnetic beads having a particle size of 1 μm.
And a fifth scheme: and removing bovine serum albumin in the working reagent 1, the working reagent 2, the working reagent 3, the working reagent 4 and the working reagent 5.
The fitting equations obtained by fitting the schemes one to five have large relative deviation, wherein R of the schemes two to four2The number of the curves is 0.7-0.9, and two intersecting curves are obtained by fitting the scheme I and the scheme V.
It should be understood that the above examples are only for clarity of illustration and are not intended to limit the embodiments. Other variations and modifications will be apparent to persons skilled in the art in light of the above description. And are neither required nor exhaustive of all embodiments. And obvious variations or modifications therefrom are within the scope of the invention.
Claims (10)
1. A method for quantitatively determining hyaluronidase in a sample, comprising the steps of:
a capturing step: mixing a sample with a working reagent 1 containing magnetic beads 1, and then incubating to obtain an incubation liquid 1; a hyaluronidase antibody is coupled on the magnetic beads 1;
combining steps: mixing the incubation liquid 1 with a working reagent 2 containing biotin and then incubating to obtain an incubation liquid 2; the biotin is conjugated to a hyaluronidase antibody;
a marking step: mixing the incubation liquid 2 with a working reagent 3 containing streptavidin, and then incubating to obtain an incubation liquid 3; the streptavidin is coupled with a fluorescent label;
a resuspension step: after the magnetic beads 1 in the incubation liquid 3 are collected and washed, the magnetic beads 1 are resuspended in the working reagent 4 to obtain a resuspension liquid;
a detection step: and measuring the fluorescence intensity value of the resuspension, and calculating the concentration of the hyaluronidase in the sample according to the relation between the fluorescence intensity value and the concentration of the hyaluronidase.
2. The method of claim 1, wherein prior to the capturing step, further comprising a pre-processing step; the pretreatment steps are as follows: mixing a sample with a working reagent 5 containing magnetic beads 2, incubating, and removing the magnetic beads 2 from the sample after the incubation is finished to obtain a pretreated sample; streptavidin is coupled on the magnetic beads 2.
3. The method of claim 1 or 2, wherein the fluorescent label is FITC; the fluorescence intensity values of the resuspension were determined as follows: and (3) measuring the fluorescence intensity value of the resuspension at the excitation wavelength of 485nm and the emission wavelength of 528nm by using a microplate reader.
4. The method of claim 3, wherein the calculation is: bringing the fluorescence intensity value into a fitting equation obtained by fitting according to the relation between the fluorescence intensity value and the hyaluronidase concentration; the fitting equation is as follows:
in the fitting equation, x is the concentration of hyaluronidase in the sample in ng/mL, and y is the fluorescence intensity value of the resuspension.
5. The method according to any one of claims 1 to 4, wherein the magnetic beads 1 are at least one of tosyl magnetic beads, carboxyl magnetic beads, amino magnetic beads, hydroxyl magnetic beads or epoxy magnetic beads.
6. The method according to any one of claims 1 to 5, wherein the tosyl magnetic beads have a particle size of 0.5 to 2 μm.
7. The method of any one of claims 1 to 6, wherein the coupling ratio of the magnetic tosyl beads to the hyaluronidase antibody is 1 mg: 5-30 μ g.
8. The method of any one of claims 1 to 7, wherein the components of working reagent 1, working reagent 2, working reagent 3, working reagent 4 and/or working reagent 5 comprise a buffer, a salt and a protein protectant.
9. A kit for quantitatively determining hyaluronidase in a sample is characterized by comprising a working reagent 1 containing magnetic beads 1, a working reagent 2 containing biotin and a working reagent 3 containing streptavidin; a hyaluronidase antibody is coupled on the magnetic beads 1; the biotin is conjugated to a hyaluronidase antibody; the streptavidin is coupled with a fluorescent label.
10. Use of the method of any one of claims 1 to 8 or the kit of any one of claims 9 in a hyaluronidase assay.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202111370793.2A CN113933507A (en) | 2021-11-18 | 2021-11-18 | Hyaluronidase quantitative determination method based on biotin-streptavidin system |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202111370793.2A CN113933507A (en) | 2021-11-18 | 2021-11-18 | Hyaluronidase quantitative determination method based on biotin-streptavidin system |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| CN113933507A true CN113933507A (en) | 2022-01-14 |
Family
ID=79287027
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN202111370793.2A Pending CN113933507A (en) | 2021-11-18 | 2021-11-18 | Hyaluronidase quantitative determination method based on biotin-streptavidin system |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN113933507A (en) |
Citations (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US20170131269A1 (en) * | 2015-11-09 | 2017-05-11 | Bio-Rad Laboratories, Inc. | Assays using avidin and biotin |
| CN110146692A (en) * | 2019-05-28 | 2019-08-20 | 迪瑞医疗科技股份有限公司 | One kind being based on acridinium ester chemiluminescent, Streptavidin MagneSphere-biotin iodine system and detection kit |
| CN110954691A (en) * | 2019-12-17 | 2020-04-03 | 丹娜（天津）生物科技有限公司 | Sample pretreatment reagent and application thereof |
| CN111239414A (en) * | 2020-01-21 | 2020-06-05 | 上海生物芯片有限公司 | Kit for detecting IL-6 and method for detecting IL-6 |
| CN112285353A (en) * | 2020-10-22 | 2021-01-29 | 武汉生之源生物科技股份有限公司 | Method for improving anti-biotin interference capability and sensitivity of chemiluminescence kit of streptavidin-biotin reaction system |
-
2021
- 2021-11-18 CN CN202111370793.2A patent/CN113933507A/en active Pending
Patent Citations (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US20170131269A1 (en) * | 2015-11-09 | 2017-05-11 | Bio-Rad Laboratories, Inc. | Assays using avidin and biotin |
| CN110146692A (en) * | 2019-05-28 | 2019-08-20 | 迪瑞医疗科技股份有限公司 | One kind being based on acridinium ester chemiluminescent, Streptavidin MagneSphere-biotin iodine system and detection kit |
| CN110954691A (en) * | 2019-12-17 | 2020-04-03 | 丹娜（天津）生物科技有限公司 | Sample pretreatment reagent and application thereof |
| CN111239414A (en) * | 2020-01-21 | 2020-06-05 | 上海生物芯片有限公司 | Kit for detecting IL-6 and method for detecting IL-6 |
| CN112285353A (en) * | 2020-10-22 | 2021-01-29 | 武汉生之源生物科技股份有限公司 | Method for improving anti-biotin interference capability and sensitivity of chemiluminescence kit of streptavidin-biotin reaction system |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| CN104634974B (en) | Detection, the discriminating conduct of intrahepatic cholangiocarcinoma | |
| EP3026119A1 (en) | Use of he4 and other biochemical markers for assessment of ovarian cancers | |
| US20210322513A1 (en) | Method for detecting cancer cells, reagent for introducing substance into cancer cells, and composition for treating cancer | |
| Giwercman et al. | A monoclonal antibody as a marker for carcinoma in situ germ cells of the human adult testis | |
| EP2833137B1 (en) | Tissue staining method | |
| Namikawa et al. | Neuroendocrine carcinoma of the stomach: clinicopathological and immunohistochemical evaluation | |
| van Casteren et al. | Noninvasive detection of testicular carcinoma in situ in semen using OCT3/4 | |
| Balley et al. | An immunocytochemical study of α‐lactalbumin in human breast tissue | |
| Ono et al. | Overexpression of heat shock protein 27 in squamous cell carcinoma of the uterine cervix: a proteomic analysis using archival formalin-fixed, paraffin-embedded tissues | |
| Jin et al. | Diagnostic value of ER, PR, FR and HER-2-targeted molecular probes for magnetic resonance imaging in patients with breast cancer | |
| CN113933507A (en) | Hyaluronidase quantitative determination method based on biotin-streptavidin system | |
| EP4027141A1 (en) | Method for measuring lectin-associated substance, kit for measuring lectin-associated substance, and blocked labeled lectin used in same | |
| JP7361543B2 (en) | AFP-L3 measurement method, AFP-L3 measurement kit, and blocked labeled lectin used therefor | |
| ASHITAKA et al. | 3, 5, 3'-triiodo-L-thyronine binding sites in nuclei of human trophoblastic cells | |
| Pradier et al. | Monitoring of therapy in head and neck patients during the radiotherapy by measurement of Cyfra 21-1 | |
| CN108279307A (en) | Protection and the heparin tube for stablizing gastrin-releasing peptide precursor in serum or blood plasma | |
| CN106928352B (en) | Monoclonal antibody of anti-PSG 3 protein, hybridoma cell strain and application thereof | |
| CN111610331B (en) | Serological detection test strip for screening early esophageal cancer | |
| CN113252906A (en) | Detection method of paraneoplastic syndrome related marker antibody | |
| EP0007214A1 (en) | Product for detecting the presence of cancerous or malignant tumor cells | |
| CN109852666B (en) | Specific target spot and diagnostic reagent for liver cancer diagnosis | |
| Yudhistira et al. | Biotinylated Fluorescent Polymeric Nanoparticles for Enhanced Immunostaining | |
| CN113884675B (en) | Tumor exosome detection reagent and detection method | |
| González‐Cámpora et al. | Dipeptidyl aminopeptidase IV in the cytologic diagnosis of thyroid carcinoma | |
| Yonezawa et al. | Lectin histochemistry in a case of ulcerative colitis complicating rectal carcinoma |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| PB01 | Publication | ||
| PB01 | Publication | ||
| SE01 | Entry into force of request for substantive examination | ||
| SE01 | Entry into force of request for substantive examination |