CN113759115A - 用于膀胱癌检测的标志物及检测试剂盒 - Google Patents
用于膀胱癌检测的标志物及检测试剂盒 Download PDFInfo
- Publication number
- CN113759115A CN113759115A CN202110746113.6A CN202110746113A CN113759115A CN 113759115 A CN113759115 A CN 113759115A CN 202110746113 A CN202110746113 A CN 202110746113A CN 113759115 A CN113759115 A CN 113759115A
- Authority
- CN
- China
- Prior art keywords
- antibody
- bladder cancer
- detection
- acridinium ester
- labeled
- 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
- 208000007097 Urinary Bladder Neoplasms Diseases 0.000 title claims abstract description 58
- 238000001514 detection method Methods 0.000 title claims abstract description 56
- 206010005003 Bladder cancer Diseases 0.000 title claims abstract description 43
- 201000005112 urinary bladder cancer Diseases 0.000 title claims abstract description 43
- 239000003550 marker Substances 0.000 title claims abstract description 19
- 102100036961 Nuclear mitotic apparatus protein 1 Human genes 0.000 claims abstract description 47
- 108010036112 nuclear matrix protein 22 Proteins 0.000 claims abstract description 47
- RXNXLAHQOVLMIE-UHFFFAOYSA-N phenyl 10-methylacridin-10-ium-9-carboxylate Chemical compound C12=CC=CC=C2[N+](C)=C2C=CC=CC2=C1C(=O)OC1=CC=CC=C1 RXNXLAHQOVLMIE-UHFFFAOYSA-N 0.000 claims abstract description 44
- 102000012406 Carcinoembryonic Antigen Human genes 0.000 claims abstract description 37
- 108010022366 Carcinoembryonic Antigen Proteins 0.000 claims abstract description 37
- 239000000427 antigen Substances 0.000 claims abstract description 22
- 102000036639 antigens Human genes 0.000 claims abstract description 20
- 108091007433 antigens Proteins 0.000 claims abstract description 20
- 239000007788 liquid Substances 0.000 claims abstract description 16
- 230000005284 excitation Effects 0.000 claims abstract description 14
- 210000002700 urine Anatomy 0.000 claims abstract description 13
- 102100033420 Keratin, type I cytoskeletal 19 Human genes 0.000 claims abstract description 8
- 108010036226 antigen CYFRA21.1 Proteins 0.000 claims abstract description 8
- 239000012634 fragment Substances 0.000 claims abstract description 8
- 108010066302 Keratin-19 Proteins 0.000 claims abstract description 6
- 239000006249 magnetic particle Substances 0.000 claims description 20
- 238000005406 washing Methods 0.000 claims description 18
- 239000011859 microparticle Substances 0.000 claims description 17
- 238000002360 preparation method Methods 0.000 claims description 17
- 238000002156 mixing Methods 0.000 claims description 12
- 238000012360 testing method Methods 0.000 claims description 12
- 238000011534 incubation Methods 0.000 claims description 10
- HEMHJVSKTPXQMS-UHFFFAOYSA-M Sodium hydroxide Chemical compound [OH-].[Na+] HEMHJVSKTPXQMS-UHFFFAOYSA-M 0.000 claims description 9
- 239000000872 buffer Substances 0.000 claims description 9
- 239000003153 chemical reaction reagent Substances 0.000 claims description 6
- 238000004020 luminiscence type Methods 0.000 claims description 6
- 238000011088 calibration curve Methods 0.000 claims description 4
- 229920001213 Polysorbate 20 Polymers 0.000 claims description 3
- 239000013504 Triton X-100 Substances 0.000 claims description 3
- 229920004890 Triton X-100 Polymers 0.000 claims description 3
- 239000000256 polyoxyethylene sorbitan monolaurate Substances 0.000 claims description 3
- 235000010486 polyoxyethylene sorbitan monolaurate Nutrition 0.000 claims description 3
- 229910017604 nitric acid Inorganic materials 0.000 claims description 2
- 239000012530 fluid Substances 0.000 claims 1
- 206010028980 Neoplasm Diseases 0.000 abstract description 12
- 230000035945 sensitivity Effects 0.000 abstract description 9
- 238000012216 screening Methods 0.000 abstract description 7
- 230000000694 effects Effects 0.000 abstract description 4
- 230000002980 postoperative effect Effects 0.000 abstract description 3
- 239000003054 catalyst Substances 0.000 abstract description 2
- 150000001875 compounds Chemical class 0.000 abstract description 2
- 230000036046 immunoreaction Effects 0.000 abstract description 2
- 239000011248 coating agent Substances 0.000 abstract 1
- 238000000576 coating method Methods 0.000 abstract 1
- 239000000243 solution Substances 0.000 description 29
- 239000000523 sample Substances 0.000 description 19
- 238000000034 method Methods 0.000 description 14
- 239000008055 phosphate buffer solution Substances 0.000 description 12
- 241001529936 Murinae Species 0.000 description 9
- 239000000203 mixture Substances 0.000 description 8
- 238000003745 diagnosis Methods 0.000 description 7
- -1 acridine ester Chemical class 0.000 description 6
- 239000011324 bead Substances 0.000 description 6
- 210000004027 cell Anatomy 0.000 description 6
- 238000006243 chemical reaction Methods 0.000 description 5
- 230000000875 corresponding effect Effects 0.000 description 5
- 238000009472 formulation Methods 0.000 description 5
- 108091003079 Bovine Serum Albumin Proteins 0.000 description 4
- 230000002494 anti-cea effect Effects 0.000 description 4
- DZBUGLKDJFMEHC-UHFFFAOYSA-N benzoquinolinylidene Natural products C1=CC=CC2=CC3=CC=CC=C3N=C21 DZBUGLKDJFMEHC-UHFFFAOYSA-N 0.000 description 4
- 229940098773 bovine serum albumin Drugs 0.000 description 4
- PEDCQBHIVMGVHV-UHFFFAOYSA-N Glycerine Chemical compound OCC(O)CO PEDCQBHIVMGVHV-UHFFFAOYSA-N 0.000 description 3
- 239000007853 buffer solution Substances 0.000 description 3
- 201000011510 cancer Diseases 0.000 description 3
- 239000003085 diluting agent Substances 0.000 description 3
- 210000001035 gastrointestinal tract Anatomy 0.000 description 3
- 238000012544 monitoring process Methods 0.000 description 3
- 238000004393 prognosis Methods 0.000 description 3
- 108090000623 proteins and genes Proteins 0.000 description 3
- 102000004169 proteins and genes Human genes 0.000 description 3
- 210000002966 serum Anatomy 0.000 description 3
- 239000006228 supernatant Substances 0.000 description 3
- 239000000439 tumor marker Substances 0.000 description 3
- 101000998011 Homo sapiens Keratin, type I cytoskeletal 19 Proteins 0.000 description 2
- 102000011782 Keratins Human genes 0.000 description 2
- 108010076876 Keratins Proteins 0.000 description 2
- 238000002574 cystoscopy Methods 0.000 description 2
- 230000014509 gene expression Effects 0.000 description 2
- 238000003384 imaging method Methods 0.000 description 2
- 238000003018 immunoassay Methods 0.000 description 2
- 210000004185 liver Anatomy 0.000 description 2
- 238000012986 modification Methods 0.000 description 2
- 230000004048 modification Effects 0.000 description 2
- 230000010355 oscillation Effects 0.000 description 2
- 210000000496 pancreas Anatomy 0.000 description 2
- 210000001519 tissue Anatomy 0.000 description 2
- 230000002485 urinary effect Effects 0.000 description 2
- 239000012224 working solution Substances 0.000 description 2
- SXGZJKUKBWWHRA-UHFFFAOYSA-N 2-(N-morpholiniumyl)ethanesulfonate Chemical compound [O-]S(=O)(=O)CC[NH+]1CCOCC1 SXGZJKUKBWWHRA-UHFFFAOYSA-N 0.000 description 1
- QYYMDNHUJFIDDQ-UHFFFAOYSA-N 5-chloro-2-methyl-1,2-thiazol-3-one;2-methyl-1,2-thiazol-3-one Chemical compound CN1SC=CC1=O.CN1SC(Cl)=CC1=O QYYMDNHUJFIDDQ-UHFFFAOYSA-N 0.000 description 1
- 206010006187 Breast cancer Diseases 0.000 description 1
- 208000026310 Breast neoplasm Diseases 0.000 description 1
- 102000008186 Collagen Human genes 0.000 description 1
- 108010035532 Collagen Proteins 0.000 description 1
- 102000016550 Complement Factor H Human genes 0.000 description 1
- 108010053085 Complement Factor H Proteins 0.000 description 1
- 230000004543 DNA replication Effects 0.000 description 1
- 102000003886 Glycoproteins Human genes 0.000 description 1
- 108090000288 Glycoproteins Proteins 0.000 description 1
- 101000994460 Homo sapiens Keratin, type I cytoskeletal 20 Proteins 0.000 description 1
- 102100032700 Keratin, type I cytoskeletal 20 Human genes 0.000 description 1
- 206010058467 Lung neoplasm malignant Diseases 0.000 description 1
- 108010035916 Nuclear Matrix-Associated Proteins Proteins 0.000 description 1
- 102000008297 Nuclear Matrix-Associated Proteins Human genes 0.000 description 1
- 229910019142 PO4 Inorganic materials 0.000 description 1
- 108091005804 Peptidases Proteins 0.000 description 1
- 239000004365 Protease Substances 0.000 description 1
- 230000006819 RNA synthesis Effects 0.000 description 1
- 102100037486 Reverse transcriptase/ribonuclease H Human genes 0.000 description 1
- 230000002378 acidificating effect Effects 0.000 description 1
- 230000003213 activating effect Effects 0.000 description 1
- 230000004913 activation Effects 0.000 description 1
- 230000001640 apoptogenic effect Effects 0.000 description 1
- 210000002469 basement membrane Anatomy 0.000 description 1
- 230000009286 beneficial effect Effects 0.000 description 1
- 230000033228 biological regulation Effects 0.000 description 1
- 230000000740 bleeding effect Effects 0.000 description 1
- 230000000903 blocking effect Effects 0.000 description 1
- 210000004369 blood Anatomy 0.000 description 1
- 239000008280 blood Substances 0.000 description 1
- 244000309466 calf Species 0.000 description 1
- 230000015556 catabolic process Effects 0.000 description 1
- 230000021164 cell adhesion Effects 0.000 description 1
- 229920001436 collagen Polymers 0.000 description 1
- 230000002596 correlated effect Effects 0.000 description 1
- 230000008878 coupling Effects 0.000 description 1
- 238000010168 coupling process Methods 0.000 description 1
- 238000005859 coupling reaction Methods 0.000 description 1
- 230000002380 cytological effect Effects 0.000 description 1
- 210000004292 cytoskeleton Anatomy 0.000 description 1
- 238000006731 degradation reaction Methods 0.000 description 1
- 238000011161 development Methods 0.000 description 1
- 238000000502 dialysis Methods 0.000 description 1
- 201000010099 disease Diseases 0.000 description 1
- 208000037265 diseases, disorders, signs and symptoms Diseases 0.000 description 1
- 210000002919 epithelial cell Anatomy 0.000 description 1
- 210000003754 fetus Anatomy 0.000 description 1
- 239000010419 fine particle Substances 0.000 description 1
- 208000035474 group of disease Diseases 0.000 description 1
- 230000006872 improvement Effects 0.000 description 1
- 208000015181 infectious disease Diseases 0.000 description 1
- 238000007689 inspection Methods 0.000 description 1
- 210000003963 intermediate filament Anatomy 0.000 description 1
- 201000005202 lung cancer Diseases 0.000 description 1
- 208000020816 lung neoplasm Diseases 0.000 description 1
- 230000003211 malignant effect Effects 0.000 description 1
- 239000011259 mixed solution Substances 0.000 description 1
- 239000003147 molecular marker Substances 0.000 description 1
- 239000002245 particle Substances 0.000 description 1
- NBIIXXVUZAFLBC-UHFFFAOYSA-K phosphate Chemical compound [O-]P([O-])([O-])=O NBIIXXVUZAFLBC-UHFFFAOYSA-K 0.000 description 1
- 239000010452 phosphate Substances 0.000 description 1
- 239000008363 phosphate buffer Substances 0.000 description 1
- 230000008569 process Effects 0.000 description 1
- 239000000047 product Substances 0.000 description 1
- 238000007789 sealing Methods 0.000 description 1
- 238000006467 substitution reaction Methods 0.000 description 1
- 239000000725 suspension Substances 0.000 description 1
- 208000024891 symptom Diseases 0.000 description 1
- 238000013518 transcription Methods 0.000 description 1
- 230000035897 transcription Effects 0.000 description 1
- 238000002604 ultrasonography Methods 0.000 description 1
- 210000001635 urinary tract Anatomy 0.000 description 1
- 210000002229 urogenital system Anatomy 0.000 description 1
- 238000003260 vortexing Methods 0.000 description 1
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
- G01N33/574—Immunoassay; Biospecific binding assay; Materials therefor for cancer
- G01N33/57407—Specifically defined cancers
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N21/00—Investigating or analysing materials by the use of optical means, i.e. using sub-millimetre waves, infrared, visible or ultraviolet light
- G01N21/75—Systems in which material is subjected to a chemical reaction, the progress or the result of the reaction being investigated
- G01N21/76—Chemiluminescence; Bioluminescence
-
- 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
- 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
- G01N33/574—Immunoassay; Biospecific binding assay; Materials therefor for cancer
- G01N33/57473—Immunoassay; Biospecific binding assay; Materials therefor for cancer involving carcinoembryonic antigen, i.e. CEA
-
- 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
- G01N33/574—Immunoassay; Biospecific binding assay; Materials therefor for cancer
- G01N33/57484—Immunoassay; Biospecific binding assay; Materials therefor for cancer involving compounds serving as markers for tumor, cancer, neoplasia, e.g. cellular determinants, receptors, heat shock/stress proteins, A-protein, oligosaccharides, metabolites
- G01N33/57488—Immunoassay; Biospecific binding assay; Materials therefor for cancer involving compounds serving as markers for tumor, cancer, neoplasia, e.g. cellular determinants, receptors, heat shock/stress proteins, A-protein, oligosaccharides, metabolites involving compounds identifable in body fluids
-
- 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; monoclonal antibodies per se are classified with their corresponding antigens
-
- 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
- G01N2333/435—Assays involving biological materials from specific organisms or of a specific nature from animals; from humans
- G01N2333/46—Assays involving biological materials from specific organisms or of a specific nature from animals; from humans from vertebrates
- G01N2333/47—Assays involving proteins of known structure or function as defined in the subgroups
-
- 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
- G01N2333/435—Assays involving biological materials from specific organisms or of a specific nature from animals; from humans
- G01N2333/46—Assays involving biological materials from specific organisms or of a specific nature from animals; from humans from vertebrates
- G01N2333/47—Assays involving proteins of known structure or function as defined in the subgroups
- G01N2333/4701—Details
- G01N2333/4742—Keratin; Cytokeratin
Landscapes
- Health & Medical Sciences (AREA)
- Life Sciences & Earth Sciences (AREA)
- Immunology (AREA)
- Engineering & Computer Science (AREA)
- Chemical & Material Sciences (AREA)
- Urology & Nephrology (AREA)
- Hematology (AREA)
- Biomedical Technology (AREA)
- Molecular Biology (AREA)
- Physics & Mathematics (AREA)
- Analytical Chemistry (AREA)
- Cell Biology (AREA)
- Pathology (AREA)
- General Physics & Mathematics (AREA)
- General Health & Medical Sciences (AREA)
- Biochemistry (AREA)
- Food Science & Technology (AREA)
- Biotechnology (AREA)
- Medicinal Chemistry (AREA)
- Microbiology (AREA)
- Oncology (AREA)
- Hospice & Palliative Care (AREA)
- Chemical Kinetics & Catalysis (AREA)
- Plasma & Fusion (AREA)
- Investigating Or Analysing Biological Materials (AREA)
- Investigating Or Analysing Materials By The Use Of Chemical Reactions (AREA)
Abstract
本发明属于生物医学检测技术领域,具体涉及一种用于膀胱癌检测的标志物及检测试剂盒。本申请所述标志物包括核基质蛋白22(NMP22)、癌胚抗原(CEA)、膀胱肿瘤抗原(BTA)和细胞角蛋白19片段(CYFRA21‑1);所述检测试剂盒采用吖啶酯直接化学发光,用吖啶酯直接标记抗体(抗原),与待测标本相应的抗原(抗体)发生免疫反应后,形成包被抗体‑待测抗原‑吖啶酯标记抗体复合物,加入激发液,不需要催化剂即分解、发光。本发明建立了一种多肿瘤标志物的组合,进而通过多个指标的联合检测提高膀胱癌的检测特异性和敏感性,当对膀胱癌患者尿液检测时,具有较高的敏感度和特异度,可以对膀胱癌患者早期筛查、治疗效果和术后复发进行动态监测。
Description
技术领域
本发明属于生物医学检测技术领域,具体涉及一种用于膀胱癌检测的标志物及检测试剂盒。
背景技术
肿瘤是一类严重威胁人类健康,具有高发病率和高死亡率的疾病。膀胱癌是泌尿生殖系统最常见的恶性肿瘤,早期症状隐匿,患者就诊时多处于中晚期。发病率位于泌尿系统中恶性肿瘤首位,具有非常高的复发率和临床复杂性。在恶性肿瘤中,膀胱癌发病率排第六位,病死率排第四位。
目前膀胱肿瘤的诊断主要以尿液检测、影像学(KUB、IVP、CT、MRI、超声等)检查、膀胱镜检查等为主。膀胱肿瘤初次发现主要以表浅性的肿瘤为主,影像学检查对于表浅、初期的膀胱肿瘤诊断能力有限,膀胱镜检查为有创检查,有创检查费用高昂且还会造成泌尿系统感染、出血等并发症,所以这些常用的诊断膀胱肿瘤的方法并不适用于膀胱肿瘤的筛查。尿液检测是目前较为理想的检测方法,为无创标本,可提供蛋白质、RNA及 DNA来源。因此,尿液是比较理想的用于检测肿瘤分子标志物的标本。
对于膀胱癌的筛查和复查,应尽量减少病人的痛苦,降低检测的成本,减轻患者的负担。肿瘤分子标志物是一种可以判断肿瘤发生、发展甚至预后的指示物。但单一的肿瘤标志物难以特异性地诊断膀胱癌。因此建立一种能够辅助,甚至替代尿脱落细胞学检测的无创、经济、高效、准确、快捷的方法尤为重要。
发明内容
本发明的目的在于提供一种用于膀胱癌检测的标志物及检测试剂盒,旨在建立一种多肿瘤标志物的组合,用于膀胱癌的早期筛查,通过多种膀胱癌肿瘤标志物的联合应用可以提高膀胱癌的诊断准确性。
为了实现上述目的,本发明采用的技术方案是:
一种用于膀胱癌检测的标志物,所述标志物包括核基质蛋白22 (NMP22)、癌胚抗原(CEA)、膀胱肿瘤抗原(BTA)和细胞角蛋白19 片段(CYFRA21-1)。
一种上述用于膀胱癌检测的标志物在制备用于膀胱癌检测的检测试剂或检测试剂盒中的应用。
优选的,所述检测试剂盒的检测方法为化学发光免疫分析方法,具体为双抗体夹心化学发光免疫分析法。
优选的,所述检测试剂盒包括:抗体包被的磁微粒、吖啶酯标记的抗体、校准品、激发液A、激发液B和洗涤液,所述抗体为标志物对应的抗体。
优选的,所述抗体包括:NMP22抗体、CEA抗体、BTA抗体、和 CYFRA21-1抗体;更优选的,所述抗体包括:核基质蛋白22(NMP22) 单克隆抗体、癌胚抗原(CEA)单克隆抗体、膀胱肿瘤抗原(BTA)单克隆抗体和细胞角蛋白19片段(CYFRA21-1)单克隆抗体。。
优选的,所述抗体包被的磁微粒包括:NMP22抗体包被的磁微粒、CEA 抗体包被的磁微粒、BTA抗体包被的磁微粒、和CYFRA21-1抗体包被的磁微粒;更优选的,所述的磁微粒的粒径为1-3μm;
更优选的,所述抗体包被的磁微粒通过如下方法制备:取磁微粒用 EDC溶液活化后,加入相应抗体进行分子偶联;再用含牛血清白蛋白的 0.0.1mol/L磷酸盐缓冲液pH7.4进行封闭;将封闭后的抗体包被的磁微粒置于2~8℃保存备用。
优选的,所述吖啶酯标记的抗体包括:吖啶酯标记的NMP22抗体、吖啶酯标记的CEA抗体、吖啶酯标记的BTA抗体、和吖啶酯标记的 CYFRA21-1抗体;
更优选的,所述吖啶酯标记的抗体通过如下方法制备:将吖啶酯加入相应抗体溶液中,混匀,避光孵育;将反应液全部透析至0.02mol/L磷酸盐缓冲液pH 7.4中,于–20℃避光保存。
优选的,所述校准品为不同浓度的标志物校准品,包括NMP22校准品、 CEA校准品、BTA校准品、和CYFRA21-1校准品;更优选的,所述标志物的浓度为0、0.5ng/mL、5ng/mL、10ng/mL、50ng/mL、100ng/mL;更优选的,所述校准品是用校准品稀释液配制的不同浓度标志物的溶液;更优选的,所述标志物为相应的标准抗原。
优选的,所述激发液A:为0.5%的H2O2和0.1mol/L HNO3的溶液;所述激发液B:为0.35mmol/L的NaOH和3%Triton X-100的溶液;所述洗涤液为:含有1%Tween-20的20×PBS缓冲液(pH7.0-8.0),使用时按1: 20倍稀释使用。
优选的,所述检测试剂盒的检测包括如下步骤:
(1)加样:取检测样本和抗体包被的磁微粒于孵育杯内混匀并孵育;
(2)加入吖啶酯标记的抗体试剂:将吖啶酯标记的抗体加入步骤(1) 的孵育杯内继续孵育;
(3)洗涤:用洗涤液洗涤;
(4)发光:依次加入激发液A和激发液B,进行发光处理;
(5)读值:测定检测样本的相对发光强度,根据校准曲线,计算出检测样本中待测标志物的浓度。
优选的,所述检测试剂盒的检测包括如下步骤:
(1)加样:分别取50μl检测样本和50μl抗体包被的磁微粒于孵育杯内混匀,37℃孵育10min;
(2)加入吖啶酯标记的抗体试剂:取50μl吖啶酯标记的抗体,加入步骤(1)的孵育杯内,37℃孵育20min;
(3)洗涤:用洗涤液洗涤2遍;
(4)发光:依次加入50μl激发液A和激发液B,混匀;
(5)读值:测定检测样本的相对发光强度,根据校准曲线,计算出检测样本中待测标志物的浓度。
优选的,所述步骤(1)中的检测样本为校准品或待测尿液。
优选的,所述步骤(1)中的抗体包被的磁微粒为NMP22抗体包被的磁微粒、CEA抗体包被的磁微粒、BTA抗体包被的磁微粒、和CYFRA21-1 抗体包被的磁微粒的混合。
优选的,所述步骤(2)中的吖啶酯标记的抗体为吖啶酯标记的NMP22 抗体、吖啶酯标记的CEA抗体、吖啶酯标记的BTA抗体、和吖啶酯标记的CYFRA21-1抗体的混合。
核基质蛋白22(NMP22,nuclear matrix protein 22):是细胞核基质蛋白成员之一,特异性的存在于尿路移行细胞中,NMP22在恶性移行细胞中的含量约为正常细胞的80倍。NMP22主要参与DNA的复制及转录、 RNA合成和基因表达调控,可通过检测凋亡细胞释放出的蛋白量来测定其含量。NMP22含量与肿瘤大小、分期、分级呈正相关。NMP22是一种新型肿瘤标志物。
膀胱肿瘤抗原(BTA,bladder tumor antigen),又称膀胱肿瘤相关抗原(BTAA),又被称为补体因子H相关蛋白,膀胱肿瘤在发生生长的过程中会产生一种蛋白水解酶,它能够降解细胞基底膜的各种成分,进而分解形成各种糖蛋白、胶原片段和蛋白多糖,膀胱肿瘤抗原对于膀胱肿瘤检测的特异性和敏感性分别为77%和60%。
癌胚抗原(carcinoembryonic antigen,CEA)是一种参与细胞黏附的酸性糖蛋白,胚胎期主要存在于胎儿的胃肠道、胰腺和肝脏,出生后组织内含量很低。在正常人胃肠道、胰腺和肝脏中也有少量表达。当发生消化道肿瘤、乳腺癌、肺癌等疾病时,血清CEA水平可显著升高。因此CEA是一种广谱肿瘤标记物。但CEA在膀胱癌特异度比较高。因此尿及血清中CEA 的检测亦被应用于膀胱癌的诊断和监测。
细胞角蛋白19片段(Cytokeratin-19-fragment CYFRA21-1,CK19), CK是构成上皮细胞细胞骨架的中间丝组成成分,具有高度的细胞和组织特异性,CK至少有20多种,其中与膀胱癌诊断、检测复发及预后关系最相关的主要有CK19、CK20。CYFRA21-1正常情况下其含量极低,当肿瘤产生时,蛋白酶的激活加速了细胞角蛋白的降解,大量细胞角蛋白片段得以释放,CYFRA21-1在血、尿中的含量均有所升高,CYFRA21-1可作为一种无创的膀胱癌诊断检测指标。
本发明建立一种多肿瘤标志物的组合,进而通过多个指标的联合检测提高膀胱癌的检测特异性和敏感性。
与现有技术相比,本发明的有益效果是:
(1)本发明提供了一种用于膀胱癌检测的标志物及检测试剂盒,该标志物及其试剂盒较为操作简便、廉价、高效,且属于无创的手段,可以用来诊断膀胱癌以及监测膀胱癌的复发。
(2)本发明提供的试剂盒灵敏度高、特异度强,并且便于群体性筛查的无创性检查,建立了一种方便、快速、敏感性高并且无创、普通人群较易接受的检测方法,对膀胱癌的及时发现、临床预后以及复发检测方面都具有重要的作用。
(3)本发明提供的试剂盒可以用于联合检测核基质蛋白22(NMP22)、膀胱肿瘤抗原(BTA)、癌胚抗原(CEA)、细胞角蛋白19片段(CYFRA21-1)。
(4)本发明提供的试剂盒采用吖啶酯直接化学发光,用吖啶酯直接标记抗体(抗原),与待测标本相应的抗原(抗体)发生免疫反应后,形成包被抗体-待测抗原-吖啶酯标记抗体复合物,加入激发液,不需要催化剂即分解、发光。收集、记录单位时间内所产生的光子能,这部分光的积分与待测抗原的量成正比,根据建立的标准曲线计算待测抗原的量。
(5)本发明提供的试剂盒当当对膀胱癌患者尿液NMP22、BTA、 CEA、CYFRA21四者联合检测时,具有较高的敏感度和特异度,可以对膀胱癌患者早期筛查、治疗效果和术后复发进行动态监测。
具体实施方式
下面通过具体实施例,对本发明的技术方案作进一步的具体说明。
实施例1
1.制备抗体包被的磁微粒
1.1鼠抗NMP22抗体包被的磁微粒制备
(1)取200μL磁珠溶液(50mg/mL),用0.01M PBS缓冲液(p H=7.4)清洗2次,最后用1m L PBS缓冲液重新悬浮磁珠;
(2)在步骤(1)得到的磁珠溶液中加入100μL的EDC溶液,涡旋混匀后于室温振荡反应2h;所述室温为15-40℃,本实施例中为25℃;EDC 溶液:0.01M MES((2-(N-吗啉代)乙烷磺酸))(PH=6.0)溶液配制5mg/ml 的EDC溶液(现配现用),避光备用;
(3)弃上清,用0.01M PBS缓冲液(p H=7.4)清洗3次;用1m L PBS缓冲液重新悬浮磁珠;
(4)加入纯化浓缩的鼠抗NMP22单克隆抗体50~800μg(本实施例中加入400μg),涡旋混匀后于室温振荡反应24h;
(5)用0.01M PBS缓冲液(p H=7.4)清洗3次,加入0.5mL 10%牛血清白蛋白(BSA)磷酸盐溶液,涡旋混匀,37℃振荡反应2h,封闭磁珠;
(6)弃上清,用0.0.1M PBS缓冲液pH7.4溶液清洗3遍,最后悬浮于20mL含有1%BSA、0.1%ProClin 300的PBS pH7.4溶液中,使磁珠的终浓度为0.5mg/mL,于4℃保存备用。
1.2鼠抗CEA抗体包被的磁微粒制备
方法同鼠抗NMP22抗体包被的磁微粒制备,替换其中抗体为鼠抗CEA 抗体。
1.3鼠抗BTA抗体包被的磁微粒制备
方法同鼠抗NMP22抗体包被的磁微粒制备,替换其中抗体为鼠抗BTA 抗体。
1.4鼠抗CYFRA21-1抗体包被的磁微粒制备
方法同鼠抗NMP22抗体包被的磁微粒制备,替换其中抗体为鼠抗 CYFRA21-1抗体。
2.吖啶酯标记的抗体的制备
2.1吖啶酯标记的鼠抗NMP22抗体的制备
取一定量的鼠抗NMP22单克隆抗体,用0.05mol/L pH 9.6PBS溶液调整鼠抗NMP22抗体浓度为1mg/mL,取一定量的吖啶酯,将NMP22抗体与吖啶酯按1:(5~20)摩尔比例进行混合(本实施例中摩尔比为1:10),室温避光震荡反应30min,将反应液移至透析袋,用0.02mol/L pH7.4的PBS 缓冲液4℃透析3次,去除游离吖啶酯,最后加入等体积甘油,放置于-20℃下保存待用。
2.2吖啶酯标记的鼠抗CEA抗体的制备
方法同吖啶酯标记的鼠抗NMP22抗体的制备,替换其中抗体为鼠抗 CEA抗体。
2.3吖啶酯标记的鼠抗BTA抗体的制备
方法同吖啶酯标记的鼠抗NMP22抗体的制备,替换其中抗体为鼠抗 BTA抗体。
2.4吖啶酯标记的鼠抗CYFRA21-1抗体的指标
方法同吖啶酯标记的鼠抗NMP22抗体的制备,替换其中抗体为鼠抗 CYFRA21-1抗体。
3.校准品的配制
校准品稀释液:配制含10%灭活新生牛血清的0.02mol/L pH7.4磷酸盐缓冲液。
3.1NMP22校准品的配制
将NMP22抗原用校准品稀释液稀释,分别配制浓度为:0、0.5ng/mL、 5ng/mL、10ng/mL、50ng/mL、100ng/mL的NMP22校准品。
3.2CEA校准品的配制
方法同NMP22校准品的配制。
3.3BTA校准品的配制
方法同NMP22校准品的配制。
3.4CYFRA21-1校准品的配制
方法同NMP22校准品的配制。
4.激发液A:为0.5%的H2O2和0.1mol/LHNO3的溶液;激发液B:为 0.35mmol/L的NaOH和3%Triton X-100的溶液;洗涤液为:含有 1%Tween-20的20×PBS缓冲液(pH7.0-8.0)。
5.检测步骤
(1)首先检测前准备工作:将试剂盒与待检测的样本从所储存的环境中取出,置于室温环境中大约30分钟;将尿液于通用离心机中2500rpm-3000rpm离心10-15min,吸取上清用于检测,得到待检测尿液样品;
(2)加样:分别取适量的NMP22抗体包被的磁微粒、CEA抗体包被的磁微粒、BTA抗体包被的磁微粒、CYFRA21-1抗体包被的磁微粒至 1×PBS缓冲液中,配制成混合磁微粒包被抗体的悬浮液。
分别取50μl检测样本(校准品和待测尿液)和50μl混合磁微粒包被的抗体溶液于孵育杯内混匀,37℃孵育10min;
(3)加入吖啶酯标记的抗体试剂:分别取适量的吖啶酯标记的NMP22 抗体、吖啶酯标记的CEA抗体、吖啶酯标记的BTA抗体、吖啶酯标记的 CYFRA21-1抗体至1×PBS缓冲液中,配制成混合的吖啶酯标记的抗体工作液,混合液中各吖啶酯标记抗体量均为:0.03μg/ml。
取50μl混合的吖啶酯标记的抗体工作液,加入步骤(2)的孵育杯内, 37℃孵育20min;
(4)洗涤:用洗涤液洗涤2遍;
(5)发光:依次加入50μl激发液A和50μl激发液B,混匀;
(6)读值:测定样本的相对发光强度,根据校准曲线,分别计算出样品中NMP22抗原、CEA抗原、BTA抗原、CYFRA21-1抗原的浓度,实验结果如下:
不同检测项目用于膀胱癌诊断的灵敏度、特异性
检测项目 | 灵敏度 | 特异性 |
NMP22单检 | 84.3% | 55% |
BTA单检 | 70% | 80.7% |
CEA单检 | 78.83 | 28.47 |
CYFRA21-1单检 | 76.6% | 85% |
NMP22+BTA | 71.9% | 64% |
NMP22+CEA | 81.75% | 20.95% |
NMP22+CYFRA21-1 | 82.8% | 35% |
NMP22+BTA+CEA | 90.3% | 80% |
NMP22+BTA+CYFRA21-1 | 93.7% | 55.2% |
NMP22+CEA+CYFRA21-1 | 96.13% | 50% |
NMP22+BTA+CEA+CYFRA21-1 | 98.4% | 80% |
结果表示,当对膀胱癌患者尿液NMP22、BTA、CEA、CYFRA21 四者联合检测时,具有较高的敏感度和特异度,可以对膀胱癌患者早期筛查、治疗效果和术后复发进行动态监测。
最后应说明的是:以上所述仅为本发明的优选实施例,不用于限制本发明,尽管参照前述实施例对本发明进行了详细的说明,对于本领域的技术人员来说,其依然可以对前述各实施例所记载的技术方案进行修改,或者对其中部分技术特征进行等同替换。凡在本发明的精神和原则之内,所作的任何修改、等同替换、改进等,均应包含在本发明的保护范围之内。
Claims (10)
1.一种用于膀胱癌检测的标志物,其特征在于,所述标志物包括核基质蛋白22(NMP22)、癌胚抗原(CEA)、膀胱肿瘤抗原(BTA)和细胞角蛋白19片段(CYFRA21-1)。
2.一种权利要求1所述的用于膀胱癌检测的标志物在制备用于膀胱癌检测的检测试剂或检测试剂盒中的应用。
3.根据权利要求2所述一种用于膀胱癌检测的检测试剂盒,其特征在于,所述检测试剂盒包括:抗体包被的磁微粒、吖啶酯标记的抗体、校准品、激发液A、激发液B和洗涤液,所述抗体为标志物对应的抗体。
4.根据权利要求3所述一种用于膀胱癌检测的检测试剂盒,其特征在于,所述抗体包括:NMP22抗体、CEA抗体、BTA抗体、和CYFRA21-1抗体。
5.根据权利要求3所述一种用于膀胱癌检测的检测试剂盒,其特征在于,所述抗体包被的磁微粒包括:NMP22抗体包被的磁微粒、CEA抗体包被的磁微粒、BTA抗体包被的磁微粒、和CYFRA21-1抗体包被的磁微粒。
6.根据权利要求3所述一种用于膀胱癌检测的检测试剂盒,其特征在于,所述吖啶酯标记的抗体包括:吖啶酯标记的NMP22抗体、吖啶酯标记的CEA抗体、吖啶酯标记的BTA抗体、和吖啶酯标记的CYFRA21-1抗体。
7.根据权利要求3所述一种用于膀胱癌检测的检测试剂盒,其特征在于,所述校准品为不同浓度的标志物校准品,包括NMP22校准品、CEA校准品、BTA校准品、和CYFRA21-1校准品。
8.根据权利要求3所述一种用于膀胱癌检测的检测试剂盒,其特征在于,所述激发液A:为0.5%的H2O2和0.1mol/L的HNO3的溶液;所述激发液B:为0.35mmol/L的NaOH和3%TritonX-100的溶液;所述洗涤液为:含有1%Tween-20的20×PBS缓冲液,pH7.0-8.0。
9.根据权利要求3所述一种用于膀胱癌检测的检测试剂盒,其特征在于,所述检测试剂盒的检测包括如下步骤:
(1)加样:取检测样本和抗体包被的磁微粒于孵育杯内混匀并孵育;
(2)加入吖啶酯标记的抗体试剂:将吖啶酯标记的抗体加入步骤(1)的孵育杯内继续孵育;
(3)洗涤:用洗涤液洗涤;
(4)发光:依次加入激发液A和激发液B,进行发光处理;
(5)读值:测定检测样本的相对发光强度,根据校准曲线,计算出检测样本中待测标志物的浓度。
10.根据权利要求9所述一种用于膀胱癌检测的检测试剂盒,其特征在于,所述步骤(1)中的检测样本为校准品或待测尿液。
Priority Applications (1)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
CN202110746113.6A CN113759115A (zh) | 2021-06-30 | 2021-06-30 | 用于膀胱癌检测的标志物及检测试剂盒 |
Applications Claiming Priority (1)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
CN202110746113.6A CN113759115A (zh) | 2021-06-30 | 2021-06-30 | 用于膀胱癌检测的标志物及检测试剂盒 |
Publications (1)
Publication Number | Publication Date |
---|---|
CN113759115A true CN113759115A (zh) | 2021-12-07 |
Family
ID=78787532
Family Applications (1)
Application Number | Title | Priority Date | Filing Date |
---|---|---|---|
CN202110746113.6A Pending CN113759115A (zh) | 2021-06-30 | 2021-06-30 | 用于膀胱癌检测的标志物及检测试剂盒 |
Country Status (1)
Country | Link |
---|---|
CN (1) | CN113759115A (zh) |
Cited By (1)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
CN116482369A (zh) * | 2023-06-13 | 2023-07-25 | 天津市协和医药科技集团有限公司 | 一种癌胚抗原检测试剂盒 |
Citations (8)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
CN103492590A (zh) * | 2011-02-22 | 2014-01-01 | 卡里斯生命科学卢森堡控股有限责任公司 | 循环生物标志物 |
US20140072987A1 (en) * | 2012-09-12 | 2014-03-13 | Randox Laboratories Ltd. | Methods of detecing bladder cancer |
CN104239707A (zh) * | 2014-09-03 | 2014-12-24 | 贾明宏 | 一种疾病快速检测与信息采集的方法 |
CN105445475A (zh) * | 2015-11-18 | 2016-03-30 | 济南大学 | 一种三抗体双通道同时检测人核基质蛋白和癌胚抗原免疫传感器的制备方法及应用 |
CN106645756A (zh) * | 2016-12-30 | 2017-05-10 | 广州市达瑞生物技术股份有限公司 | 一种检测nmp22的试剂盒及其制备方法 |
CN108414756A (zh) * | 2017-10-25 | 2018-08-17 | 深圳市罗湖区人民医院 | 一种同时检测尿液中四个膀胱癌标志物的蛋白芯片的制备方法 |
CN108593922A (zh) * | 2018-05-03 | 2018-09-28 | 常州领航量子生物医疗科技有限公司 | 一种测定膀胱癌的联合检测试剂盒及其制备方法 |
TW202016545A (zh) * | 2018-06-21 | 2020-05-01 | 中國醫藥大學 | 泌尿道上皮細胞癌生物標記及其應用 |
-
2021
- 2021-06-30 CN CN202110746113.6A patent/CN113759115A/zh active Pending
Patent Citations (8)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
CN103492590A (zh) * | 2011-02-22 | 2014-01-01 | 卡里斯生命科学卢森堡控股有限责任公司 | 循环生物标志物 |
US20140072987A1 (en) * | 2012-09-12 | 2014-03-13 | Randox Laboratories Ltd. | Methods of detecing bladder cancer |
CN104239707A (zh) * | 2014-09-03 | 2014-12-24 | 贾明宏 | 一种疾病快速检测与信息采集的方法 |
CN105445475A (zh) * | 2015-11-18 | 2016-03-30 | 济南大学 | 一种三抗体双通道同时检测人核基质蛋白和癌胚抗原免疫传感器的制备方法及应用 |
CN106645756A (zh) * | 2016-12-30 | 2017-05-10 | 广州市达瑞生物技术股份有限公司 | 一种检测nmp22的试剂盒及其制备方法 |
CN108414756A (zh) * | 2017-10-25 | 2018-08-17 | 深圳市罗湖区人民医院 | 一种同时检测尿液中四个膀胱癌标志物的蛋白芯片的制备方法 |
CN108593922A (zh) * | 2018-05-03 | 2018-09-28 | 常州领航量子生物医疗科技有限公司 | 一种测定膀胱癌的联合检测试剂盒及其制备方法 |
TW202016545A (zh) * | 2018-06-21 | 2020-05-01 | 中國醫藥大學 | 泌尿道上皮細胞癌生物標記及其應用 |
Non-Patent Citations (3)
Title |
---|
Л. А. ДЕРЖАВЕЦ: "Информативность опухолевых маркеров для оценки степени распространенности рака мочевого пузыря", 《ДЕРЖАВЕЦ》, pages 129 * |
张秉琪等编著: "《肿瘤标志物临床手册》", 31 July 2008, 人民军医出版社, pages: 193 * |
黄邦荣主编: "《裴氏实用肿瘤学 中西医结合》", 31 August 2013, 甘肃科学技术出版社, pages: 342 * |
Cited By (2)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
CN116482369A (zh) * | 2023-06-13 | 2023-07-25 | 天津市协和医药科技集团有限公司 | 一种癌胚抗原检测试剂盒 |
CN116482369B (zh) * | 2023-06-13 | 2023-08-25 | 天津市协和医药科技集团有限公司 | 一种癌胚抗原检测试剂盒 |
Similar Documents
Publication | Publication Date | Title |
---|---|---|
WO2016127318A1 (zh) | 心肌肌钙蛋白i超敏检测试剂盒及其超敏检测方法 | |
CN108008132B (zh) | 一种联合检测卵巢癌肿瘤标志物he4和ca125的试剂盒及其制备方法和应用 | |
WO2016127301A1 (zh) | rT3化学发光免疫检测试剂盒及其检测方法和应用 | |
US20210190780A1 (en) | Serum thymidine kinase 1 detection kit based on automatic chemiluminescence analyzer | |
WO2021088730A1 (zh) | 游离前列腺特异性抗原检测试剂盒及其制备方法 | |
CN111175491A (zh) | 一种sBCMA磁微粒化学发光免疫检测试剂盒及其制备方法和应用 | |
CN111089958A (zh) | 一种基于葡聚糖信号放大的p16INK4a化学发光试剂盒 | |
CN111983243A (zh) | 氨基末端脑利钠肽前体测定试剂盒、制备方法及检测方法 | |
CN109187971A (zh) | 神经元特异性烯醇化酶化学发光免疫检测试剂盒及其制备方法 | |
CN101368961B (zh) | 尿膀胱癌抗原化学发光免疫分析定量测定试剂盒及其制备方法 | |
CN113759115A (zh) | 用于膀胱癌检测的标志物及检测试剂盒 | |
CN111157725A (zh) | 一种人Legumain化学发光检测试剂盒及其应用 | |
CN111381034A (zh) | 一种gpc-3抗体包被磁珠及其制备方法、试剂盒 | |
CN113433319A (zh) | 用于宫颈癌检测的标志物及检测试剂盒 | |
CN107271692B (zh) | 一种标记特异性高亲和力重组抗体的荧光微球及其应用 | |
CN113933521A (zh) | 磁微粒化学发光检测试剂盒及其制备方法和应用 | |
CN111707825A (zh) | 联合检测肿瘤标志物mct1和mct4的试剂盒及其制备方法、应用 | |
WO2024001044A1 (zh) | 一种与肺癌相关的生物标志物组合、含其的试剂盒及其用途 | |
CN109633163B (zh) | 降钙素原/c反应蛋白二合一检测试剂盒 | |
CN104215778B (zh) | 一种c肽单克隆抗体交联磁微粒及其制备方法和包括其的检测试剂盒 | |
CN111175496A (zh) | 妊娠相关蛋白a磁微粒化学发光检测试剂盒及其使用方法 | |
CN115290897A (zh) | 一种癌胚抗原(cea)化学发光免疫测定试剂盒 | |
CN113109325A (zh) | 一种胃蛋白酶原i酶促化学发光检测试剂盒及其制备方法与应用 | |
CN113533726A (zh) | 用于检测胸苷激酶1的试剂盒及其应用 | |
CN112180104A (zh) | 一种基于抗缪勒氏管激素amh时间分辨荧光免疫分析试剂盒及其制备方法和检测方法 |
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 | ||
TA01 | Transfer of patent application right |
Effective date of registration: 20220302 Address after: 255000 room 1610, block B, polymer material industrial innovation park, No. 51, Lutai Avenue, high tech Zone, Zibo City, Shandong Province Applicant after: Shandong Guanke Biotechnology Co.,Ltd. Address before: Room 907, No. 15, Yinhu innovation center, No. 9, Fuxian Road, Yinhu street, Fuyang District, Hangzhou, Zhejiang 311422 Applicant before: Hangzhou Zhuoheng Biotechnology Co.,Ltd. |
|
TA01 | Transfer of patent application right |