CN111812326A - 一种定量检测pd-l1与pd-1结合物含量的试剂盒 - Google Patents
一种定量检测pd-l1与pd-1结合物含量的试剂盒 Download PDFInfo
- Publication number
- CN111812326A CN111812326A CN202010716591.8A CN202010716591A CN111812326A CN 111812326 A CN111812326 A CN 111812326A CN 202010716591 A CN202010716591 A CN 202010716591A CN 111812326 A CN111812326 A CN 111812326A
- Authority
- CN
- China
- Prior art keywords
- conjugate
- kit
- antibody
- reagent
- 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
- 102000008096 B7-H1 Antigen Human genes 0.000 title claims abstract description 67
- 108010074708 B7-H1 Antigen Proteins 0.000 title claims abstract description 66
- 102100040678 Programmed cell death protein 1 Human genes 0.000 claims abstract description 52
- 101710089372 Programmed cell death protein 1 Proteins 0.000 claims abstract description 52
- 239000003153 chemical reaction reagent Substances 0.000 claims abstract description 25
- 239000006249 magnetic particle Substances 0.000 claims abstract description 23
- 102000004190 Enzymes Human genes 0.000 claims abstract description 16
- 108090000790 Enzymes Proteins 0.000 claims abstract description 16
- 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 14
- 102000002260 Alkaline Phosphatase Human genes 0.000 claims abstract description 14
- 108020004774 Alkaline Phosphatase Proteins 0.000 claims abstract description 14
- 239000000758 substrate Substances 0.000 claims abstract description 12
- 239000011859 microparticle Substances 0.000 claims abstract description 10
- 239000011616 biotin Substances 0.000 claims abstract description 7
- 229960002685 biotin Drugs 0.000 claims abstract description 7
- 235000020958 biotin Nutrition 0.000 claims abstract description 7
- 230000008878 coupling Effects 0.000 claims abstract description 6
- 238000010168 coupling process Methods 0.000 claims abstract description 6
- 238000005859 coupling reaction Methods 0.000 claims abstract description 6
- 102000037865 fusion proteins Human genes 0.000 claims description 12
- 108020001507 fusion proteins Proteins 0.000 claims description 12
- 238000003908 quality control method Methods 0.000 claims description 8
- 238000001514 detection method Methods 0.000 abstract description 18
- 238000000034 method Methods 0.000 abstract description 12
- 230000008045 co-localization Effects 0.000 abstract description 4
- 238000000749 co-immunoprecipitation Methods 0.000 abstract description 3
- 238000002649 immunization Methods 0.000 abstract description 2
- 230000003053 immunization Effects 0.000 abstract description 2
- 230000008569 process Effects 0.000 abstract description 2
- 230000035484 reaction time Effects 0.000 abstract description 2
- 230000035945 sensitivity Effects 0.000 abstract description 2
- PEDCQBHIVMGVHV-UHFFFAOYSA-N Glycerine Chemical compound OCC(O)CO PEDCQBHIVMGVHV-UHFFFAOYSA-N 0.000 description 21
- LENZDBCJOHFCAS-UHFFFAOYSA-N tris Chemical compound OCC(N)(CO)CO LENZDBCJOHFCAS-UHFFFAOYSA-N 0.000 description 17
- 239000000243 solution Substances 0.000 description 13
- 239000007983 Tris buffer Substances 0.000 description 11
- 229920001213 Polysorbate 20 Polymers 0.000 description 10
- 239000000256 polyoxyethylene sorbitan monolaurate Substances 0.000 description 10
- 235000010486 polyoxyethylene sorbitan monolaurate Nutrition 0.000 description 10
- FAPWRFPIFSIZLT-UHFFFAOYSA-M Sodium chloride Chemical compound [Na+].[Cl-] FAPWRFPIFSIZLT-UHFFFAOYSA-M 0.000 description 8
- 206010028980 Neoplasm Diseases 0.000 description 6
- 239000007853 buffer solution Substances 0.000 description 6
- 239000011780 sodium chloride Substances 0.000 description 6
- 239000000126 substance Substances 0.000 description 6
- HDTRYLNUVZCQOY-UHFFFAOYSA-N α-D-glucopyranosyl-α-D-glucopyranoside Natural products OC1C(O)C(O)C(CO)OC1OC1C(O)C(O)C(O)C(CO)O1 HDTRYLNUVZCQOY-UHFFFAOYSA-N 0.000 description 5
- 108091003079 Bovine Serum Albumin Proteins 0.000 description 5
- HDTRYLNUVZCQOY-WSWWMNSNSA-N Trehalose Natural products O[C@@H]1[C@@H](O)[C@@H](O)[C@@H](CO)O[C@@H]1O[C@@H]1[C@H](O)[C@@H](O)[C@@H](O)[C@@H](CO)O1 HDTRYLNUVZCQOY-WSWWMNSNSA-N 0.000 description 5
- HDTRYLNUVZCQOY-LIZSDCNHSA-N alpha,alpha-trehalose Chemical compound O[C@@H]1[C@@H](O)[C@H](O)[C@@H](CO)O[C@@H]1O[C@@H]1[C@H](O)[C@@H](O)[C@H](O)[C@@H](CO)O1 HDTRYLNUVZCQOY-LIZSDCNHSA-N 0.000 description 5
- 239000011324 bead Substances 0.000 description 5
- 229940098773 bovine serum albumin Drugs 0.000 description 5
- 238000006243 chemical reaction Methods 0.000 description 5
- 239000008363 phosphate buffer Substances 0.000 description 5
- 238000002360 preparation method Methods 0.000 description 5
- 102000004169 proteins and genes Human genes 0.000 description 5
- 108090000623 proteins and genes Proteins 0.000 description 5
- 239000007836 KH2PO4 Substances 0.000 description 4
- 239000007987 MES buffer Substances 0.000 description 4
- 241000699666 Mus <mouse, genus> Species 0.000 description 4
- XYIPYISRNJUPBA-UHFFFAOYSA-N [3-(3'-methoxyspiro[adamantane-2,4'-dioxetane]-3'-yl)phenyl] dihydrogen phosphate Chemical compound O1OC2(C3CC4CC(C3)CC2C4)C1(OC)C1=CC=CC(OP(O)(O)=O)=C1 XYIPYISRNJUPBA-UHFFFAOYSA-N 0.000 description 4
- 239000000872 buffer Substances 0.000 description 4
- BNIILDVGGAEEIG-UHFFFAOYSA-L disodium hydrogen phosphate Chemical compound [Na+].[Na+].OP([O-])([O-])=O BNIILDVGGAEEIG-UHFFFAOYSA-L 0.000 description 4
- 229910000397 disodium phosphate Inorganic materials 0.000 description 4
- FYFFGSSZFBZTAH-UHFFFAOYSA-N methylaminomethanetriol Chemical compound CNC(O)(O)O FYFFGSSZFBZTAH-UHFFFAOYSA-N 0.000 description 4
- 229910000402 monopotassium phosphate Inorganic materials 0.000 description 4
- JQWHASGSAFIOCM-UHFFFAOYSA-M sodium periodate Chemical compound [Na+].[O-]I(=O)(=O)=O JQWHASGSAFIOCM-UHFFFAOYSA-M 0.000 description 4
- 210000001519 tissue Anatomy 0.000 description 4
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Chemical compound O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 description 4
- LLIANSAISVOLHR-GBCQHVBFSA-N 5-[(3as,4s,6ar)-2-oxidanylidene-1,3,3a,4,6,6a-hexahydrothieno[3,4-d]imidazol-4-yl]pentanoic acid Chemical compound N1C(=O)N[C@@H]2[C@H](CCCCC(=O)O)SC[C@@H]21.N1C(=O)N[C@@H]2[C@H](CCCCC(=O)O)SC[C@@H]21 LLIANSAISVOLHR-GBCQHVBFSA-N 0.000 description 3
- LYCAIKOWRPUZTN-UHFFFAOYSA-N Ethylene glycol Chemical compound OCCO LYCAIKOWRPUZTN-UHFFFAOYSA-N 0.000 description 3
- 210000001744 T-lymphocyte Anatomy 0.000 description 3
- 230000008901 benefit Effects 0.000 description 3
- 238000010586 diagram Methods 0.000 description 3
- 239000012153 distilled water Substances 0.000 description 3
- 238000002156 mixing Methods 0.000 description 3
- 210000004881 tumor cell Anatomy 0.000 description 3
- FWBHETKCLVMNFS-UHFFFAOYSA-N 4',6-Diamino-2-phenylindol Chemical compound C1=CC(C(=N)N)=CC=C1C1=CC2=CC=C(C(N)=N)C=C2N1 FWBHETKCLVMNFS-UHFFFAOYSA-N 0.000 description 2
- 206010006187 Breast cancer Diseases 0.000 description 2
- 208000026310 Breast neoplasm Diseases 0.000 description 2
- 229940076838 Immune checkpoint inhibitor Drugs 0.000 description 2
- 108091008026 Inhibitory immune checkpoint proteins Proteins 0.000 description 2
- 102000037984 Inhibitory immune checkpoint proteins Human genes 0.000 description 2
- CDBYLPFSWZWCQE-UHFFFAOYSA-L Sodium Carbonate Chemical compound [Na+].[Na+].[O-]C([O-])=O CDBYLPFSWZWCQE-UHFFFAOYSA-L 0.000 description 2
- ORILYTVJVMAKLC-UHFFFAOYSA-N adamantane Chemical compound C1C(C2)CC3CC1CC2C3 ORILYTVJVMAKLC-UHFFFAOYSA-N 0.000 description 2
- 239000011230 binding agent Substances 0.000 description 2
- 238000004140 cleaning Methods 0.000 description 2
- 210000000805 cytoplasm Anatomy 0.000 description 2
- 238000010494 dissociation reaction Methods 0.000 description 2
- 230000005593 dissociations Effects 0.000 description 2
- 230000000694 effects Effects 0.000 description 2
- 238000005516 engineering process Methods 0.000 description 2
- 230000028993 immune response Effects 0.000 description 2
- 239000012274 immune-checkpoint protein inhibitor Substances 0.000 description 2
- 238000010166 immunofluorescence Methods 0.000 description 2
- 238000005259 measurement Methods 0.000 description 2
- 125000001997 phenyl group Chemical group [H]C1=C([H])C([H])=C(*)C([H])=C1[H] 0.000 description 2
- 230000035755 proliferation Effects 0.000 description 2
- 239000003223 protective agent Substances 0.000 description 2
- 238000011160 research Methods 0.000 description 2
- -1 salt ions Chemical class 0.000 description 2
- 239000002904 solvent Substances 0.000 description 2
- 239000012086 standard solution Substances 0.000 description 2
- 238000012360 testing method Methods 0.000 description 2
- 238000005406 washing Methods 0.000 description 2
- SGHZXLIDFTYFHQ-UHFFFAOYSA-L Brilliant Blue Chemical compound [Na+].[Na+].C=1C=C(C(=C2C=CC(C=C2)=[N+](CC)CC=2C=C(C=CC=2)S([O-])(=O)=O)C=2C(=CC=CC=2)S([O-])(=O)=O)C=CC=1N(CC)CC1=CC=CC(S([O-])(=O)=O)=C1 SGHZXLIDFTYFHQ-UHFFFAOYSA-L 0.000 description 1
- OFOVRTMJTCMPLI-UHFFFAOYSA-N C1(=CC=CC=C1)C12C(C3(CC(CC(C1)C3)C2)OC)=C Chemical class C1(=CC=CC=C1)C12C(C3(CC(CC(C1)C3)C2)OC)=C OFOVRTMJTCMPLI-UHFFFAOYSA-N 0.000 description 1
- 102000008203 CTLA-4 Antigen Human genes 0.000 description 1
- 108010021064 CTLA-4 Antigen Proteins 0.000 description 1
- 229940045513 CTLA4 antagonist Drugs 0.000 description 1
- 102000004127 Cytokines Human genes 0.000 description 1
- 108090000695 Cytokines Proteins 0.000 description 1
- SXRSQZLOMIGNAQ-UHFFFAOYSA-N Glutaraldehyde Chemical compound O=CCCCC=O SXRSQZLOMIGNAQ-UHFFFAOYSA-N 0.000 description 1
- 238000003747 Grignard reaction Methods 0.000 description 1
- 239000007818 Grignard reagent Substances 0.000 description 1
- 101000868279 Homo sapiens Leukocyte surface antigen CD47 Proteins 0.000 description 1
- 101000914484 Homo sapiens T-lymphocyte activation antigen CD80 Proteins 0.000 description 1
- 102100032913 Leukocyte surface antigen CD47 Human genes 0.000 description 1
- 101100519207 Mus musculus Pdcd1 gene Proteins 0.000 description 1
- 101001117316 Mus musculus Programmed cell death 1 ligand 1 Proteins 0.000 description 1
- 241000699670 Mus sp. Species 0.000 description 1
- 101150036449 SIRPA gene Proteins 0.000 description 1
- 108010090804 Streptavidin Proteins 0.000 description 1
- 102100027222 T-lymphocyte activation antigen CD80 Human genes 0.000 description 1
- 230000009471 action Effects 0.000 description 1
- 230000004913 activation Effects 0.000 description 1
- VGBBJZYOAOCLLS-UHFFFAOYSA-N adamantane-2-carbaldehyde Chemical compound C1C(C2)CC3CC1C(C=O)C2C3 VGBBJZYOAOCLLS-UHFFFAOYSA-N 0.000 description 1
- 238000005054 agglomeration Methods 0.000 description 1
- 230000002776 aggregation Effects 0.000 description 1
- 238000005804 alkylation reaction Methods 0.000 description 1
- 238000003556 assay Methods 0.000 description 1
- 210000003719 b-lymphocyte Anatomy 0.000 description 1
- 230000009286 beneficial effect Effects 0.000 description 1
- 239000006177 biological buffer Substances 0.000 description 1
- 230000000903 blocking effect Effects 0.000 description 1
- 239000011203 carbon fibre reinforced carbon Substances 0.000 description 1
- 238000006555 catalytic reaction Methods 0.000 description 1
- 210000000170 cell membrane Anatomy 0.000 description 1
- 238000002659 cell therapy Methods 0.000 description 1
- 238000002038 chemiluminescence detection Methods 0.000 description 1
- 238000000975 co-precipitation Methods 0.000 description 1
- 239000011248 coating agent Substances 0.000 description 1
- 238000000576 coating method Methods 0.000 description 1
- 150000001875 compounds Chemical class 0.000 description 1
- 210000004443 dendritic cell Anatomy 0.000 description 1
- 238000010511 deprotection reaction Methods 0.000 description 1
- 238000000502 dialysis Methods 0.000 description 1
- 238000010790 dilution Methods 0.000 description 1
- 239000012895 dilution Substances 0.000 description 1
- UJMYYOYPZCSYDQ-UHFFFAOYSA-N ethanesulfonic acid morpholine hydrate Chemical compound O.C(C)S(=O)(=O)O.N1CCOCC1 UJMYYOYPZCSYDQ-UHFFFAOYSA-N 0.000 description 1
- 230000017188 evasion or tolerance of host immune response Effects 0.000 description 1
- 238000002474 experimental method Methods 0.000 description 1
- 238000011049 filling Methods 0.000 description 1
- 239000010419 fine particle Substances 0.000 description 1
- 150000004795 grignard reagents Chemical class 0.000 description 1
- 229940126533 immune checkpoint blocker Drugs 0.000 description 1
- 230000001900 immune effect Effects 0.000 description 1
- 230000036039 immunity Effects 0.000 description 1
- 238000009169 immunotherapy Methods 0.000 description 1
- 230000006872 improvement Effects 0.000 description 1
- 238000011534 incubation Methods 0.000 description 1
- 239000003446 ligand Substances 0.000 description 1
- 239000007788 liquid Substances 0.000 description 1
- 239000000696 magnetic material Substances 0.000 description 1
- 230000014759 maintenance of location Effects 0.000 description 1
- 210000004216 mammary stem cell Anatomy 0.000 description 1
- 238000004949 mass spectrometry Methods 0.000 description 1
- 239000012528 membrane Substances 0.000 description 1
- 210000002901 mesenchymal stem cell Anatomy 0.000 description 1
- 238000012986 modification Methods 0.000 description 1
- 230000004048 modification Effects 0.000 description 1
- 210000001616 monocyte Anatomy 0.000 description 1
- 210000000822 natural killer cell Anatomy 0.000 description 1
- 230000010355 oscillation Effects 0.000 description 1
- 230000003647 oxidation Effects 0.000 description 1
- 238000007254 oxidation reaction Methods 0.000 description 1
- 239000002245 particle Substances 0.000 description 1
- 238000007539 photo-oxidation reaction Methods 0.000 description 1
- 238000004321 preservation Methods 0.000 description 1
- 238000011002 quantification Methods 0.000 description 1
- 230000028327 secretion Effects 0.000 description 1
- 230000019491 signal transduction Effects 0.000 description 1
- 229910000033 sodium borohydride Inorganic materials 0.000 description 1
- 239000012279 sodium borohydride Substances 0.000 description 1
- 229910000029 sodium carbonate Inorganic materials 0.000 description 1
- 238000013112 stability test Methods 0.000 description 1
- 238000003860 storage Methods 0.000 description 1
- 238000006467 substitution reaction Methods 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
- G01N33/574—Immunoassay; Biospecific binding assay; Materials therefor for cancer
-
- 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
- G01N33/68—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids
- G01N33/6872—Intracellular protein regulatory factors and their receptors, e.g. including ion channels
Landscapes
- Health & Medical Sciences (AREA)
- Life Sciences & Earth Sciences (AREA)
- Immunology (AREA)
- Engineering & Computer Science (AREA)
- Chemical & Material Sciences (AREA)
- Molecular Biology (AREA)
- Urology & Nephrology (AREA)
- Biomedical Technology (AREA)
- Hematology (AREA)
- Physics & Mathematics (AREA)
- General Physics & Mathematics (AREA)
- General Health & Medical Sciences (AREA)
- Pathology (AREA)
- Biochemistry (AREA)
- Cell Biology (AREA)
- Analytical Chemistry (AREA)
- Medicinal Chemistry (AREA)
- Microbiology (AREA)
- Biotechnology (AREA)
- Food Science & Technology (AREA)
- Chemical Kinetics & Catalysis (AREA)
- Plasma & Fusion (AREA)
- Proteomics, Peptides & Aminoacids (AREA)
- Hospice & Palliative Care (AREA)
- Oncology (AREA)
- Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)
Abstract
本发明公开了一种定量检测PD‑L1与PD‑1结合物含量的试剂盒,包括磁微粒试剂、酶标二抗试剂、化学发光底物;所述磁微粒试剂包括链亲和素‑磁性微粒与生物素标记的PD L1抗体的结合物,标记为M‑SA/Bio‑Anti‑PD L1;所述酶标二抗试剂包括碱性磷酸酶与PD 1抗体偶联结合物,标记为AP‑Anti‑PD 1;所述化学发光底物包括:3‑(2‑螺旋金刚烷)‑4‑甲氧基‑4‑(3‑磷氧酰)‑苯基‑1,2‑二氧环乙烷二钠盐。采用本发明提供的试剂盒进行检测,完成所有流程出结果时间为20min,相比免疫共沉淀,免疫共定位缩短了反应时间;且具有较高的灵敏度、重复性和稳定性。
Description
技术领域
本发明涉及检验技术领域,具体涉及一种定量检测PD-L1与PD-1结合物含量的试剂盒。
背景技术
目前肿瘤免疫疗法的主要两大领域分别是:细胞治疗、以及以PD-1/PD-L1、 CTLA-4/CD80,CD47/SIRPα为代表的免疫检查点抑制剂。程序性死亡受体(Programmed CellDeath-1,PD-1)主要表达在活化的T/B淋巴细胞、NK细胞、单核细胞、树突状细胞以及间充质干细胞膜以及胞质内,以T细胞为例,PD-1可与其配体PD-L1(高表达在肿瘤细胞中) 结合从而抑制T细胞的活化、增殖和细胞因子的分泌,负性调控免疫应答,使肿瘤细胞发生免疫逃逸现象。免疫检查点抑制剂通过阻断PD-1/PD-L1信号通路,可重新激活T细胞,增强免疫应答反应,从而抑制肿瘤细胞的增殖等。
免疫检查点阻断剂的联用被广泛推荐,这也说明了PD-1/PD-L1在肿瘤治疗中的重要性,目前科研实验都在小鼠上注射PD-1抗体或者PD-L1抗体,然后通过检测PD-1和PD-L1结合物的含量来判定疗效,由于现有检测结合物的方法例如免疫共沉淀、免疫共定位、蛋白质谱等方法均不能准确定量,因此急需一种能检测小鼠PD-1与PD-L1结合物含量的定量检测方法。
发明内容
本发明所要解决的技术问题是:,本发明提供了解决上述问题的一种定量检测PD-L1与 PD-1结合物含量的试剂盒,基于磁微粒化学发光检测原理来实现PD-L1与PD-1结合物的定量检测。
本发明通过下述技术方案实现:
一种定量检测PD-L1与PD-1结合物含量的试剂盒,包括磁微粒试剂、酶标二抗试剂、化学发光底物;所述磁微粒试剂包括链亲和素-磁性微粒与生物素标记的PD L1抗体的结合物,标记为M-SA/Bio-Anti-PD L1;所述酶标二抗试剂包括碱性磷酸酶与PD 1抗体偶联结合物,标记为AP-Anti-PD 1;所述化学发光底物包括:3-(2-螺旋金刚烷)-4-甲氧基-4-(3-磷氧酰)- 苯基-1,2-二氧环乙烷二钠盐。
传统的双抗夹心法只检测样本中的一种物质,例如检测样本中的PD-L1,图1磁微粒上标记的是PD-L1抗体1以及碱性磷酸酶上标记的是PD-L1的抗体2,PD-L1的抗体1和抗体2均属于同一种但是有着不同结构域的抗体。本发明提供的试剂盒的,如图2所示,可以同时检测样本里的两种不同物质,并且可以定量这两种结合物的含量。与传统的双抗夹心法有很大的区别。
进一步优选,所述磁性微粒的直径为500nm~2500nm,具有超顺磁性。
进一步优选,所述磁微粒试剂中结合物的M-SA/Bio-Anti-PD L1的浓度为 0.05μg/mL-5μg/mL。
具体地,所述磁微粒试剂中结合物的M-SA/Bio-Anti-PD L1是包被有PD-L1抗体磁性微粒和缓冲液配制而成,其中PD-L1抗体是Biotin标记的PD-L1抗体。M-SA/Bio-Anti-PDL1 的浓度为0.05μg/mL-5μg/mL。其缓冲液包括:MES缓冲液,中文名一水吗啉乙磺酸;主要用于生物缓冲剂;以及PBS-磷酸盐缓冲液(phoshpate buffer saline),一般作为溶剂,起溶解保护试剂的作用,它是生物化学研究中使用最为广泛的一种缓冲液,主要成分为Na2HPO4、 KH2PO4、NaCl和KCl,由于Na2HPO4、KH2PO4它们有二级解离,缓冲的pH值范围很广,而NaCl和KCl主要作用为增加盐离子浓度;还有浓度为0.01M-0.1M的三羟甲基氨基甲烷(Tris);所述三羟甲基氨基甲烷(Tris)中含有0.1M-5mM的Mg2+、0.01M-1mM的Zn2+、质量分数为0.5%-5%的牛血清白蛋白(BSA),质量分数为0.05%-0.5%的吐温20(Tween-20)、质量分数为0.5%-5%的海藻糖和质量分数为0.5%-5%的丙三醇。
进一步优选,所述酶标二抗试剂中,结合物AP-Anti-PD 1的浓度为0.05μg/mL-1.5μg/mL。具体地,合物AP-Anti-PD 1是由碱性磷酸酶标记的PD-1抗体以及缓冲液配制而成;其中 AP-Anti-PD 1的浓度为0.05μg/mL-1.5μg/mL。其缓冲液包括:PBS-磷酸盐缓冲液(phoshpate buffer saline),一般作为溶剂,起溶解保护试剂的作用,它是生物化学研究中使用最为广泛的一种缓冲液,主要成分为Na2HPO4、KH2PO4、NaCl和KCl,由于Na2HPO4、KH2PO4它们有二级解离,缓冲的pH值范围很广,而NaCl和KCl主要作用为增加盐离子浓度。
进一步优选,还包括化学发光底物,所述化学发光底物包括3-(2-螺旋金刚烷)-4-甲氧基 -4-(3-磷氧酰)-苯基-1,2-二氧环乙烷二钠盐。本发明(AP)为碱性磷酸酶;(SA)为链酶亲合素。化学发光底物(AMPPD)配制方法为:金刚烷-2-醛和取代苯基的格氏试剂经格氏反应、氧化制备取代苯基金刚烷-2-酮中间体,该中间体再经氧-烷基化构造碳-碳双键,酚羟基脱保护两步反应得到关键中间体取代苯基甲氧基甲叉基金刚烷,然后通过常规的光氧化反应得到 AMPPD。
进一步优选,还包括校准品和质控品;所述校准品包括PD-1和PD-L1融合蛋白,所述质控品包括PD-1和PD-L1融合蛋白。
具体地,所述校准品,构建的PD-1和PD-L1融合蛋白,用于校准曲线。校准品的制备方法为:构建的PD-1和PD-L1融合蛋白,用浓度为0.01M-0.1M的三羟甲基氨基甲烷(Tris)缓冲液稀释,分别制备成400ng/mL、200ng/mL、50ng/mL、20ng/mL、5ng/mL、0ng/mL的梯度标准液;所述三羟甲基氨基甲烷(Tris)中含有0.1M-5mM的Mg2+、0.01M-1mM的Zn2+、质量分数为0.5%-5%的牛血清白蛋白(BSA),质量分数为0.05%-0.5%的吐温20(Tween-20)、质量分数为0.5%-5%的海藻糖和质量分数为0.5%-5%的丙三醇。
所述质控品,构建的PD-1和PD-L1融合蛋白。构建的PD-1和PD-L1融合蛋白,然后用浓度为0.01M-0.1M的三羟甲基氨基甲烷(Tris);所述三羟甲基氨基甲烷(Tris)中含有0.1M-5mM的Mg2+、0.01M-1mM的Zn2+、质量分数为0.5%-5%的牛血清白蛋白(BSA),质量分数为0.05%-0.5%的吐温20(Tween-20)、质量分数为0.5%-5%的海藻糖和质量分数为0.5%-5%的丙三醇。
本发明具有如下的优点和有益效果:
1.采用本发明提供的试剂盒进行检测,完成所有流程出结果时间为20min,相比免疫共沉淀,免疫共定位缩短了反应时间;
2.本发明采用碱性磷酸酶(AP)-金刚烷(AMPPD)***,灵敏度高;
3.本发明为全自动封闭操作***,可靠性高,稳定性好,检测结果重复性好;
4.本发明从稀释、加样、孵育、清洗以及检测步骤实现了全自动化,避免了人为操作带来的结果偏差。
附图说明
此处所说明的附图用来提供对本发明实施例的进一步理解,构成本申请的一部分,并不构成对本发明实施例的限定。在附图中:
图1为传统的双抗夹心法的检测原理;传统的双抗夹心法只检测样本中的一种物质,例如检测样本中的PD-L1,图1磁微粒上标记的是PD-L1抗体1以及碱性磷酸酶上标记的是 PD-L1的抗体2,PD-L1的抗体1和抗体2均属于同一种但是有着不同结构域的抗体。
图2为本发明试剂盒的检测原理;可以同时检测样本里的两种不同物质,并且可以定量这两种结合物的含量。与传统的双抗夹心法有很大的区别。
图3为现有免疫荧光检测结果;图中PD-1,是绿色荧光图,表示PD-1的表达;图中PD-L1,是红色荧光图,表示PD-L1的表达;图中DAPI,是蓝色荧光图,表示DAPI染的细胞核;图中Merge,是红色荧光、绿色荧光以及蓝色荧光的叠加合图;红色荧光和绿色荧光表明PD-L1 和PD-1在小鼠乳腺细胞的细胞膜和胞质内表达,并且通过合图的橙色看出红色和绿色有荧光重叠,也说明了PD-1和PD-L1的结合。但是此方法无法检测PD-1和PD-L1结合物的含量,但是本发明的试剂盒可以检测。
图4为基于校准品绘制的标准曲线。
图5为检测对照图;表示加入dd H2O未检测出,加入含有PD-1和PD-L1的样本检测出。
图6为稳定性检测结果图。
具体实施方式
为使本发明的目的、技术方案和优点更加清楚明白,下面结合实施例和附图,对本发明作进一步的详细说明,本发明的示意性实施方式及其说明仅用于解释本发明,并不作为对本发明的限定。
实施例1
本实施例提供了一种定量检测PD-L1与PD-1结合物含量的试剂盒,包括磁微粒试剂、酶标二抗试剂、化学发光底物、清洗浓缩液、校准品和质控品;
所述磁微粒试剂为链亲和素-磁性微粒与生物素标记的PD L1抗体的结合物,标记为 M-SA/Bio-Anti-PD L1。具体地,配制前,选取直径为500nm的磁微粒,具有超顺磁性;利用已有成熟的偶联技术,将生物素(Biotin)与抗体PD L1(Anti-PD L1)进行偶联;将生物素标记的Anti-PD L1与链亲和素-磁性微粒(SA-M)进行结合,最终获得一种包被有Anti-PDL1 的磁微粒(M-SA/Bio-Anti-PD L1),磁微粒试剂中结合物M-SA/Bio-Anti-PD L1的浓度为 5μg/mL。
所述酶标二抗试剂为碱性磷酸酶与PD 1抗体偶联结合物,标记为AP-Anti-PD 1。具体地,利用已有成熟的偶联技术,将碱性磷酸酶(AP)与PD 1抗体(Anti-PD 1)进行偶联;最终获得一种酶标记Anti-PD 1的R2试剂,酶标二抗试剂中,结合物AP-Anti-PD 1的浓度为1.5μg/mL;
所述化学发光底物包括:3-(2-螺旋金刚烷)-4-甲氧基-4-(3-磷氧酰)-苯基-1,2-二氧环乙烷二钠盐,主要成分为金刚烷(AMPPD),在碱性磷酸酶的催化下释放光子。
所述校准品,构建的PD-1和PD-L1融合蛋白,用于校准曲线。校准品的制备方法为:构建的PD-1和PD-L1融合蛋白,用浓度为0.01M-0.1M的三羟甲基氨基甲烷(Tris)缓冲液稀释,分别制备成400ng/mL、200ng/mL、50ng/mL、20ng/mL、5ng/mL、0ng/mL的梯度标准液;所述三羟甲基氨基甲烷(Tris)中含有2mM的Mg2+、0.1mM的Zn2+、质量分数为3%的牛血清白蛋白(BSA),质量分数为0.09%的吐温20(Tween-20)、质量分数为1%的海藻糖和质量分数为2%的丙三醇。
所述质控品,构建的PD-1和PD-L1融合蛋白。构建的PD-1和PD-L1融合蛋白,然后用浓度为0.01M-0.1M的三羟甲基氨基甲烷(Tris);所述三羟甲基氨基甲烷(Tris)中中含有2mM的Mg2+、0.1mM的Zn2+、质量分数为3%的牛血清白蛋白(BSA),质量分数为0.09%的吐温20(Tween-20)、质量分数为1%的海藻糖和质量分数为2%的丙三醇。
本发明采用将生物素(Biotin)标记的PD L1抗体(Anti-PD L1)与链亲和素-磁性微粒(SA-M)进行结合,最终获得磁微粒M-SA/Bio-Anti-PD L1,然后加入待测样本,最后加入酶标二抗试剂进行反应。
本发明采用将生物素(Biotin)标记的PD L1抗体(Anti-PD L1)与链亲和素-磁性微粒 (SA-M)进行结合,最终获得磁微粒M-SA/Bio-Anti-PD L1,然后加入待测样本,最后加入酶标二抗试剂进行反应。
实施例2
本实施例以小鼠乳腺癌(4T1)肿瘤组织样本为例,取出小鼠的肿瘤组织,再将全蛋白提取出来,然后使用本发明检测方法对其进行后续的检测,具体步骤如下:
1.免疫磁珠制备:通过化学共沉淀方法制备,选用粒径为500nm的棕色圆形磁性微粒,用0.05mol/L的MES缓冲液进行清洗,之后再加入1.25%的戊二醛和0.05mol/L PH6.0的PBS 进行振荡反应1h,然后再用0.05mol/L的MES缓冲液进行清洗,得到活化磁珠;再吸取4mg 的PD-L1抗体于1.5ml离心管中,加入10mMBNHS 53.2ul,混匀,至室温放置反应30min。加入1M的Tris缓冲液至最终浓度为10mM,终止反应,室温放置反应10min,取出标记好的生物素抗体并测量体积,加等体积甘油-20℃保存。取出活化磁珠与PD-L1抗体,以1mg磁珠,250μg的抗体比例在25℃的条件下孵育1h,磁性分离后再加入2%的BSA封闭游离基, MES再次清洗得到免疫磁珠;
2.酶标抗体的制备:取出1mgHRP溶于0.5mL蒸馏水中,充分溶解,取0.125mL进行标记,加入新鲜配制的12mmol/L的过碘酸钠蒸馏水溶液0.125mL,混匀,2-8℃放置30min,加入32mmol/L的乙二醇蒸馏水溶液0.125mL中,混匀,室温避光放置30min以终止过碘酸钠的作用,将0.25mg的PD-1抗体加入上述溶液中,混匀,装入透析袋用0.05mol/L碳酸钠缓冲液(PH为9.5)2-8℃透析16-24h。最后将溶液取出,加入硼氢化钠溶液放置2h,用 0.02mol/LPH7.4的PBS溶液透析,最后取出离心,测量体积加等体积的甘油保存;
3.小鼠乳腺癌肿瘤组织中的PD-1和PD-L1结合物的检测:取出肿瘤组织,提取全蛋白,然后利用卡马斯亮蓝进行蛋白定量,每组都取出相同量的蛋白,然后加入已经包被PD-L1 抗体浓度为0.05μg/mL的磁微粒,再加入浓度为0.05μg/mL酶标的PD-1。
结果分析:磁微粒经过活化离心,偶联抗体后,可以在溶液中分散均匀,不团聚。在测定结合物含量之前通过免疫荧光对PD-1和PD-L1的结合进行了辅助检测,如图2所示合图的橙色荧光说明了免疫共定位,也就是PD-1和PD-L1有结合,并且通过本发明的试剂盒可以检测出PD-1和PD-L1结合的含量,如图5所示,加入dd H2O未检测出,而加入还有PD-1 和PD-L1的样本检测出的平均浓度为15.64ng/ml。
实施例3
本实施例对实施例2的检测结果进行效果测试。
1、精密度测试:分别对400ng/mL、50ng/mL、5ng/mL的质控品进行测定,每天两次,每次进行4个重复,共检测10天,每种浓度共测定80次,计算变异系数,检测试剂在 400ng/mL、50ng/mL、5ng/mL标本时的测定精密度。结果表明变异系数都在8%以内,重复性好。
表1精密度测试结果
2、稳定性:本发明的检测试剂盒(M,R2)37℃分别放置7天,测定400ng/mL、50ng/mL、 5ng/mL这三种浓度质控的信号保留率。如图6结果所示均>95%,表明试剂盒稳定好。
以上所述的具体实施方式,对本发明的目的、技术方案和有益效果进行了进一步详细说明,所应理解的是,以上所述仅为本发明的具体实施方式而已,并不用于限定本发明的保护范围,凡在本发明的精神和原则之内,所做的任何修改、等同替换、改进等,均应包含在本发明的保护范围之内。
Claims (6)
1.一种定量检测PD-L1与PD-1结合物含量的试剂盒,其特征在于,包括磁微粒试剂、酶标二抗试剂、化学发光底物;
所述磁微粒试剂包括链亲和素-磁性微粒与生物素标记的PD L1抗体的结合物,标记为M-SA/Bio-Anti-PD L1;
所述酶标二抗试剂包括碱性磷酸酶与PD 1抗体偶联结合物,标记为AP-Anti-PD 1;
所述化学发光底物包括:3-(2-螺旋金刚烷)-4-甲氧基-4-(3-磷氧酰)-苯基-1,2-二氧环乙烷二钠盐。
2.根据权利要求1所述的一种定量检测PD-L1与PD-1结合物含量的试剂盒,其特征在于,所述磁性微粒的直径为500nm~2500nm,具有超顺磁性。
3.根据权利要求1所述的一种定量检测PD-L1与PD-1结合物含量的试剂盒,其特征在于,所述磁微粒试剂中结合物的M-SA/Bio-Anti-PD L1的浓度为0.05μg/mL-5μg/mL。
4.根据权利要求1所述的一种定量检测PD-L1与PD-1结合物含量的试剂盒,其特征在于,所述酶标二抗试剂中,结合物AP-Anti-PD 1的浓度为0.05μg/mL-1.5μg/mL。
5.根据权利要求1所述的一种定量检测PD-L1与PD-1结合物含量的试剂盒,其特征在于还包括化学发光底物,所述化学发光底物包括3-(2-螺旋金刚烷)-4-甲氧基-4-(3-磷氧酰)-苯基-1,2-二氧环乙烷二钠盐。
6.根据权利要求1所述的一种定量检测PD-L1与PD-1结合物含量的试剂盒,其特征在于,还包括校准品和质控品;所述校准品包括PD-1和PD-L1融合蛋白,所述质控品包括PD-1和PD-L1融合蛋白。
Priority Applications (1)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
CN202010716591.8A CN111812326A (zh) | 2020-07-23 | 2020-07-23 | 一种定量检测pd-l1与pd-1结合物含量的试剂盒 |
Applications Claiming Priority (1)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
CN202010716591.8A CN111812326A (zh) | 2020-07-23 | 2020-07-23 | 一种定量检测pd-l1与pd-1结合物含量的试剂盒 |
Publications (1)
Publication Number | Publication Date |
---|---|
CN111812326A true CN111812326A (zh) | 2020-10-23 |
Family
ID=72862324
Family Applications (1)
Application Number | Title | Priority Date | Filing Date |
---|---|---|---|
CN202010716591.8A Pending CN111812326A (zh) | 2020-07-23 | 2020-07-23 | 一种定量检测pd-l1与pd-1结合物含量的试剂盒 |
Country Status (1)
Country | Link |
---|---|
CN (1) | CN111812326A (zh) |
Citations (13)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
WO1997039132A1 (en) * | 1996-04-16 | 1997-10-23 | University Of Miami | Stabilized preparations of human troponins and modifications thereof, diagnostic assay methods and assay kits |
CN101935354A (zh) * | 2010-07-08 | 2011-01-05 | 温州医学院 | 人心肌肌钙蛋白t/人心肌肌钙蛋白i融合蛋白 |
CN104990912A (zh) * | 2015-06-26 | 2015-10-21 | 苏州浩欧博生物医药有限公司 | 一种碱性磷酸酶的酶促化学发光底物 |
CN105929156A (zh) * | 2016-04-20 | 2016-09-07 | 北京中航赛维生物科技有限公司 | 一种抗双链DNA抗体IgG的磁微粒化学发光定量测定试剂盒及制备和检测方法 |
US20160305947A1 (en) * | 2013-12-10 | 2016-10-20 | Merck Sharp & Dohme Corp. | Immunohistochemical proximity assay for pd-1 positive cells and pd-ligand positive cells in tumor tissue |
WO2017050855A1 (en) * | 2015-09-22 | 2017-03-30 | Institut Gustave Roussy | A scoring method for predicting the efficiency of a treatment with anti-pd-1 and/or anti-pd-l1 monoclonal antibodies |
CN107192817A (zh) * | 2017-05-22 | 2017-09-22 | 广州华弘生物科技有限公司 | 一种用于检测pd‑l1的快速检测试剂盒及其检测方法 |
CN107656071A (zh) * | 2017-11-17 | 2018-02-02 | 南通伊仕生物技术股份有限公司 | 一种NT‑ProBNP检测试剂盒及其使用方法 |
CN108593907A (zh) * | 2018-05-21 | 2018-09-28 | 苏州佑君环境科技有限公司 | 一种羧基化磁性粒子的稀释液及其制备方法 |
CN109613248A (zh) * | 2018-12-26 | 2019-04-12 | 郑州安图生物工程股份有限公司 | 检测脂蛋白磷脂酶a2蛋白浓度的试剂盒 |
CN109781999A (zh) * | 2019-02-27 | 2019-05-21 | 华中科技大学 | 一种pd-l1外泌体的磁免疫化学发光检测方法 |
WO2019235420A1 (ja) * | 2018-06-04 | 2019-12-12 | 中外製薬株式会社 | 複合体を検出する方法 |
CN110836973A (zh) * | 2019-09-09 | 2020-02-25 | 浙江博实生物科技有限公司 | 检测肌钙蛋白及复合物的双标记试剂盒及制备与检测方法 |
-
2020
- 2020-07-23 CN CN202010716591.8A patent/CN111812326A/zh active Pending
Patent Citations (13)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
WO1997039132A1 (en) * | 1996-04-16 | 1997-10-23 | University Of Miami | Stabilized preparations of human troponins and modifications thereof, diagnostic assay methods and assay kits |
CN101935354A (zh) * | 2010-07-08 | 2011-01-05 | 温州医学院 | 人心肌肌钙蛋白t/人心肌肌钙蛋白i融合蛋白 |
US20160305947A1 (en) * | 2013-12-10 | 2016-10-20 | Merck Sharp & Dohme Corp. | Immunohistochemical proximity assay for pd-1 positive cells and pd-ligand positive cells in tumor tissue |
CN104990912A (zh) * | 2015-06-26 | 2015-10-21 | 苏州浩欧博生物医药有限公司 | 一种碱性磷酸酶的酶促化学发光底物 |
WO2017050855A1 (en) * | 2015-09-22 | 2017-03-30 | Institut Gustave Roussy | A scoring method for predicting the efficiency of a treatment with anti-pd-1 and/or anti-pd-l1 monoclonal antibodies |
CN105929156A (zh) * | 2016-04-20 | 2016-09-07 | 北京中航赛维生物科技有限公司 | 一种抗双链DNA抗体IgG的磁微粒化学发光定量测定试剂盒及制备和检测方法 |
CN107192817A (zh) * | 2017-05-22 | 2017-09-22 | 广州华弘生物科技有限公司 | 一种用于检测pd‑l1的快速检测试剂盒及其检测方法 |
CN107656071A (zh) * | 2017-11-17 | 2018-02-02 | 南通伊仕生物技术股份有限公司 | 一种NT‑ProBNP检测试剂盒及其使用方法 |
CN108593907A (zh) * | 2018-05-21 | 2018-09-28 | 苏州佑君环境科技有限公司 | 一种羧基化磁性粒子的稀释液及其制备方法 |
WO2019235420A1 (ja) * | 2018-06-04 | 2019-12-12 | 中外製薬株式会社 | 複合体を検出する方法 |
CN109613248A (zh) * | 2018-12-26 | 2019-04-12 | 郑州安图生物工程股份有限公司 | 检测脂蛋白磷脂酶a2蛋白浓度的试剂盒 |
CN109781999A (zh) * | 2019-02-27 | 2019-05-21 | 华中科技大学 | 一种pd-l1外泌体的磁免疫化学发光检测方法 |
CN110836973A (zh) * | 2019-09-09 | 2020-02-25 | 浙江博实生物科技有限公司 | 检测肌钙蛋白及复合物的双标记试剂盒及制备与检测方法 |
Non-Patent Citations (1)
Title |
---|
龚隆财等: "cTnI-linker-TnC融合蛋白的原核表达及鉴定", 《中国生物工程杂志》 * |
Similar Documents
Publication | Publication Date | Title |
---|---|---|
CN100464189C (zh) | 磁分离直接化学发光试剂及用该试剂的测试方法 | |
EP2574926B1 (en) | Chromatographic kit and chromatography method | |
JPH0562302B2 (zh) | ||
EP3258266B1 (en) | Reagent kit used for detecting gastrin-17, and preparation method and application for reagent kit | |
US20130011827A1 (en) | Methods and kits for decreasing interferences in plasma or serum containing assay samples of specific binding assays | |
CN111024956A (zh) | 一种检测ptx3的时间分辨荧光免疫层析试剂盒 | |
WO2021258618A1 (zh) | 一种生物样品检测方法及检测试剂盒 | |
CN106771239A (zh) | 血清淀粉样蛋白a/降钙素原/c‑反应蛋白三合一测定试剂盒及制法 | |
CN111579781A (zh) | 丙型肝炎病毒抗体检测试剂盒、制备方法及检测方法 | |
CN110044879A (zh) | 一种cyfra21-1磁微粒化学发光免疫定量检测试剂盒及其制备方法 | |
CN104515855A (zh) | 检测Galectin-3的纳米磁珠分选-时间分辨免疫荧光试剂盒 | |
CN109142753A (zh) | 鳞状上皮细胞癌抗原化学发光免疫检测试剂盒及其制备方法 | |
JPH0792460B2 (ja) | 抽出組成物として界面活性剤混合物を使用する歯周病に随伴する微生物検出用キットおよびその検出方法 | |
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 | |
Huang et al. | Receptor antibody time‐resolved A2 phospholipase bead immunochromatography and its application in idiopathic membranous nephropathy | |
CN111579801A (zh) | 用于抗缪勒氏管激素含量检测的单人份试剂盒及其检测方法 | |
CN111812326A (zh) | 一种定量检测pd-l1与pd-1结合物含量的试剂盒 | |
CN110618280A (zh) | 一种促甲状腺素测定试剂盒及其制备方法 | |
JPS63501819A (ja) | イムノアッセイ法およびキット | |
CN111024940B (zh) | 一种基于金磁微粒的时间分辨荧光免疫检测方法 | |
CN110780079A (zh) | 一种鳞状上皮细胞癌抗原检测试剂 | |
CN113109558A (zh) | 一种定量检测cyfra21-1的磁微粒化学发光试剂盒及其制作方法 | |
CN106596524A (zh) | 一种胰岛素抗体化学发光免疫检测试剂盒及其制备方法 | |
CN106980020A (zh) | 降钙素原和c‑反应蛋白二合一测定试剂盒及制法 | |
CN115541873B (zh) | 测定三联吡啶钌浓度的方法和组合物 |
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 | ||
RJ01 | Rejection of invention patent application after publication |
Application publication date: 20201023 |
|
RJ01 | Rejection of invention patent application after publication |