CN116718777A - 一种plgf检测试剂盒的制备及应用 - Google Patents
一种plgf检测试剂盒的制备及应用 Download PDFInfo
- Publication number
- CN116718777A CN116718777A CN202310628224.6A CN202310628224A CN116718777A CN 116718777 A CN116718777 A CN 116718777A CN 202310628224 A CN202310628224 A CN 202310628224A CN 116718777 A CN116718777 A CN 116718777A
- Authority
- CN
- China
- Prior art keywords
- plgf
- antibody
- kit
- solution
- detection kit
- 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.)
- Granted
Links
- 238000001514 detection method Methods 0.000 title claims abstract description 33
- 238000002360 preparation method Methods 0.000 title claims abstract description 15
- 101150062285 PGF gene Proteins 0.000 title claims abstract 23
- 102100035194 Placenta growth factor Human genes 0.000 title claims abstract 23
- 239000011324 bead Substances 0.000 claims abstract description 31
- 239000003085 diluting agent Substances 0.000 claims abstract description 15
- 238000003908 quality control method Methods 0.000 claims abstract description 15
- KQFCNGKUXYNDPF-UHFFFAOYSA-N 3-[9-[[4-(2,5-dioxopyrrolidin-1-yl)oxy-4-oxobutyl]-(4-methylphenyl)sulfonylcarbamoyl]acridin-10-ium-10-yl]propane-1-sulfonate Chemical compound C1=CC(C)=CC=C1S(=O)(=O)N(C(=O)C=1C2=CC=CC=C2[N+](CCCS([O-])(=O)=O)=C2C=CC=CC2=1)CCCC(=O)ON1C(=O)CCC1=O KQFCNGKUXYNDPF-UHFFFAOYSA-N 0.000 claims abstract description 10
- 239000012224 working solution Substances 0.000 claims abstract description 8
- 201000011461 pre-eclampsia Diseases 0.000 claims description 19
- 239000000872 buffer Substances 0.000 claims description 12
- 239000000243 solution Substances 0.000 claims description 11
- 239000007853 buffer solution Substances 0.000 claims description 8
- 238000000034 method Methods 0.000 claims description 8
- QAPSNMNOIOSXSQ-YNEHKIRRSA-N 1-[(2r,4s,5r)-4-[tert-butyl(dimethyl)silyl]oxy-5-(hydroxymethyl)oxolan-2-yl]-5-methylpyrimidine-2,4-dione Chemical compound O=C1NC(=O)C(C)=CN1[C@@H]1O[C@H](CO)[C@@H](O[Si](C)(C)C(C)(C)C)C1 QAPSNMNOIOSXSQ-YNEHKIRRSA-N 0.000 claims description 7
- 238000006243 chemical reaction Methods 0.000 claims description 7
- KDXKERNSBIXSRK-UHFFFAOYSA-N lysine Chemical compound NCCCCC(N)C(O)=O KDXKERNSBIXSRK-UHFFFAOYSA-N 0.000 claims description 6
- 239000000725 suspension Substances 0.000 claims description 6
- 239000000463 material Substances 0.000 claims description 4
- 239000010413 mother solution Substances 0.000 claims description 4
- 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 4
- BTBUEUYNUDRHOZ-UHFFFAOYSA-N Borate Chemical compound [O-]B([O-])[O-] BTBUEUYNUDRHOZ-UHFFFAOYSA-N 0.000 claims description 3
- 229920005654 Sephadex Polymers 0.000 claims description 3
- 239000012507 Sephadex™ Substances 0.000 claims description 3
- 239000011248 coating agent Substances 0.000 claims description 3
- 238000000576 coating method Methods 0.000 claims description 3
- 239000012895 dilution Substances 0.000 claims description 3
- 238000010790 dilution Methods 0.000 claims description 3
- 239000000203 mixture Substances 0.000 claims description 3
- 239000003755 preservative agent Substances 0.000 claims description 3
- 230000002335 preservative effect Effects 0.000 claims description 3
- 238000003756 stirring Methods 0.000 claims description 3
- 238000011033 desalting Methods 0.000 claims description 2
- 239000012149 elution buffer Substances 0.000 claims description 2
- 238000002372 labelling Methods 0.000 claims description 2
- 239000000126 substance Substances 0.000 claims description 2
- 238000003149 assay kit Methods 0.000 claims 2
- 239000012530 fluid Substances 0.000 claims 1
- 230000035945 sensitivity Effects 0.000 abstract description 8
- 150000001251 acridines Chemical class 0.000 abstract description 2
- 239000003607 modifier Substances 0.000 abstract description 2
- 238000004321 preservation Methods 0.000 abstract description 2
- 230000035484 reaction time Effects 0.000 abstract description 2
- 108010082093 Placenta Growth Factor Proteins 0.000 description 43
- 102000003666 Placenta Growth Factor Human genes 0.000 description 43
- 239000000523 sample Substances 0.000 description 15
- 239000000427 antigen Substances 0.000 description 13
- 108091007433 antigens Proteins 0.000 description 13
- 102000036639 antigens Human genes 0.000 description 13
- 241000699670 Mus sp. Species 0.000 description 12
- 239000000047 product Substances 0.000 description 11
- 108090000623 proteins and genes Proteins 0.000 description 11
- 210000004027 cell Anatomy 0.000 description 8
- 102000004169 proteins and genes Human genes 0.000 description 8
- 238000012360 testing method Methods 0.000 description 8
- 239000007788 liquid Substances 0.000 description 7
- 230000035935 pregnancy Effects 0.000 description 7
- 206010003445 Ascites Diseases 0.000 description 6
- FAPWRFPIFSIZLT-UHFFFAOYSA-M Sodium chloride Chemical compound [Na+].[Cl-] FAPWRFPIFSIZLT-UHFFFAOYSA-M 0.000 description 6
- 150000001413 amino acids Chemical group 0.000 description 6
- 239000003153 chemical reaction reagent Substances 0.000 description 6
- 230000014509 gene expression Effects 0.000 description 6
- 238000004020 luminiscence type Methods 0.000 description 6
- 239000002671 adjuvant Substances 0.000 description 5
- 230000006698 induction Effects 0.000 description 5
- 239000006228 supernatant Substances 0.000 description 5
- 210000002993 trophoblast Anatomy 0.000 description 5
- QKNYBSVHEMOAJP-UHFFFAOYSA-N 2-amino-2-(hydroxymethyl)propane-1,3-diol;hydron;chloride Chemical compound Cl.OCC(N)(CO)CO QKNYBSVHEMOAJP-UHFFFAOYSA-N 0.000 description 4
- 230000004927 fusion Effects 0.000 description 4
- 230000003053 immunization Effects 0.000 description 4
- 238000004519 manufacturing process Methods 0.000 description 4
- 239000013612 plasmid Substances 0.000 description 4
- 239000012898 sample dilution Substances 0.000 description 4
- QTBSBXVTEAMEQO-UHFFFAOYSA-N Acetic acid Chemical compound CC(O)=O QTBSBXVTEAMEQO-UHFFFAOYSA-N 0.000 description 3
- 241000894006 Bacteria Species 0.000 description 3
- PEDCQBHIVMGVHV-UHFFFAOYSA-N Glycerine Chemical compound OCC(O)CO PEDCQBHIVMGVHV-UHFFFAOYSA-N 0.000 description 3
- 241000699666 Mus <mouse, genus> Species 0.000 description 3
- HEMHJVSKTPXQMS-UHFFFAOYSA-M Sodium hydroxide Chemical compound [OH-].[Na+] HEMHJVSKTPXQMS-UHFFFAOYSA-M 0.000 description 3
- 238000004458 analytical method Methods 0.000 description 3
- 230000036772 blood pressure Effects 0.000 description 3
- 208000037265 diseases, disorders, signs and symptoms Diseases 0.000 description 3
- 238000002649 immunization Methods 0.000 description 3
- 230000006872 improvement Effects 0.000 description 3
- 239000007924 injection Substances 0.000 description 3
- 238000002347 injection Methods 0.000 description 3
- 238000002156 mixing Methods 0.000 description 3
- 238000012986 modification Methods 0.000 description 3
- 230000004048 modification Effects 0.000 description 3
- 238000005457 optimization Methods 0.000 description 3
- 210000002826 placenta Anatomy 0.000 description 3
- 238000012216 screening Methods 0.000 description 3
- 239000011780 sodium chloride Substances 0.000 description 3
- 108020004705 Codon Proteins 0.000 description 2
- 238000002965 ELISA Methods 0.000 description 2
- 241000588724 Escherichia coli Species 0.000 description 2
- 206010020772 Hypertension Diseases 0.000 description 2
- 206010035226 Plasma cell myeloma Diseases 0.000 description 2
- QAOWNCQODCNURD-UHFFFAOYSA-N Sulfuric acid Chemical compound OS(O)(=O)=O QAOWNCQODCNURD-UHFFFAOYSA-N 0.000 description 2
- 230000009471 action Effects 0.000 description 2
- 239000012496 blank sample Substances 0.000 description 2
- 210000004369 blood Anatomy 0.000 description 2
- 239000008280 blood Substances 0.000 description 2
- 239000003795 chemical substances by application Substances 0.000 description 2
- 238000012258 culturing Methods 0.000 description 2
- 230000006378 damage Effects 0.000 description 2
- 238000013461 design Methods 0.000 description 2
- 238000011161 development Methods 0.000 description 2
- 201000010099 disease Diseases 0.000 description 2
- 230000001804 emulsifying effect Effects 0.000 description 2
- 230000005284 excitation Effects 0.000 description 2
- 230000036541 health Effects 0.000 description 2
- 210000004408 hybridoma Anatomy 0.000 description 2
- 210000004698 lymphocyte Anatomy 0.000 description 2
- 238000007885 magnetic separation Methods 0.000 description 2
- 239000002609 medium Substances 0.000 description 2
- 201000000050 myeloid neoplasm Diseases 0.000 description 2
- WWZKQHOCKIZLMA-UHFFFAOYSA-N octanoic acid Chemical compound CCCCCCCC(O)=O WWZKQHOCKIZLMA-UHFFFAOYSA-N 0.000 description 2
- 210000002966 serum Anatomy 0.000 description 2
- 238000007920 subcutaneous administration Methods 0.000 description 2
- 208000024891 symptom Diseases 0.000 description 2
- 230000009885 systemic effect Effects 0.000 description 2
- 210000003556 vascular endothelial cell Anatomy 0.000 description 2
- 238000012795 verification Methods 0.000 description 2
- 238000005406 washing Methods 0.000 description 2
- 108090001008 Avidin Proteins 0.000 description 1
- 238000011725 BALB/c mouse Methods 0.000 description 1
- 241000283707 Capra Species 0.000 description 1
- 108090000790 Enzymes Proteins 0.000 description 1
- 102000004190 Enzymes Human genes 0.000 description 1
- 101000595923 Homo sapiens Placenta growth factor Proteins 0.000 description 1
- 208000000091 Maternal Death Diseases 0.000 description 1
- 206010029113 Neovascularisation Diseases 0.000 description 1
- 229910019142 PO4 Inorganic materials 0.000 description 1
- 229920001030 Polyethylene Glycol 4000 Polymers 0.000 description 1
- 208000004880 Polyuria Diseases 0.000 description 1
- 208000005392 Spasm Diseases 0.000 description 1
- 101710120037 Toxin CcdB Proteins 0.000 description 1
- 102000003425 Tyrosinase Human genes 0.000 description 1
- 108060008724 Tyrosinase Proteins 0.000 description 1
- 108010073929 Vascular Endothelial Growth Factor A Proteins 0.000 description 1
- 108010053096 Vascular Endothelial Growth Factor Receptor-1 Proteins 0.000 description 1
- 102000005789 Vascular Endothelial Growth Factors Human genes 0.000 description 1
- 108010019530 Vascular Endothelial Growth Factors Proteins 0.000 description 1
- 102100033178 Vascular endothelial growth factor receptor 1 Human genes 0.000 description 1
- 238000000246 agarose gel electrophoresis Methods 0.000 description 1
- 125000003277 amino group Chemical group 0.000 description 1
- 238000004164 analytical calibration Methods 0.000 description 1
- 238000011091 antibody purification Methods 0.000 description 1
- 210000002565 arteriole Anatomy 0.000 description 1
- QVGXLLKOCUKJST-UHFFFAOYSA-N atomic oxygen Chemical compound [O] QVGXLLKOCUKJST-UHFFFAOYSA-N 0.000 description 1
- 230000003305 autocrine Effects 0.000 description 1
- 238000010009 beating Methods 0.000 description 1
- 230000015572 biosynthetic process Effects 0.000 description 1
- 229960002685 biotin Drugs 0.000 description 1
- YBJHBAHKTGYVGT-ZKWXMUAHSA-N biotin Natural products N1C(=O)N[C@@H]2[C@H](CCCCC(=O)O)SC[C@@H]21 YBJHBAHKTGYVGT-ZKWXMUAHSA-N 0.000 description 1
- 235000020958 biotin Nutrition 0.000 description 1
- 239000011616 biotin Substances 0.000 description 1
- 238000010241 blood sampling Methods 0.000 description 1
- 210000004204 blood vessel Anatomy 0.000 description 1
- 230000005779 cell damage Effects 0.000 description 1
- 230000003915 cell function Effects 0.000 description 1
- 208000037887 cell injury Diseases 0.000 description 1
- 230000001684 chronic effect Effects 0.000 description 1
- 238000003776 cleavage reaction Methods 0.000 description 1
- 230000034994 death Effects 0.000 description 1
- 231100000517 death Toxicity 0.000 description 1
- 206010012601 diabetes mellitus Diseases 0.000 description 1
- 238000002405 diagnostic procedure Methods 0.000 description 1
- 239000012470 diluted sample Substances 0.000 description 1
- 238000007865 diluting Methods 0.000 description 1
- 238000003113 dilution method Methods 0.000 description 1
- 230000035619 diuresis Effects 0.000 description 1
- 230000000694 effects Effects 0.000 description 1
- 238000001962 electrophoresis Methods 0.000 description 1
- 210000002889 endothelial cell Anatomy 0.000 description 1
- 230000007613 environmental effect Effects 0.000 description 1
- 238000001976 enzyme digestion Methods 0.000 description 1
- 238000011156 evaluation Methods 0.000 description 1
- 238000002474 experimental method Methods 0.000 description 1
- 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 description 1
- 239000000499 gel Substances 0.000 description 1
- 238000001502 gel electrophoresis Methods 0.000 description 1
- 208000035474 group of disease Diseases 0.000 description 1
- 239000001963 growth medium Substances 0.000 description 1
- 238000003018 immunoassay Methods 0.000 description 1
- 239000012535 impurity Substances 0.000 description 1
- 210000003000 inclusion body Anatomy 0.000 description 1
- 238000011534 incubation Methods 0.000 description 1
- 230000001939 inductive effect Effects 0.000 description 1
- 238000007912 intraperitoneal administration Methods 0.000 description 1
- 239000007928 intraperitoneal injection Substances 0.000 description 1
- BPHPUYQFMNQIOC-NXRLNHOXSA-N isopropyl beta-D-thiogalactopyranoside Chemical compound CC(C)S[C@@H]1O[C@H](CO)[C@H](O)[C@H](O)[C@H]1O BPHPUYQFMNQIOC-NXRLNHOXSA-N 0.000 description 1
- 239000003446 ligand Substances 0.000 description 1
- 239000006249 magnetic particle Substances 0.000 description 1
- 239000003550 marker Substances 0.000 description 1
- 230000008774 maternal effect Effects 0.000 description 1
- 238000005259 measurement Methods 0.000 description 1
- 239000011259 mixed solution Substances 0.000 description 1
- 238000012544 monitoring process Methods 0.000 description 1
- 239000012452 mother liquor Substances 0.000 description 1
- 230000008816 organ damage Effects 0.000 description 1
- 229910052760 oxygen Inorganic materials 0.000 description 1
- 239000001301 oxygen Substances 0.000 description 1
- 238000012856 packing Methods 0.000 description 1
- 230000003076 paracrine Effects 0.000 description 1
- 230000007170 pathology Effects 0.000 description 1
- 230000004796 pathophysiological change Effects 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
- 229920002401 polyacrylamide Polymers 0.000 description 1
- 238000002264 polyacrylamide gel electrophoresis Methods 0.000 description 1
- 229920001223 polyethylene glycol Polymers 0.000 description 1
- 230000008569 process Effects 0.000 description 1
- 230000009465 prokaryotic expression Effects 0.000 description 1
- 230000009257 reactivity Effects 0.000 description 1
- 108020003175 receptors Proteins 0.000 description 1
- 238000000611 regression analysis Methods 0.000 description 1
- 230000001105 regulatory effect Effects 0.000 description 1
- 108091008146 restriction endonucleases Proteins 0.000 description 1
- 230000007017 scission Effects 0.000 description 1
- 230000028327 secretion Effects 0.000 description 1
- 238000011895 specific detection Methods 0.000 description 1
- 210000004989 spleen cell Anatomy 0.000 description 1
- 239000007929 subcutaneous injection Substances 0.000 description 1
- 238000010254 subcutaneous injection Methods 0.000 description 1
- 238000002636 symptomatic treatment Methods 0.000 description 1
- 238000003786 synthesis reaction Methods 0.000 description 1
- 150000003573 thiols Chemical class 0.000 description 1
- 230000006444 vascular growth Effects 0.000 description 1
- 210000003462 vein Anatomy 0.000 description 1
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 description 1
- 230000036266 weeks of gestation Effects 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
- G01N33/68—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids
- G01N33/6863—Cytokines, i.e. immune system proteins modifying a biological response such as cell growth proliferation or differentiation, e.g. TNF, CNF, GM-CSF, lymphotoxin, MIF or their receptors
-
- 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
- G01N33/54333—Modification of conditions of immunological binding reaction, e.g. use of more than one type of particle, use of chemical agents to improve binding, choice of incubation time or application of magnetic field during binding reaction
-
- 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
- G01N33/58—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving labelled substances
- G01N33/582—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving labelled substances 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
- G01N33/68—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids
- G01N33/689—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids related to pregnancy or the gonads
-
- Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
- Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
- Y02A—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE
- Y02A50/00—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE in human health protection, e.g. against extreme weather
- Y02A50/30—Against vector-borne diseases, e.g. mosquito-borne, fly-borne, tick-borne or waterborne diseases whose impact is exacerbated by climate change
Landscapes
- Health & Medical Sciences (AREA)
- Life Sciences & Earth Sciences (AREA)
- Engineering & Computer Science (AREA)
- Immunology (AREA)
- Chemical & Material Sciences (AREA)
- Molecular Biology (AREA)
- Urology & Nephrology (AREA)
- Biomedical Technology (AREA)
- Hematology (AREA)
- Cell Biology (AREA)
- Physics & Mathematics (AREA)
- Biochemistry (AREA)
- Biotechnology (AREA)
- Food Science & Technology (AREA)
- Medicinal Chemistry (AREA)
- Pathology (AREA)
- Analytical Chemistry (AREA)
- Microbiology (AREA)
- General Health & Medical Sciences (AREA)
- General Physics & Mathematics (AREA)
- Proteomics, Peptides & Aminoacids (AREA)
- Chemical Kinetics & Catalysis (AREA)
- Gynecology & Obstetrics (AREA)
- Pregnancy & Childbirth (AREA)
- Reproductive Health (AREA)
- Peptides Or Proteins (AREA)
Abstract
本发明公开了一种PLGF检测试剂盒的制备及应用,具体涉及生物检测领域。所述试剂盒包括抗体包被磁珠工作液、PLGF标记抗体、PLGF校准品、PLGF质控品、样品稀释液。本发明的试剂盒选择吖啶盐NSP‑SA‑NHS修饰物与人源化抗PLGF抗体Fab段偶联,稳定性高,提升了检测系统的灵敏度、检测范围以及保存时间并缩短了反应时间;本试剂盒线性范围达到2.44~10000pg/mL。
Description
技术领域
本发明涉及生物检测领域,具体涉及一种PLGF检测试剂盒的制备及应用。
背景技术
子痫前期是一种常见的产科并发症,在全部妊娠中的发生率在2%-8%;由于慢性高血压,糖尿病等相关疾病患者的增加,子痫前期的发病率自1990年以来呈上升趋势。全世界范围内,子痫前期是导致孕产妇和新生儿死亡的重要原因,每年约有5万-7.5万孕妇因子痫前期死亡,在发达国家约42%的孕产妇死亡由子痫前期导致;尤其对于初次妊娠的孕妇,其子痫前期的发病风险显著高于有妊娠史的孕妇。
子痫前期是妊娠与高血压并存的一组疾病,既有母体及胎盘的基础病理,也有环境因素。子痫前期的基本病理生理变化为血管内皮细胞受损、全身细小动脉痉挛,继而可能导致全身各系统、脏器损伤,导致一系列严重并发症,严重威胁母婴健康。目前子痫前期的治疗方法主要为对症治疗,包括解痉、降压、利尿、合理扩容、终止妊娠;然而,这些方法并不能从根本上治疗子痫前期。只有终止妊娠才能解除疾病对母体的短期危害。如果能在孕中期(孕13周至孕27周)识别子痫前期风险并对高危患者予以预防,可以有效降低孕产妇不良结局的发生概率。
目前,子痫前期的诊断依据为孕妇血压、以及其它临床症状。然而临床实践表明,血压、临床指标对鉴别子痫前期相关不良结局的敏感性、特异性较低,不能尽早、及时、准确鉴别子痫前期不良结局。而孕妇血液中的可溶性fms-like酪氨酸激酶-1(sFlt-1)与胎盘生长因子(PlGF)两种蛋白的比值可以更好反映胎盘血管的生长情况,早于血压升高、以及临床症状。
胎盘生长因子(PLGF)主要由合体滋养层细胞合成,可与位于滋养层细胞和血管内皮细胞的酪氨酸酶受体结合,是一个对滋养层细胞功能有自分泌作用和对血管生长有旁分泌作用的蛋白。PLGF对滋养层细胞和内皮细胞功能有独特的调节作用,能够促进新生血管生成。检测孕妇血液PLGF水平在临床上可用于识别胎盘合体滋养层细胞存在供氧压力,对子痫前期进行鉴别和治疗监测。
发明内容
为此,本发明提供一种PLGF检测试剂盒的制备及应用,以解决现有鉴别子痫前期疾病效果不佳的问题。
为了实现上述目的,本发明提供如下技术方案:
根据本发明一方面提供的一种PLGF检测试剂盒,所述试剂盒包括抗体包被磁珠工作液、PLGF标记抗体、PLGF校准品、PLGF质控品、样品稀释液。
进一步的,所述包被磁珠工作液为包被抗体A的磁珠悬液;
所述PLGF标记抗体是通过NSP-SA-NHS溶液对抗体B标记获得;
抗体A和抗体B分别为PLGF-Fab 1和PLGF-Fab 2,均由轻链和重链组成;PLGF-Fab1轻链如SEQ ID NO.1,重链如SEQ ID NO.2所示;PLGF-Fab 2轻链如SEQ ID NO.3,重链如SEQ ID NO.4所示。
进一步的,所述磁珠与抗体A的质量比为50:1~150:1;所述抗体包被磁珠工作液中的磁珠为甲苯磺酰活化的磁珠;所述抗体B与NSP-SA-NHS的物质的量1:5~1:20。
进一步的,所述包被抗体A的磁珠悬液的方法为取磁珠母液,重悬后加入硼酸盐缓冲液和抗体A振荡混匀;然后加入缓冲液TBS-T稀释即得。
进一步的,所述PLGF标记抗体的制备方法为抗体B用CBS缓冲液稀释后,加入NSP-SA-NHS溶液,搅拌均匀后经过反应得到标记物,再加入DL-赖氨酸后使用Sephadex G-25脱盐,洗脱缓冲体系为TBS-T缓冲液。
进一步的,所述PLGF校准品和PLGF质控品均由样品稀释液配制而成,PLGF校准品的浓度范围为1.22-10000pg/mL;PLGF质控品的浓度为20pg/ml和2500pg/mL。
进一步的,所述样品稀释液为pH 6.5-9.0的PBS缓冲液。
进一步的,所述品稀释液中还含有BSA和防腐剂;优选的防腐剂选用Proclin-300;浓度BSA:5-20g/L和Proclin-300:1-10ml/L。
进一步的,TBS-T缓冲液为0.06M Tris-HCl缓冲液,0.6M NaCl,0.05%BSA,0.5%吐温-20,0.1%Proclin300,pH 7.5。
根据本发明另一方面提供的一种PLGF检测试剂盒在制备鉴别和治疗子痫前期产品中的应用。
本发明具有如下优点:
1.本发明的试剂盒选择吖啶盐NSP-SA-NHS修饰物与人源化抗PLGF抗体Fab段偶联,稳定性高,提升了检测系统的灵敏度、检测范围以及保存时间并缩短了反应时间;本试剂盒线性范围达到2.44~10000pg/mL。
2.本发明试剂盒优选的甲苯磺酰活化的磁珠,它可以同时与配体的巯基和氨基反应且无需缩合剂。这类磁珠对蛋白的结合效率更高,不需要对抗体进行交联来提升抗体与磁珠的结合能力。由于基础信号值的提升,无需借助异硫氰酸荧光素+对应抗体或者亲和素+生物素连接物对信号进行放大。基于上述两点,成功简化了生产工艺,提升了生产效率。
3.本发明通过筛选具有临床应用价值的、反应性强的抗体对,并对检测的反应体系如包被浓度等进行了系列优化,建立了对人胎盘生长因子检测的体系,并制备成可商用的检测试剂盒。该检测试剂盒操作过程简单,相比现有的试剂盒具有耗时短、灵敏度高、线性范围宽、精密度好、抗干扰性强等优点,能够精准反映样品中PlGF含量,在子痫前期鉴别和治疗等方面具有巨大的临床应用前景。
附图说明
为了更清楚地说明本发明的实施方式或现有技术中的技术方案,下面将对实施方式或现有技术描述中所需要使用的附图作简单地介绍。显而易见地,下面描述中的附图仅仅是示例性的,对于本领域普通技术人员来讲,在不付出创造性劳动的前提下,还可以根据提供的附图引伸获得其它的实施附图。
本说明书所绘示的结构、比例、大小等,均仅用以配合说明书所揭示的内容,以供熟悉此技术的人士了解与阅读,并非用以限定本发明可实施的限定条件,故不具技术上的实质意义,任何结构的修饰、比例关系的改变或大小的调整,在不影响本发明所能产生的功效及所能达成的目的下,均应仍落在本发明所揭示的技术内容得能涵盖的范围内。
图1为本发明实施例1提供的一种凝胶电泳鉴定结果图;其中,1-M;2-Hpal,Xhol酶切质粒;
图2为本发明实施例1提供的37℃诱导蛋白聚丙烯酰胺凝胶电泳分析结果图;其中,M-marker;1-未诱导全菌;2-诱导全菌;3-37℃破碎上清;4-37℃破碎沉。
具体实施方式
以下由特定的具体实施例说明本发明的实施方式,熟悉此技术的人士可由本说明书所揭露的内容轻易地了解本发明的其他优点及功效,显然,所描述的实施例是本发明一部分实施例,而不是全部的实施例。基于本发明中的实施例,本领域普通技术人员在没有做出创造性劳动前提下所获得的所有其他实施例,都属于本发明保护的范围。
实施例1
1.重组抗原及单克隆抗体制备
PLGF原核表达:
根据Uniprot(https://www.uniprot.org)上PLGF氨基酸序列,进行密码子优化(https://climsprod.genewiz.com.cn/Toolbox/CodonOptimization)预测蛋白性质(https://web.expasy.org/protparam/)包括等电点、理论分子量、亲疏水性、半衰期等。金斯瑞完成基因合成并连接在由Youbio公司提供的pET-28a(+)载体上。
重组氨基酸序列为如SEQ ID NO.5所示:
MPVMRLFPCFLQLLAGLALPAVPPQQWALSAGNGSSEVEVVPFQEVWGRS
YCRALERVDVVSEYPSEVEHMFSPSCVSLLRCTGCCGDENLHCVPVETANV
TMQLLKIRSGDRPSYVELTFSQHVRCECRPLREKMKPERRRPKGRGKRRREKQRPTDCHLCGDAVPRR*
构建载体验证:在构建好的质粒加入Hpal和Xhol进行双酶切,用1.5%琼脂糖凝胶电泳鉴定结果,结果如图1所示,结果在500bp左右可以看见条带,与预期大小一致验证成功,说明构建质粒中成功插入目的基因。将重组质粒转化到DE3大肠杆菌表达菌株,并37℃培养12h后采用甘油法保存菌株。
2.制备大肠杆菌表达的PLGF抗原。
PLGF诱导表达:
将含有重组质粒的表达菌株接种到LB培养基中经不同温度培养,使用IPTG诱导不同时间表达后收集菌泥进行超声破碎获得破碎上清和破碎沉淀采用15%,10%的聚丙烯酰胺凝胶电泳分析结果如图2所示,经37℃,4℃和16℃进行PLGF的诱导表达结果发现目的蛋白均在包涵体中但16℃的目的蛋白表达量多所以最终确定诱导条件为16℃,诱导时间为48h。
3.制备抗体:使用重组PLGF抗原,免疫小鼠,获得一对抗体。
具体为:取50ug重组抗原与等体积的弗氏完全佐剂充分乳化后对适龄小鼠进行多点皮下注射;首次免疫后2周后,取30μg抗原与等体积的弗氏不完全佐剂充分混合乳化后对小鼠进行皮下多点注射;二次免疫1周后,取30μg抗原与等体积的弗氏不完全佐剂充分乳化后对小鼠进行皮下多点注射,注射7天后对小鼠尾静脉采血测效价;对于三次免疫效价复合的小鼠,取30μg抗原进行腹腔注射作为加强免疫。
将符合要求的小鼠处死取淋巴细胞。以50%PEG 4000作为融合剂,在37℃水浴条件下,骨髓瘤细胞和淋巴细胞两种细胞融合比例为1:3-1:5,将50%PEG在1min中加入到混匀且弃上清的脾脏细胞与骨髓瘤细胞细胞团中,震荡1min后静止2min,然后加入10ml无血清1640培养基。离心弃上清,用添加有HAT的1640培养基重悬细胞。融合后将细胞放置到CO2培养箱中37度培养7-9天,观察融合状态。10天开始采用间接ELISA板进行筛选。对初步筛选的阳性克隆挑选阳性强和稀薄数较多的细胞株利用有限稀释法进行4次亚克隆。通过上述筛选过程,获得两株高亲和力和且能稳定分泌抗体的细胞株,相应的抗体株分别编码为PLGF-Fab 1和PLGF-Fab 2。
选取6-8周生长发育正常的BALB/c小鼠,每只小鼠首先腹腔注射0.5ml弗氏不完全佐剂;10天后进行腹腔注射2×106个杂交瘤细胞。小鼠接种杂交瘤细胞7-9天后可产生腹水,观察小鼠健康状况及腹水生产量;在腹水生产量较多而小鼠健康较差时处死小鼠,收集相应的腹水。
用3倍体积的50mM醋酸缓冲液(pH=4.0)稀释收集的小鼠腹水。在混合溶液中加入终浓度为3%的正辛酸沉淀腹水杂质。14500rpm低温4℃离心30min,取上清溶液。用1M的氢氧化钠溶液调节pH至7.4。将溶液通过平衡后的proteinA/G层析柱,使用磷酸盐溶液冲洗后利用0.1M Gly-Hcl(pH=2.8)溶液洗脱,洗脱产物用1M tris-HCL(pH=9.0)调节pH至7.5。
4.人源化抗体制备
两种人源化PLGF抗体Fab,分别是PLGF-Fab 1和PLGF-Fab 2(本实施例中,抗体A为PLGF-Fab 1、抗体B为PLGF-Fab 2)
包括:
(1)以PLGF重组抗原对小鼠进行免疫,获得与抗原亲和力强的鼠源单克隆抗体细胞系后进行抗体可变区序列测序。
(2)根据得到的可变区序列进行免疫基因数据库IMGT(www.imgt.org)分析,参照人源化抗体模板进行嵌合抗体设计。将优化后的序列借助Hind III和Xho I酶切位点构建至pcDNA3.1(+)载体并转染至HEK293T细胞系,随后进行嵌合抗体表达。
PLGF-Fab 1轻链氨基酸序列为序列表中SEQ ID NO.1所示;
AspIleGluLeuThrGlnSerProAlaSerLeuAlaValSerLeuGlyGlnArgAlaThrIleSerCysArgAlaSerGluSerValAspSerTyrGlyAsnSerPheMetHisTrpTyrGlnGlnLysProGlyGlnProProLysLeuLeuIleTyrLeuAlaSerAsnLeuGluSerGlyValProAlaArgPheSerGlySerGlySerArgThrAspPheThrLeuThrIleAspProValGluAlaAspAspAlaAlaThrTyrTyrCysGlnGlnAsnAsnGluAspProTyrThrPheGlyGlyGlyThrLysLeuGluIleLys。
PLGF-Fab 1重链氨基酸序列为序列表中SEQ ID NO.2所示:
GluValGlnLeuGlnGluSerGlyProGlyLeuValLysProSerGlnSerLeuSerLeuThrCysThrValThrGlyTyrSerIleThrSerAspTyrAlaTrpAsnTrpIleArgGlnLeuProGlyAsnLysLeuGluTrpMetGlyTyrIleSerTyrSerGlySerThrSerTyrAsnProSerLeuLysSerArgIleSerIleThrArgAspThrSerLysAsnGlnPhePheLeuGlnLeuAsnSerValThrThrGluAspThrAlaThrTyrTyrCysAlaArgArgGlyGlyIleTyrTyrTyrPheAspTyrTrpGlyGlnGlyThrProLeuThrValSerSer。
PLGF-Fab 2轻链氨基酸序列为序列表中SEQ ID NO.3所示:
AspIleGlnLeuThrGlnProProSerTyrLeuAlaAlaSerProGlyGluThrIleThrIleAsnCysArgAlaSerLysSerIleSerLysTyrLeuAlaTrpTyrGlnGluLysProGlyLysThrAsnLysLeuLeuIleTyrSerGlySerThrLeuGlnSerGlyIleProSerArgPheSerGlySerGlySerGlyThrAspPheThrLeuThrIleSerSerLeuGluProGluAspPheAlaMetTyrTyrCysGlnGlnHisAsnGluTyrProTyrThrPheGlyGlyGlyThrLysLeuGluIleLys。
PLGF-Fab 2重链氨基酸序列为序列表中SEQ ID NO.4所示:
GluValGlnLeuValGluSerGlyGlyGlyLeuValGlnProLysGlySerLeuLysLeuSerCysAlaAlaSerGlyPheThrPheAsnThrTyrAlaMetAsnTrpValArgGlnAlaProGlyLysGlyLeuGluTrpValAlaArgIleArgSerLysSerAsnAsnTyrAlaThrTyrTyrAlaAspSerValLysAspArgPheThrIleSerArgAspAspSerGlnSerIleLeuTyrLeuGlnMetAsnAsnLeuLysThrGluAspThrValMetTyrTyrCysValArgHisAspTyrTyrGlySerSerTyrPheAspTyrTrpGlyGlnGlyThrThrLeuThrValSerSer。
(3)借助Protein G填料进行抗体纯化。根据BCA法对蛋白浓度进行检测,结果见表1。
表1
5.抗体效价检测
酶标板每孔包被100ng PLGF重组抗原,加入倍比稀释的PLGFAb 37℃温育1小时。温育结束后加入带有HRP标记的羊抗人二抗,最后加入TMB显色,加入2M硫酸终止反应,置酶标仪波长于450nM并进行检测。检测结果见表2。
表2
效价 | 01批 | 02批 | 03批 |
PLGFab-1 | 5000000 | 5000000 | 5000000 |
PLGFab-2 | 2500000 | 2500000 | 2500000 |
6.磁微粒检测试剂制备
抗体包被磁珠工作液的制备方法:为取磁珠母液(Thermo Dynabeads TM M-280Tosylactivated,货号30110D,原始浓度100mg/mL)200μL,重悬(用移液枪轻柔吹打,使其充分悬浮)后加入0.1M硼酸盐缓冲液(BBS,pH 9.5)165μL和抗体A200μg振荡混匀;然后加入缓冲液TBS-T(0.06M Tris-HCl缓冲液,0.6M NaCl,0.05%BSA,0.5%吐温-20,0.1%Proclin300,pH 7.5)。磁珠:抗体A质量比为100:1。磁珠为甲苯磺酰活化的磁珠。
本实施例中PLGF标记抗体的制备方法为:取1mg抗体B用CBS缓冲液稀释至2mg/ml,加入16.5μL 4mM NSP-SA-NHS溶液,搅拌均匀后经过反应得到标记物,加入200μl 5%DL-赖氨酸后使用Sephadex G-25脱盐,洗脱缓冲体系为TBS-T(0.06M Tris-HCl缓冲液,0.6MNaCl,0.05%BSA,0.5%吐温-20,0.1%Proclin300,pH 7.5)。本实施例中抗体B与NSP-SA-NHS的物质的量1:10。
PLGF校准品和PLGF质控品均由样品稀释液配制而成,PLGF校准品的浓度范围为1.22-10000pg/mL;PLGF质控品的浓度为20pg/ml和2500pg/mL,具体的,是用样品稀释液缓冲液将血管内皮生长因子抗原配置成相应的浓度;样品稀释液为pH 6.5-9.0的PBS缓冲液,且每升样品稀释液还含有5-20g BSA和1-10ml Proclin-300。
然后,将上述步骤的抗体包被磁珠工作液、PLGF标记抗体、PLGF校准品、PLGF质控品、样品稀释液分别进行分装。
实施例2
本实施例提供PLGF检测的应用。
1.具体检测方法:
1)以全自动化学发光免疫分析仪为检测工具。检测前,所有样本、校准品和质控品需平衡至室温(18℃-26℃)。
2)扫描试剂包二维码自动获取测试所需参数,扫码后将试剂包放于项目对应试剂位。
3)使用PLGF校准品进行仪器定标并使用PLGF质控品进行质量控制,确定质控品测定浓度在质控范围后进行目标样本检测。
4)机器自动向反应杯中依次加入待测样品20μL和4倍体积的样品稀释液并进行充分混匀,随后依次加入100μL磁珠工作液和100μLPLGF标记抗体,充分混匀并于37℃孵育15min后进行磁分离。
5)磁分离后弃掉上清,并加入300μL稀释后的浓缩洗液,重复清洗3次。
6)将反应杯送入暗室,各加入100μL发光激发液A与100μL发光激发液B进行发光反应,并记录发光值。
7)根据试剂包二维码测得的发光值自动计算为待测样本浓度值。
经测定该试剂盒的主要产品性能指标为:
将罗氏诊断测试赋值血清,重复三次进行检测,计算相对偏差。经验证,本试剂盒准确度指标检测结果见表3。
表3
结论:测量结果的相对偏差在±10%范围内,且3次结果都符合要求,以上3批试剂盒的准确度与罗氏产品相当。
2.试剂盒的精密度试验
依据美国临床实验室标准化委员会(NCCLS/CLSI)文件EP5-A2方案进行,批内精密度和实验室间精密度采用多因素整合的嵌套设计进行实验,不同操作者,不同设备,不同地点,每天上、下午各检测一次,每个样本重复检测2次,连续检测20天,每台设备各收集80个数据结果,计算其批内精密度和实验室间精密度。经验证,本试剂盒精密度指标检测结果如表4所示。
表4
结论:不同操作者,不同设备,不同地点,试剂盒对低、高浓度样本重复进行检测,其批间精密度和实验室间精密度CV均<15%,说明本试剂盒符合精密度性能评估要求。
3.试剂盒的线性区间
选择1例PLGF高浓度样本(补充PLGF重组抗原的基础血清),采用稀释液稀释13个浓度水平,每个稀释后的样本重复测试3次,计算其均值。依据结果逐渐减少浓度点计算相应的线性关系,确定试剂盒的最宽线性范围。经验证,本试剂盒精密度指标检测结果如表5所示。
表5
结论:通过试剂盒对线性区间的评价,在[2.44~10000]pg/mL的范围内相关系数r不低于0.9900且无离群值;对此线性区间采用多项式回归分析,二次方程与三次方程中的b2和b3中Sig.均>0.05,与零无显著性差异,故数据组具线性。因此本试剂盒线性范围为[2.44~10000]pg/mL。
4.试剂盒的灵敏度试验
用试剂盒检测空白样本重复检测20次,进行灵敏度确定。经验证,本试剂灵敏度指标检测结果如表6所示。
表6
结论:测定的空白样本平均发光值为364.70,将值带入反应曲线,得灵敏度为0.44pg/mL,符合要求。
虽然,上文中已经用一般性说明及具体实施例对本发明作了详尽的描述,但在本发明基础上,可以对之作一些修改或改进,这对本领域技术人员而言是显而易见的。因此,在不偏离本发明精神的基础上所做的这些修改或改进,均属于本发明要求保护的范围。
Claims (9)
1.一种PLGF检测试剂盒,其特征在于,所述试剂盒包括抗体包被磁珠工作液、PLGF标记抗体、PLGF校准品、PLGF质控品、样品稀释液。
2.根据权利要求1所述一种PLGF检测试剂盒,其特征在于,所述包被磁珠工作液为包被抗体A的磁珠悬液;
所述PLGF标记抗体是通过NSP-SA-NHS溶液对抗体B标记获得;
抗体A和抗体B分别为PLGF-Fab1和PLGF-Fab2,均由轻链和重链组成;PLGF-Fab1轻链如SEQ ID NO.1,重链如SEQ ID NO.2所示;PLGF-Fab2轻链如SEQ ID NO.3,重链如SEQ IDNO.4所示。
3.根据权利要求2所述一种PLGF检测试剂盒,其特征在于,所述磁珠与抗体A的质量比为50:1~150:1;所述抗体包被磁珠工作液中的磁珠为甲苯磺酰活化的磁珠;所述抗体B与NSP-SA-NHS的物质的量1:5~1:20。
4.根据权利要求2所述一种PLGF检测试剂盒,其特征在于,所述包被抗体A的磁珠悬液的方法为取磁珠母液,重悬后加入硼酸盐缓冲液和抗体A振荡混匀;然后加入缓冲液TBS-T稀释即得。
5.根据权利要求2所述一种PLGF检测试剂盒,其特征在于,所述PLGF标记抗体的制备方法为抗体B用CBS缓冲液稀释后,加入NSP-SA-NHS溶液,搅拌均匀后经过反应得到标记物,再加入DL-赖氨酸后使用SephadexG-25脱盐,洗脱缓冲体系为TBS-T缓冲液。
6.根据权利要求1所述一种PLGF检测试剂盒,其特征在于,所述PLGF校准品和PLGF质控品均由样品稀释液配制而成,PLGF校准品的浓度范围为1.22-10000pg/mL;PLGF质控品的浓度为20pg/ml和2500pg/mL。
7.据权利要求1所述一种PLGF检测试剂盒,其特征在于,所述样品稀释液为pH6.5-9.0的PBS缓冲液。
8.据权利要求1所述一种PLGF检测试剂盒,其特征在于,所述品稀释液中还含有BSA和防腐剂。
9.一种PLGF检测试剂盒在制备鉴别和治疗子痫前期产品中的应用。
Priority Applications (1)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
CN202310628224.6A CN116718777B (zh) | 2023-05-30 | 2023-05-30 | 一种plgf检测试剂盒的制备及应用 |
Applications Claiming Priority (1)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
CN202310628224.6A CN116718777B (zh) | 2023-05-30 | 2023-05-30 | 一种plgf检测试剂盒的制备及应用 |
Publications (2)
Publication Number | Publication Date |
---|---|
CN116718777A true CN116718777A (zh) | 2023-09-08 |
CN116718777B CN116718777B (zh) | 2024-02-13 |
Family
ID=87874462
Family Applications (1)
Application Number | Title | Priority Date | Filing Date |
---|---|---|---|
CN202310628224.6A Active CN116718777B (zh) | 2023-05-30 | 2023-05-30 | 一种plgf检测试剂盒的制备及应用 |
Country Status (1)
Country | Link |
---|---|
CN (1) | CN116718777B (zh) |
Citations (3)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
CN112710851A (zh) * | 2020-12-29 | 2021-04-27 | 苏州百志生物科技有限公司 | 一种检测人体液中胎盘生长因子含量的试剂盒 |
CN113999310A (zh) * | 2020-12-30 | 2022-02-01 | 江苏普若维生物技术有限责任公司 | 一种plgf单克隆抗体、试剂盒、其制备方法和应用 |
CN114252594A (zh) * | 2021-11-22 | 2022-03-29 | 广州万孚生物技术股份有限公司 | 一种胎盘生长因子检测试剂盒及其制备方法与应用 |
-
2023
- 2023-05-30 CN CN202310628224.6A patent/CN116718777B/zh active Active
Patent Citations (3)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
CN112710851A (zh) * | 2020-12-29 | 2021-04-27 | 苏州百志生物科技有限公司 | 一种检测人体液中胎盘生长因子含量的试剂盒 |
CN113999310A (zh) * | 2020-12-30 | 2022-02-01 | 江苏普若维生物技术有限责任公司 | 一种plgf单克隆抗体、试剂盒、其制备方法和应用 |
CN114252594A (zh) * | 2021-11-22 | 2022-03-29 | 广州万孚生物技术股份有限公司 | 一种胎盘生长因子检测试剂盒及其制备方法与应用 |
Also Published As
Publication number | Publication date |
---|---|
CN116718777B (zh) | 2024-02-13 |
Similar Documents
Publication | Publication Date | Title |
---|---|---|
US10830771B2 (en) | PIVKA-II assay method and method for manufacturing reagent or kit for PIVKA-II immunoassay | |
CN109580959B (zh) | 一种检测肝素结合性表皮生长因子的elisa试剂盒 | |
CN114019174B (zh) | 一种试剂盒及其在血管内皮生长因子检测中的应用 | |
CN104711279B (zh) | 一种血管内皮生长因子检测试剂盒及其原料制备 | |
CN114507287A (zh) | 一种抗cldn18.2重组兔单克隆抗体及其应用 | |
CN114989303B (zh) | 一种抗cd56重组兔单克隆抗体及其应用 | |
CN114516919B (zh) | 一种抗pla2r重组兔单克隆抗体及其应用 | |
CN113801226A (zh) | 抗人PlGF鼠源单克隆抗体及应用 | |
CN116284382A (zh) | 抗降钙素原抗体及其应用 | |
EP2884277B1 (en) | Method and immunoassay reagent for measuring PIVKA-II | |
CN108845141B (zh) | 一种cst1磁微粒化学发光免疫分析检测试剂盒及检测方法 | |
CN116718777B (zh) | 一种plgf检测试剂盒的制备及应用 | |
CN110618270B (zh) | 一种用于定量测定粪便中幽门螺杆菌抗原试剂的制备方法 | |
CN116773813B (zh) | 一种vegfr1检测试剂盒的制备及应用 | |
CN112358546B (zh) | 杂交瘤细胞株9c1、plgf-1单克隆抗体及其应用 | |
CN112646036B (zh) | 一种抗cd23特异性单克隆抗体及其应用 | |
CN111273028B (zh) | rhTSG-6直接竞争ELISA法定量检测试剂盒及其使用方法和应用 | |
CN114736300B (zh) | 一种抗her2重组兔单克隆抗体及其应用 | |
CN114213534B (zh) | 抗人PlGF鼠源单克隆抗体及应用 | |
CN114573700B (zh) | 糖类抗原ca19-9的检测试剂盒 | |
CN116203243B (zh) | 一种pinp单克隆抗体、含有其的试剂盒及其应用 | |
CN114507286B (zh) | 一种抗pd-l1重组兔单克隆抗体及其应用 | |
JPH09274038A (ja) | 糖化ヘモグロビンの測定方法 | |
CN111323601A (zh) | 用于检测尿液中微量触珠蛋白的化学发光试剂盒及方法 | |
CN117701577A (zh) | 抗CLDN18.2全人源单克隆抗体Fab2片段的抗体对、试剂盒和应用 |
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 | ||
CB02 | Change of applicant information | ||
CB02 | Change of applicant information |
Address after: Room 201-1, floor 2, building 2, yard 5, Yongfeng Road, Haidian District, Beijing 100094 Applicant after: Beijing Jianping Jinxing Biopharmaceutical Co.,Ltd. Address before: Room 201-1, floor 2, building 2, yard 5, Yongfeng Road, Haidian District, Beijing 100094 Applicant before: Beijing Jianping Jinxing Medical Instrument Co.,Ltd. |
|
GR01 | Patent grant | ||
GR01 | Patent grant |