CN116380882A - Renin magnetic particle chemiluminescence detection kit, preparation method and application - Google Patents
Renin magnetic particle chemiluminescence detection kit, preparation method and application Download PDFInfo
- Publication number
- CN116380882A CN116380882A CN202310315221.7A CN202310315221A CN116380882A CN 116380882 A CN116380882 A CN 116380882A CN 202310315221 A CN202310315221 A CN 202310315221A CN 116380882 A CN116380882 A CN 116380882A
- Authority
- CN
- China
- Prior art keywords
- renin
- active site
- antibody
- monoclonal antibody
- detection
- 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
- 108090000783 Renin Proteins 0.000 title claims abstract description 189
- 102100028255 Renin Human genes 0.000 title claims abstract description 182
- 239000006249 magnetic particle Substances 0.000 title claims abstract description 24
- 238000002360 preparation method Methods 0.000 title claims abstract description 20
- 238000002038 chemiluminescence detection Methods 0.000 title claims abstract description 17
- 238000001514 detection method Methods 0.000 claims description 78
- 238000006243 chemical reaction Methods 0.000 claims description 29
- 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 description 28
- 102000004160 Phosphoric Monoester Hydrolases Human genes 0.000 claims description 28
- 108090000608 Phosphoric Monoester Hydrolases Proteins 0.000 claims description 28
- 239000000243 solution Substances 0.000 claims description 26
- 238000000034 method Methods 0.000 claims description 25
- 239000011859 microparticle Substances 0.000 claims description 19
- 239000011324 bead Substances 0.000 claims description 17
- 108010090804 Streptavidin Proteins 0.000 claims description 16
- 229960002685 biotin Drugs 0.000 claims description 16
- 239000011616 biotin Substances 0.000 claims description 16
- 235000020958 biotin Nutrition 0.000 claims description 14
- 239000006166 lysate Substances 0.000 claims description 14
- 102000002260 Alkaline Phosphatase Human genes 0.000 claims description 12
- 108020004774 Alkaline Phosphatase Proteins 0.000 claims description 12
- 238000002156 mixing Methods 0.000 claims description 12
- 239000007853 buffer solution Substances 0.000 claims description 11
- 239000000758 substrate Substances 0.000 claims description 9
- IAZDPXIOMUYVGZ-UHFFFAOYSA-N Dimethylsulphoxide Chemical compound CS(C)=O IAZDPXIOMUYVGZ-UHFFFAOYSA-N 0.000 claims description 8
- 229920001213 Polysorbate 20 Polymers 0.000 claims description 8
- 150000002148 esters Chemical class 0.000 claims description 8
- 239000000256 polyoxyethylene sorbitan monolaurate Substances 0.000 claims description 8
- 235000010486 polyoxyethylene sorbitan monolaurate Nutrition 0.000 claims description 8
- 239000003755 preservative agent Substances 0.000 claims description 8
- 230000002255 enzymatic effect Effects 0.000 claims description 7
- 230000002335 preservative effect Effects 0.000 claims description 7
- 208000016998 Conn syndrome Diseases 0.000 claims description 5
- 230000003213 activating effect Effects 0.000 claims description 5
- 238000011156 evaluation Methods 0.000 claims description 5
- 230000003907 kidney function Effects 0.000 claims description 5
- 239000007788 liquid Substances 0.000 claims description 5
- 208000013846 primary aldosteronism Diseases 0.000 claims description 5
- LENZDBCJOHFCAS-UHFFFAOYSA-N tris Chemical compound OCC(N)(CO)CO LENZDBCJOHFCAS-UHFFFAOYSA-N 0.000 claims description 5
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 claims description 5
- 239000003795 chemical substances by application Substances 0.000 claims description 4
- 239000000203 mixture Substances 0.000 claims description 4
- 239000004094 surface-active agent Substances 0.000 claims description 4
- 238000003149 assay kit Methods 0.000 claims description 3
- 239000011248 coating agent Substances 0.000 claims description 2
- 238000000576 coating method Methods 0.000 claims description 2
- 239000011259 mixed solution Substances 0.000 claims description 2
- 238000007789 sealing Methods 0.000 claims description 2
- 229960000281 trometamol Drugs 0.000 claims description 2
- 125000003275 alpha amino acid group Chemical group 0.000 claims 8
- 239000000523 sample Substances 0.000 description 35
- 239000000427 antigen Substances 0.000 description 19
- 102000036639 antigens Human genes 0.000 description 19
- 108091007433 antigens Proteins 0.000 description 19
- 239000003153 chemical reaction reagent Substances 0.000 description 19
- 238000012360 testing method Methods 0.000 description 18
- 239000012224 working solution Substances 0.000 description 17
- FAPWRFPIFSIZLT-UHFFFAOYSA-M Sodium chloride Chemical compound [Na+].[Cl-] FAPWRFPIFSIZLT-UHFFFAOYSA-M 0.000 description 12
- 230000035945 sensitivity Effects 0.000 description 12
- 150000001413 amino acids Chemical class 0.000 description 11
- 239000000872 buffer Substances 0.000 description 10
- 210000002381 plasma Anatomy 0.000 description 9
- 238000003018 immunoassay Methods 0.000 description 8
- CUKWUWBLQQDQAC-VEQWQPCFSA-N (3s)-3-amino-4-[[(2s)-1-[[(2s)-1-[[(2s)-1-[[(2s,3s)-1-[[(2s)-1-[(2s)-2-[[(1s)-1-carboxyethyl]carbamoyl]pyrrolidin-1-yl]-3-(1h-imidazol-5-yl)-1-oxopropan-2-yl]amino]-3-methyl-1-oxopentan-2-yl]amino]-3-(4-hydroxyphenyl)-1-oxopropan-2-yl]amino]-3-methyl-1-ox Chemical compound C([C@@H](C(=O)N[C@@H]([C@@H](C)CC)C(=O)N[C@@H](CC=1NC=NC=1)C(=O)N1[C@@H](CCC1)C(=O)N[C@@H](C)C(O)=O)NC(=O)[C@@H](NC(=O)[C@H](CCCN=C(N)N)NC(=O)[C@@H](N)CC(O)=O)C(C)C)C1=CC=C(O)C=C1 CUKWUWBLQQDQAC-VEQWQPCFSA-N 0.000 description 6
- 102000005862 Angiotensin II Human genes 0.000 description 6
- 101800000733 Angiotensin-2 Proteins 0.000 description 6
- 108091003079 Bovine Serum Albumin Proteins 0.000 description 6
- 229950006323 angiotensin ii Drugs 0.000 description 6
- 229940098773 bovine serum albumin Drugs 0.000 description 6
- 238000004140 cleaning Methods 0.000 description 6
- 239000011780 sodium chloride Substances 0.000 description 6
- 239000007987 MES buffer Substances 0.000 description 5
- 238000004090 dissolution Methods 0.000 description 5
- 230000000694 effects Effects 0.000 description 5
- 238000007885 magnetic separation Methods 0.000 description 5
- 239000000047 product Substances 0.000 description 5
- 101800000734 Angiotensin-1 Proteins 0.000 description 4
- 102400000344 Angiotensin-1 Human genes 0.000 description 4
- 102000004881 Angiotensinogen Human genes 0.000 description 4
- 108090001067 Angiotensinogen Proteins 0.000 description 4
- 239000004593 Epoxy Substances 0.000 description 4
- ORILYTVJVMAKLC-UHFFFAOYSA-N adamantane Chemical compound C1C(C2)CC3CC1CC2C3 ORILYTVJVMAKLC-UHFFFAOYSA-N 0.000 description 4
- ORWYRWWVDCYOMK-HBZPZAIKSA-N angiotensin I Chemical compound C([C@@H](C(=O)N[C@@H]([C@@H](C)CC)C(=O)N[C@@H](CC=1NC=NC=1)C(=O)N1[C@@H](CCC1)C(=O)N[C@@H](CC=1C=CC=CC=1)C(=O)N[C@@H](CC=1NC=NC=1)C(=O)N[C@@H](CC(C)C)C(O)=O)NC(=O)[C@@H](NC(=O)[C@H](CCCN=C(N)N)NC(=O)[C@@H](N)CC(O)=O)C(C)C)C1=CC=C(O)C=C1 ORWYRWWVDCYOMK-HBZPZAIKSA-N 0.000 description 4
- 239000002131 composite material Substances 0.000 description 4
- BTBUEUYNUDRHOZ-UHFFFAOYSA-N Borate Chemical compound [O-]B([O-])[O-] BTBUEUYNUDRHOZ-UHFFFAOYSA-N 0.000 description 3
- 239000012190 activator Substances 0.000 description 3
- 230000004071 biological effect Effects 0.000 description 3
- 230000000903 blocking effect Effects 0.000 description 3
- 210000004369 blood Anatomy 0.000 description 3
- 239000008280 blood Substances 0.000 description 3
- 150000001875 compounds Chemical group 0.000 description 3
- 238000007865 diluting Methods 0.000 description 3
- 201000010099 disease Diseases 0.000 description 3
- 208000037265 diseases, disorders, signs and symptoms Diseases 0.000 description 3
- 210000002966 serum Anatomy 0.000 description 3
- 239000011734 sodium Substances 0.000 description 3
- 239000000126 substance Substances 0.000 description 3
- QMMRCKSBBNJCMR-KMZPNFOHSA-N Angiotensin III Chemical compound C([C@@H](C(=O)N[C@@H]([C@@H](C)CC)C(=O)N[C@@H](CC=1NC=NC=1)C(=O)N1[C@@H](CCC1)C(=O)N[C@@H](CC=1C=CC=CC=1)C(O)=O)NC(=O)[C@@H](NC(=O)[C@@H](N)CCCN=C(N)N)C(C)C)C1=CC=C(O)C=C1 QMMRCKSBBNJCMR-KMZPNFOHSA-N 0.000 description 2
- 101800000738 Angiotensin-3 Proteins 0.000 description 2
- 102400000348 Angiotensin-3 Human genes 0.000 description 2
- 102000015427 Angiotensins Human genes 0.000 description 2
- 108010064733 Angiotensins Proteins 0.000 description 2
- LZZYPRNAOMGNLH-UHFFFAOYSA-M Cetrimonium bromide Chemical compound [Br-].CCCCCCCCCCCCCCCC[N+](C)(C)C LZZYPRNAOMGNLH-UHFFFAOYSA-M 0.000 description 2
- DHMQDGOQFOQNFH-UHFFFAOYSA-N Glycine Chemical compound NCC(O)=O DHMQDGOQFOQNFH-UHFFFAOYSA-N 0.000 description 2
- 230000004913 activation Effects 0.000 description 2
- 230000003321 amplification Effects 0.000 description 2
- 230000036772 blood pressure Effects 0.000 description 2
- 238000004132 cross linking Methods 0.000 description 2
- 125000004122 cyclic group Chemical group 0.000 description 2
- 238000011161 development Methods 0.000 description 2
- 238000009826 distribution Methods 0.000 description 2
- 210000003734 kidney Anatomy 0.000 description 2
- 239000000463 material Substances 0.000 description 2
- 230000004060 metabolic process Effects 0.000 description 2
- 238000003199 nucleic acid amplification method Methods 0.000 description 2
- 230000035790 physiological processes and functions Effects 0.000 description 2
- 239000008213 purified water Substances 0.000 description 2
- 239000000376 reactant Substances 0.000 description 2
- 230000009257 reactivity Effects 0.000 description 2
- 238000011084 recovery Methods 0.000 description 2
- 230000001105 regulatory effect Effects 0.000 description 2
- 239000002461 renin inhibitor Substances 0.000 description 2
- 229940086526 renin-inhibitors Drugs 0.000 description 2
- 229910001415 sodium ion Inorganic materials 0.000 description 2
- KZNICNPSHKQLFF-UHFFFAOYSA-N succinimide Chemical compound O=C1CCC(=O)N1 KZNICNPSHKQLFF-UHFFFAOYSA-N 0.000 description 2
- ATGUDZODTABURZ-UHFFFAOYSA-N thiolan-2-ylideneazanium;chloride Chemical compound Cl.N=C1CCCS1 ATGUDZODTABURZ-UHFFFAOYSA-N 0.000 description 2
- 238000005406 washing Methods 0.000 description 2
- 238000005303 weighing Methods 0.000 description 2
- UUUHXMGGBIUAPW-UHFFFAOYSA-N 1-[1-[2-[[5-amino-2-[[1-[5-(diaminomethylideneamino)-2-[[1-[3-(1h-indol-3-yl)-2-[(5-oxopyrrolidine-2-carbonyl)amino]propanoyl]pyrrolidine-2-carbonyl]amino]pentanoyl]pyrrolidine-2-carbonyl]amino]-5-oxopentanoyl]amino]-3-methylpentanoyl]pyrrolidine-2-carbon Chemical compound C1CCC(C(=O)N2C(CCC2)C(O)=O)N1C(=O)C(C(C)CC)NC(=O)C(CCC(N)=O)NC(=O)C1CCCN1C(=O)C(CCCN=C(N)N)NC(=O)C1CCCN1C(=O)C(CC=1C2=CC=CC=C2NC=1)NC(=O)C1CCC(=O)N1 UUUHXMGGBIUAPW-UHFFFAOYSA-N 0.000 description 1
- PQSUYGKTWSAVDQ-ZVIOFETBSA-N Aldosterone Chemical compound C([C@@]1([C@@H](C(=O)CO)CC[C@H]1[C@@H]1CC2)C=O)[C@H](O)[C@@H]1[C@]1(C)C2=CC(=O)CC1 PQSUYGKTWSAVDQ-ZVIOFETBSA-N 0.000 description 1
- PQSUYGKTWSAVDQ-UHFFFAOYSA-N Aldosterone Natural products C1CC2C3CCC(C(=O)CO)C3(C=O)CC(O)C2C2(C)C1=CC(=O)CC2 PQSUYGKTWSAVDQ-UHFFFAOYSA-N 0.000 description 1
- 102000004400 Aminopeptidases Human genes 0.000 description 1
- 108090000915 Aminopeptidases Proteins 0.000 description 1
- KCXVZYZYPLLWCC-UHFFFAOYSA-N EDTA Chemical compound OC(=O)CN(CC(O)=O)CCN(CC(O)=O)CC(O)=O KCXVZYZYPLLWCC-UHFFFAOYSA-N 0.000 description 1
- 238000002965 ELISA Methods 0.000 description 1
- 108010059378 Endopeptidases Proteins 0.000 description 1
- 102000005593 Endopeptidases Human genes 0.000 description 1
- 102000004190 Enzymes Human genes 0.000 description 1
- 108090000790 Enzymes Proteins 0.000 description 1
- 239000004471 Glycine Substances 0.000 description 1
- 241000282412 Homo Species 0.000 description 1
- 101000945318 Homo sapiens Calponin-1 Proteins 0.000 description 1
- 101000652736 Homo sapiens Transgelin Proteins 0.000 description 1
- 108091005804 Peptidases Proteins 0.000 description 1
- 102000035195 Peptidases Human genes 0.000 description 1
- 102000004270 Peptidyl-Dipeptidase A Human genes 0.000 description 1
- 108090000882 Peptidyl-Dipeptidase A Proteins 0.000 description 1
- ZLMJMSJWJFRBEC-UHFFFAOYSA-N Potassium Chemical compound [K] ZLMJMSJWJFRBEC-UHFFFAOYSA-N 0.000 description 1
- 229920005654 Sephadex Polymers 0.000 description 1
- 239000012507 Sephadex™ Substances 0.000 description 1
- 102100031013 Transgelin Human genes 0.000 description 1
- 239000007983 Tris buffer Substances 0.000 description 1
- 230000009471 action Effects 0.000 description 1
- 210000004404 adrenal cortex Anatomy 0.000 description 1
- 229960002478 aldosterone Drugs 0.000 description 1
- 230000000890 antigenic effect Effects 0.000 description 1
- 230000004872 arterial blood pressure Effects 0.000 description 1
- 238000003556 assay Methods 0.000 description 1
- 230000009286 beneficial effect Effects 0.000 description 1
- 108010051210 beta-Fructofuranosidase Proteins 0.000 description 1
- 239000012496 blank sample Substances 0.000 description 1
- 230000003197 catalytic effect Effects 0.000 description 1
- 238000007796 conventional method Methods 0.000 description 1
- 238000005336 cracking Methods 0.000 description 1
- NZNMSOFKMUBTKW-UHFFFAOYSA-N cyclohexanecarboxylic acid Chemical compound OC(=O)C1CCCCC1 NZNMSOFKMUBTKW-UHFFFAOYSA-N 0.000 description 1
- 238000003748 differential diagnosis Methods 0.000 description 1
- 231100000673 dose–response relationship Toxicity 0.000 description 1
- 238000005516 engineering process Methods 0.000 description 1
- 239000012467 final product Substances 0.000 description 1
- 239000012530 fluid Substances 0.000 description 1
- 230000001434 glomerular Effects 0.000 description 1
- 210000002503 granulosa cell Anatomy 0.000 description 1
- 235000011073 invertase Nutrition 0.000 description 1
- 239000001573 invertase Substances 0.000 description 1
- 210000004185 liver Anatomy 0.000 description 1
- 210000004072 lung Anatomy 0.000 description 1
- 230000002934 lysing effect Effects 0.000 description 1
- 238000005259 measurement Methods 0.000 description 1
- 238000012544 monitoring process Methods 0.000 description 1
- 210000005036 nerve Anatomy 0.000 description 1
- 230000004962 physiological condition Effects 0.000 description 1
- 230000001766 physiological effect Effects 0.000 description 1
- 229920000136 polysorbate Polymers 0.000 description 1
- BITYAPCSNKJESK-UHFFFAOYSA-N potassiosodium Chemical compound [Na].[K] BITYAPCSNKJESK-UHFFFAOYSA-N 0.000 description 1
- 239000011591 potassium Substances 0.000 description 1
- 229910001414 potassium ion Inorganic materials 0.000 description 1
- 238000003127 radioimmunoassay Methods 0.000 description 1
- 230000009103 reabsorption Effects 0.000 description 1
- 210000002796 renal vein Anatomy 0.000 description 1
- 230000036454 renin-angiotensin system Effects 0.000 description 1
- 238000012216 screening Methods 0.000 description 1
- 230000028327 secretion Effects 0.000 description 1
- 238000001179 sorption measurement Methods 0.000 description 1
- 229960002317 succinimide Drugs 0.000 description 1
- JJAHTWIKCUJRDK-UHFFFAOYSA-N succinimidyl 4-(N-maleimidomethyl)cyclohexane-1-carboxylate Chemical compound C1CC(CN2C(C=CC2=O)=O)CCC1C(=O)ON1C(=O)CCC1=O JJAHTWIKCUJRDK-UHFFFAOYSA-N 0.000 description 1
- 125000000020 sulfo group Chemical group O=S(=O)([*])O[H] 0.000 description 1
- 230000002889 sympathetic effect Effects 0.000 description 1
- 230000008685 targeting Effects 0.000 description 1
- 238000010998 test method Methods 0.000 description 1
- 210000001519 tissue Anatomy 0.000 description 1
- 210000005239 tubule Anatomy 0.000 description 1
Images
Classifications
-
- 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
- G01N33/577—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
-
- 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)
- Immunology (AREA)
- Chemical & Material Sciences (AREA)
- Engineering & Computer Science (AREA)
- Physics & Mathematics (AREA)
- Urology & Nephrology (AREA)
- Molecular Biology (AREA)
- Hematology (AREA)
- Biomedical Technology (AREA)
- General Physics & Mathematics (AREA)
- General Health & Medical Sciences (AREA)
- Pathology (AREA)
- Biochemistry (AREA)
- Analytical Chemistry (AREA)
- Biotechnology (AREA)
- Cell Biology (AREA)
- Microbiology (AREA)
- Chemical Kinetics & Catalysis (AREA)
- Food Science & Technology (AREA)
- Medicinal Chemistry (AREA)
- Plasma & Fusion (AREA)
- Investigating Or Analysing Materials By The Use Of Chemical Reactions (AREA)
Abstract
The invention belongs to the technical field of biology, and particularly relates to a renin magnetic particle chemiluminescence detection kit, a preparation method and application thereof.
Description
Technical Field
The invention belongs to the technical field of magnetic particle chemiluminescence detection kits, and particularly relates to a renin magnetic particle chemiluminescence detection kit, a preparation method and application.
Background
Renin (Renin), also known as Renin, is a proteolytic enzyme released by periglomerular granulosa cells of the glomerular apparatus (also known as the periglomerular complex) and catalyzes the conversion of Renin to angiotensin I, a component of the Renin-angiotensin-aldosterone system. The content of renin in normal human body is low, mainly in the form of renin, and the renin comprises active renin and inactive renin, wherein the active renin content is only one tenth of the inactive renin content. Renin is 66 amino acids more than renin, and under normal physiological conditions, the active binding site of renin is in a closed state and has no catalytic ability. When arterial blood pressure is reduced, circulation is reduced, and sympathetic nerves are excited, renin is activated to open renin or renin under the action of endopeptidase, and both active sites are opened to have physiological activity. The renin enters blood through renal veins after secretion, can catalyze the liver to secrete angiotensinogen entering blood plasma to be converted into angiotensin I, and the angiotensin I is formed within a few seconds and is degraded by angiotensin converting enzyme in blood and lung tissues to generate angiotensin II, so that the amount of aldosterone secreted by the spherical zone of adrenal cortex stimulated by the angiotensin II is increased, and the reabsorption of renal tubules on NaCl and water is increased. Angiotensin II can also be hydrolyzed by aminopeptidases to angiotensin III. All three kinds of angiotensin have biological activity, wherein the biological activity of angiotensin II and angiotensin III is strong, and the concentration of angiotensin II in blood plasma is low, so that the biological activity of angiotensin II is the strongest. Renin and invertase, etc. are often present in plasma, whereas the release of "active renin" (herein referring to renin and active site-opened renin, the same applies hereinafter) is a critical condition determining the concentration of angiotensin in plasma, which plays an important role in regulating the blood pressure and the metabolism of potassium and sodium ions in humans and the normal physiological functions of the kidneys.
Renin is one of the most commonly used screening indexes of primary aldosteronism, and has been widely applied to clinic, and especially the clinic development of measuring the content of renin can greatly improve the detection rate of the disease so as to enable part of patients to be diagnosed and treated early.
The existing detection methods for renin on the market mainly comprise two methods, namely renin activity and renin concentration, and the renin activity is detected as a traditional method, and the principle is that the activity renin level in blood plasma is indirectly detected by measuring the amount of converting angiotensinogen into angiotensin I in unit volume per unit time, and the activity renin level is influenced by the concentration of the angiotensinogen, sample pretreatment, culture time, pH value or other factors, so that detection results of detection reagents of different operators and different manufacturers are greatly different. The detection of the renin concentration is a newly established method for detecting the renin content in recent years, and the method is not influenced by the concentration of the angiotensinogen, is simple and convenient to operate, has high detection speed, can realize high-flux detection, has good stability and repeatability, and is easy to standardize.
The detection of the concentration of renin adopts the principle of specific reaction between antigen and antibody, and common detection methods include an enzyme-linked immunosorbent assay, a radioimmunoassay and a chemiluminescent immunoassay. However, considering that the content of renin in the human body is extremely low, the sensitivity of the renin detection kit used clinically at present is low, and the renin content detection between low-value samples is not differentiated, and is limited by the development difficulty of specific antibodies against 'active renin', most of antibodies used in renin detection reagents are antibodies against total renin (comprising renin and active site open renin and active site closed renin), but not antibodies against renin and active site open renin. The content of inactive renin in human body is far higher than that of renin and active renin, and the inactive renin has no help to the evaluation of kidney function and the differential diagnosis of related diseases, so that if the detection is carried out by adopting an antibody aiming at total renin, the detection of active renin can be interfered, and the accuracy of the renin determination result in clinical application is affected. The renin detection reagent which has high sensitivity and can accurately detect the active renin is lacking in the market at present, and particularly the full-automatic detection reagent which has stable test result of a low-value sample and high precision is lacking.
Renin can regulate human blood pressure and potassium sodium ion metabolism and maintain kidney normal physiological function, and has great application potential in preventing, monitoring and treating primary aldosteronism, kidney function evaluation and other diseases. In order to overcome the inherent drawbacks of the prior art, it is highly desirable to establish a methodology that allows for accurate detection of "active renin" and enables large-scale detection of renin with satisfactory sensitivity.
Disclosure of Invention
Aiming at the problems existing in the prior art, the invention aims to provide a renin magnetic particle chemiluminescence detection kit, a preparation method and application thereof, wherein the kit can specifically detect 'active renin', namely renin and renin with an opened active site, and has higher detection sensitivity, accuracy, precision and lower detection limit.
Based on the above purpose, the technical scheme adopted by the invention is as follows:
in a first aspect, the present invention provides an anti-renin active site antibody that specifically recognizes renin and active site-open renin, the anti-renin active site antibody specifically recognizes amino acid sequences from position 25 to position 50 in the amino acid sequence of a renin molecule and from position 91 to position 116 in the amino acid sequence of an active site-open renin molecule.
Preferably, the amino acid sequence of the anti-renin active site antibody is: GIGTPPQTFKVVFDTGSSNVWVPSSK (SEQ ID NO. 1).
The anti-renin active site antibody provided by the invention can be specifically combined with active renin, namely renin and renin with an open active site, is not combined with inactive renin and renin with a closed active site, and has higher recognition specificity for the active renin.
In a second aspect, the invention provides the use of an anti-renin active site antibody as described above in renin and active site open renin assays.
In a third aspect, the present invention provides a renin magnetic particle chemiluminescent detection kit for detection of renin and active site-open renin, the kit comprising a biotin-labeled anti-renin active site antibody as described above.
The kit can specifically bind renin and renin with an open active site by utilizing the anti-renin active site antibody, but does not bind with inactive renin and renin with an blocked active site, so that the detection specificity of the kit on the active renin is greatly improved.
Preferably, the kit further comprises streptavidin-alkaline phosphatase conjugate, anti-renin monoclonal antibody coated magnetic microparticles, enzymatic luminescent substrate and sample lysate.
The detection principle of the kit of the invention is as follows: the renin antigen in the sample to be detected is cracked by utilizing sample cracking liquid, so that antigen sites of the renin antigen in the sample are fully exposed, the renin antigen in the sample reacts with an anti-renin monoclonal antibody coated on magnetic microparticles to form a magnetic microparticle-renin monoclonal antibody-renin antigen complex, the magnetic microparticle-renin monoclonal antibody-renin antigen complex reacts with a biotin-marked anti-renin active site antibody to form a magnetic microparticle-renin monoclonal antibody-renin antigen-renin active site antibody-biotin complex, the complex reacts with a streptavidin-alkaline phosphatase connector to form a magnetic microparticle-renin monoclonal antibody-renin antigen-renin active site antibody-biotin-streptavidin-alkaline phosphatase complex, the RLU value of the complex is detected, and the concentration of 'active renin' in the sample to be detected is calculated based on a standard curve of the renin concentration and the RLU value.
Meanwhile, the kit realizes the composite crosslinking of a plurality of magnetic particle-antibody-antigen-antibody-biotin-streptavidin-alkaline phosphatase compound structures within a certain space range based on the principle that one streptavidin molecule is combined with four biotin molecules, thereby achieving the purpose of signal value cyclic amplification and further improving the detection sensitivity of the kit.
Preferably, the enzymatic luminescent substrate is 3- (2-spiral adamantane) -4-methoxy-4- (3-phosphoryl) -phenyl-1, 2-dioxy-cyclohexane (AMPPD).
In a fourth aspect, the invention provides a method for preparing a renin magnetic particle chemiluminescence detection kit, which comprises the steps of preparing a biotin-labeled anti-renin active site antibody, a streptavidin-alkaline phosphatase connector, anti-renin monoclonal antibody-coated magnetic particles and a sample lysate;
the preparation method of the biotin-labeled anti-renin active site antibody comprises the following steps:
(1) Dissolving biotin-N-succinimidyl ester in dimethyl sulfoxide to prepare biotin-N-succinimidyl ester solution;
(2) Adding the anti-renin active site antibody into biotin-N-succinimidyl ester solution for reaction to obtain a connection product, and purifying the connection product to obtain the biotin-marked anti-renin active site antibody.
Preferably, the preparation method of the anti-renin monoclonal antibody coated magnetic microparticles comprises the following steps:
(1) Mixing the anti-renin monoclonal antibody and magnetic beads in a buffer solution to obtain a mixed solution of the anti-renin monoclonal antibody and the magnetic beads;
(2) Adding (NH 4) to the mixture of anti-renin monoclonal antibody and magnetic beads 2 SO 4 The solution is subjected to magnetic bead coating anti-renin monoclonal antibody reaction, after the reaction, a sealing liquid is added to seal the magnetic beads coated with the anti-renin monoclonal antibody, and then the magnetic microparticles coated with the anti-renin monoclonal antibody are prepared through cleaning.
Preferably, the method for preparing the streptavidin-alkaline phosphatase conjugate comprises the following steps:
(1) Treating streptavidin by an activating agent to obtain activated streptavidin;
(2) Treating alkaline phosphatase with an activating agent to obtain activated alkaline phosphatase;
(3) Mixing activated or unactivated alkaline phosphatase with activated streptavidin to obtain streptavidin-alkaline phosphatase conjugate.
Preferably, the preparation method of the sample lysate comprises the following steps:
the sample lysate was prepared by dispersing Trometamol (TRIS), surfactant, tween-20 and preservative in water.
In a fifth aspect, the present invention provides an application of the above-mentioned renin magnetic particle chemiluminescence detection kit in primary aldosteronism index detection and kidney function evaluation index detection.
The kit has higher detection specificity to the renin, so that the kit has higher application prospect in primary aldosteronism index detection and kidney function evaluation index detection.
Compared with the prior art, the invention has the following beneficial effects:
(1) The present invention claims an anti-renin active site antibody that has a higher reactivity towards "active renin" such as renin and active site-open renin, but is not reactive towards active site-closed renin. The kit provided by the invention adopts the biotin-marked anti-renin active site antibody, can specifically identify the active renin, and improves the detection accuracy of the active renin.
(2) The kit provided by the invention utilizes sample lysate to lyse renin antigen in a sample to be detected, so that antigen sites of the renin antigen are fully exposed, and the renin antigen is sequentially reacted with magnetic microparticles coated by an anti-renin monoclonal antibody, a biotin-labeled anti-renin active site antibody and a streptavidin-alkaline phosphatase connector to form a magnetic microparticle-renin monoclonal antibody-renin antigen-renin active site antibody-biotin-streptavidin-alkaline phosphatase compound.
(3) The kit of the invention adopts a specific sample lysate to pretreat the blood sample to be detected, so that the antigen structure of renin in the sample to be detected is fully exposed, the binding efficiency between the renin antibody and renin is improved, and the detection sensitivity of the kit is further improved.
(4) The kit has higher detection specificity to renin and renin with an open active site, higher detection sensitivity, accuracy and precision and lower detection limit.
Drawings
FIG. 1 is a detection standard curve of an international standard of renin;
FIG. 2 is a graph comparing the effect of the kit of the present invention on the ability of the native renin antigen/active site to detect renin after it has been opened or closed.
Detailed Description
For a better description of the objects, technical solutions and advantages of the present invention, the present invention will be further described with reference to the following specific examples. It will be appreciated by persons skilled in the art that the specific embodiments described herein are for purposes of illustration only and are not intended to be limiting.
The test methods used in the examples are conventional methods unless otherwise specified; the materials, reagents and the like used, unless otherwise specified, are all commercially available.
The renin magnetic particle chemiluminescence detection kit provided by the invention is prepared by self by I, and the amino acid sequence of the renin magnetic particle chemiluminescence detection kit is GIGTPPQTFKVVFDTGSSNVWVPSSK (SEQ ID NO. 1); the remaining materials or reagents are commercially available. Wherein the full-automatic chemiluminescence immunoassay analyzer is a VIT700 full-automatic chemiluminescence immunoassay analyzer which is self-developed by Shenzhen tylode medical Co., ltd.
Example 1
The embodiment provides a renin magnetic particle chemiluminescence detection kit, which comprises the following reagents: streptavidin-alkaline phosphatase connector, biotin-marked anti-renin active site antibody, anti-renin monoclonal antibody-coated magnetic microparticle working solution, cleaning buffer solution, enzymatic luminescent substrate solution and sample lysate; the preparation method of the reagent comprises the following steps:
1. preparation method of streptavidin-alkaline phosphatase connector
This example provides two different methods for preparing streptavidin-alkaline phosphatase conjugates, specifically as follows:
(1) First preparation method of streptavidin-alkaline phosphatase conjugate
3mg of streptavidin is weighed, dissolved in 0.6mL of 0.1M PBS (pH=7.5) buffer containing 0.15% EDTA.2Na at room temperature, the final concentration after dissolution is 5mg/mL, 2mg of activator succinimide 4- (N-maleimidomethyl) cyclohexane-1-carboxylate (short: SMCC) is weighed, dissolved in 0.4mL of dimethyl sulfoxide at room temperature, and the final concentration after dissolution is 5mg/mL. Adding 60 mu LSMCC solution into the streptavidin solution, performing an activation reaction, and uniformly mixing at room temperature for 60min to obtain activated streptavidin; diluting alkaline phosphatase to 5mg/mL with 0.1M PBS (pH=7.5) buffer containing 0.15% EDTA-2 Na, weighing 2mg of activator Traut's reagent, dissolving in 0.4mL of 0.1M PBS (pH=7.5) buffer containing 0.15% EDTA-2 Na at room temperature, adding 35 mu L of Traut's reagent solution into the alkaline phosphatase solution, activating, uniformly mixing at room temperature, and reacting for 60min to obtain the activated alkaline phosphatase. And uniformly mixing the activated streptavidin with the activated alkaline phosphatase, and reacting at 4 ℃ overnight for 15 hours to obtain the streptavidin-alkaline phosphatase connector. The streptavidin-alkaline phosphatase conjugate is prepared into 0.1-5 mug/ml working solution by using 0.05M, pH =6.0 MES buffer (containing 1% of bovine serum albumin, 0.9% of sodium chloride, 0.1% of tween-20 and 0.05% of biological preservative Proclin 300) and is preserved at 2-8 ℃ for standby. The streptavidin-alkaline phosphatase working solution may also be 0.2 μg/ml, or 0.3 μg/ml, or 0.5 μg/ml, or 0.8 μg/ml, or more preferably 0.4 μg/ml.
(2) Second method for preparing streptavidin-alkaline phosphatase connector
3mg of streptavidin was weighed and dissolved in 0.30mL of 0.1M MES (pH=5.0) buffer at room temperature, the final concentration after dissolution was 10mg/mL, 3mg of activator EDC was weighed and dissolved in 0.3mL of 0.1M MES (pH=5.0) at room temperature, and the final concentration after dissolution was 10mg/mL. Adding 30 mu LEDC solution into the streptavidin solution, performing an activation reaction, and uniformly mixing at room temperature for 15min to obtain activated streptavidin; diluting alkaline phosphatase to 10mg/mL by adopting 0.30mL of 0.1M MES (pH=5.0) buffer solution, adding 0.6mL of alkaline phosphatase into the activated streptavidin solution, and uniformly mixing at room temperature for reaction for 2 hours to obtain the streptavidin-alkaline phosphatase connector. The streptavidin-alkaline phosphatase conjugate is prepared into 0.2-5 mug/ml working solution by using 0.05M, pH =6.0 MES buffer (containing 1% of bovine serum albumin, 0.9% of sodium chloride, 0.1% of tween-20 and 0.05% of biological preservative Proclin 300) and is preserved at 2-8 ℃ for standby. The streptavidin-alkaline phosphatase working solution may also be 0.5 μg/ml, or 1.0 μg/ml, or 2.0 μg/ml, or 3.0 μg/ml, or more preferably 2.5 μg/ml.
2. Preparation method of biotin-labeled anti-renin active site antibody
The amino acid sequence of the anti-renin active site antibody used in the kit is as follows: GIGTPPQTFKVVFDTGSSNVWVPSSK the anti-renin active site antibody is capable of targeting recognition of amino acid sequences 25 to 50 in the amino acid sequence of a renin molecule and recognition of amino acid sequences 91 to 116 in the amino acid sequence of an active site open renin pro-molecule.
0.5mg of anti-renin active site antibody was diluted to 1mg/ml with 0. M, pH =7.4 PBS buffer. 1mg of biotin-N-succinimidyl ester was weighed and dissolved in 1ml of dimethyl sulfoxide at room temperature, and the final concentration after dissolution was 1mg/ml. To the anti-renin active site antibody, 5. Mu.l of 10mg/ml biotin-N-succinimidyl ester was added and reacted at room temperature for 1 hour. And purifying the connection product by using a Sephadex G-25 gel chromatographic column after the reaction is finished, and obtaining a final product which is the biotin-labeled anti-renin active site antibody. The biotin-labeled anti-renin active site antibody is prepared into 0.2-3 mug/ml working solution by using PBS buffer solution (containing 0.5% of bovine serum albumin, 0.9% of sodium chloride, 0.1% of tween-20 and 0.05% of biological preservative Proclin 300) with the concentration of 0.02M, pH =7.4, and the working solution is preserved at the temperature of 2-8 ℃ for standby. The working fluid concentration of the biotin-labeled anti-renin active site antibody may also be 0.5. Mu.g/ml, or 0.8. Mu.g/ml, or 2. Mu.g/ml, or more preferably 1. Mu.g/ml.
3. Preparation method of magnetic microparticle working solution coated by anti-renin monoclonal antibody
The present example provides two different methods for preparing the working solution of magnetic microparticles coated with an anti-renin monoclonal antibody, specifically as follows:
(1) First preparation method of anti-renin monoclonal antibody coated magnetic microparticle working solution
Diluting 0.2mg of anti-renin monoclonal antibody to 1mg/ml with 0.1M, pH =9.5 borate buffer, washing 5mg of sulfo magnetic beads (purchased externally) with 0.1M, pH =9.5 borate buffer on a magnetic plate with high magnetic force for 3 times, preparing 20mg/ml with 0.1M, pH =9.5 borate buffer after washing, mixing the two, and adding 3M (NH 4 ) 2 SO 4 400 μl of the solution was mixed overnight at 37deg.C and reacted for 18h. After the reaction is completed, 0.2M,Magnetic microparticles were blocked with PBS buffer (containing 2% bovine serum albumin, 1% glycine) at ph=7.4 and mixed well for 4h at 37 ℃. After the reaction, the mixture is washed on a magnetic plate with high magnetic force for 5 times by using a PBS buffer solution with the concentration of 0. M, pH =7.4, and unbound other substances are washed away, so that the magnetic microparticles coated with the anti-renin monoclonal antibody are obtained. The above-mentioned anti-renin monoclonal antibody-sulfo magnetic bead microparticles were prepared into a working solution of 0.1-1 mg/ml using a PBS buffer solution (containing 0.5% bovine serum albumin, 0.9% sodium chloride, 0.1% tween-20, 0.05% bio-preservative Proclin 300) of 0.02M, pH =7.4, and stored at 2-8 ℃ for later use. The working solution concentration of the anti-renin monoclonal antibody-sulfo magnetic bead microparticles can also be 0.2mg/ml, or 0.3mg/ml, or 0.5mg/ml, or 0.8mg/ml, or more preferably 0.25mg/ml.
(2) Second preparation method of magnetic microparticle working solution coated by anti-renin monoclonal antibody
Epoxy-based magnetic beads were formulated into 40mg/ml epoxy-based magnetic bead adsorption working solution using 0.1M, pH =7.4 PBS buffer, to which an anti-renin monoclonal antibody was added, and 3M (NH 4 ) 2 SO 4 400 μl of the solution is reacted for 4 hours at 37 ℃, unbound other substances are washed off after the reaction is completed, and the reaction is carried out by adopting a blocking solution to carry out overnight blocking at 37 ℃, so that the epoxy magnetic beads coated with the anti-renin antibody are obtained, and the epoxy magnetic beads coated with the anti-renin antibody are prepared into the working solution of the magnetic microparticles coated with the anti-renin monoclonal antibody by using a PBS buffer solution with the concentration of 0.1M, pH =7.4. Wherein the blocking solution is TRIS buffer solution of 0.05M, pH =7.4, which further contains 1% bovine serum albumin and 0.1% tween-20.
4. Preparation method of cleaning buffer solution
Weighing Na 2 HPO 4 ·12H 2 O 14.32g、NaH 2 PO 4· 2H 2 Adding 1.56g of O, 9g of NaCl, 20.5 g of Tween, 9 2g of surfactant S and 300.5 g of preservative PC into a beaker, adding 0.8L of purified water, uniformly mixing, adjusting the pH value to 7.5, finally fixing the volume to 1L, and storing at 2-8 ℃ for later use.
5. Preparation method of enzymatic luminescent substrate liquid
100mL of adamantane AMPPD substrate solution is measured, and diluted 10 times by using a PBS buffer solution with the concentration of 0.02M, pH =9.0, and the substrate solution is used as enzymatic luminescent substrate solution and is preserved at the temperature of 2-8 ℃ for standby.
6. Preparation method of sample lysate
6.05g of TRIS, 5g of cetyl trimethyl ammonium bromide (CTAB for short) serving as a surfactant, 20 g of tween-20 and 0.5g of PC serving as a preservative are weighed into a beaker, 0.8L of purified water is added into the beaker for uniform mixing, the pH is regulated to 8.0, the volume is finally fixed to 1L, and the mixture is preserved at 2-8 ℃ for standby.
Example 2
The embodiment provides a method for detecting the content of renin in a sample by using a renin magnetic particle chemiluminescence detection kit, which specifically comprises the following steps:
(1) Absorbing 100 mu l of sample or standard to be detected and 50 mu l of sample lysate to react for 2min at 37 ℃, and lysing renin antigen in the sample to fully expose antigenic sites;
(2) Absorbing the cracked sample to 50 μl of magnetic microparticle working solution coated with anti-renin monoclonal antibody after the reaction is completed, carrying out a reaction at 37 ℃ for 2min, and reacting the renin antigen in the sample with the renin antibody coated on the magnetic microparticles, wherein the renin antigen is combined on the magnetic microparticles to form a magnetic microparticle-renin antibody-renin antigen complex structure;
(3) After the reaction is finished, adding the reactants into a magnetic field for magnetic separation, and after the magnetic separation is finished, continuously adding 50 mu l of biotin-labeled anti-renin active site antibody for reaction for 2min at 37 ℃ to form a magnetic particle-renin antibody-renin antigen-renin active site antibody-biotin composite structure;
(4) After the reaction is completed, adding the reactants into a magnetic field for magnetic separation, and after the magnetic separation is completed, continuously adding 50 mu l of streptavidin-alkaline phosphatase connector for reaction at 37 ℃ for 2min to form a magnetic particle-renin antibody-renin antigen-renin active site antibody-biotin-streptavidin-alkaline phosphatase composite structure, wherein the reaction is a round of reaction;
(5) Adding a cleaning buffer solution after the reaction is finished, introducing a magnetic field, and cleaning to remove excessive components which are not adsorbed by the magnetic field;
(6) And after the cleaning is finished, adding the enzymatic luminescent substrate liquid to react, and detecting to obtain a test result.
If the detection reaction signal value or concentration value of the magnetic particle-renin antibody-renin antigen-renin active site antibody-biotin-streptavidin-alkaline phosphatase complex in (6) cannot meet the detection requirement, the detection of steps (5) to (6) is carried out after repeating the above steps (2) to (4) by magnetic separation for a plurality of rounds of reaction, and the number of the above rounds of reaction can theoretically be counted for a plurality of times until the test requirement is met, which may be generally 1 time, 3 times, 5 times, or more preferably 2 times.
The detection is carried out on a VIT700 full-automatic chemiluminescence immunoassay analyzer which is self-developed by tylode medical limited company. The reagents in the kit can be pre-packaged into self-made reagent strips of tylode medical limited company, and pressed into films to prepare finished products. During detection, a sample to be detected or a standard substance is only required to be added into a sample hole of the reagent strip and is loaded onto a VIT700 full-automatic chemiluminescence immunoassay instrument which is self-developed by tylode medical company, so that the full-automatic detection of the renin content in the sample by the instrument can be realized.
According to the renin detection method and the detection kit, provided by the invention, a renin detection method applicable to a chemiluminescence immunoassay technology is creatively developed, the enzyme circulation gain reaction principle applied by the kit and the application of antibodies aiming at renin active sites are creative, the detection scheme is based on the principle that one streptavidin molecule is combined with four biotin molecules to realize the composite crosslinking of a plurality of magnetic particle-antibody-antigen-antibody-biotin-streptavidin-alkaline phosphatase compound structures within a certain space range, so that the aim of cyclic amplification of signal values is fulfilled, the sensitivity of a reagent is further improved, in addition, a specific sample lysate is adopted to pretreat the sample, the antigen structure of renin in the sample is fully exposed, the binding efficiency between the renin antibody and renin is improved, and the sensitivity of the reagent is further improved; the kit prepared by the invention has higher detection sensitivity for detecting renin and renin with an open active site.
Example 3
According to the kit described in the example 1 and the method for using the kit described in the example 2, a standard curve for detecting the international standard of renin WHO by using the kit of the invention is constructed.
The renin WHO international standard is prepared into standard working solutions of 0 mu IU/mL, 2.0 mu IU/mL, 5.0 mu IU/mL, 20.0 mu IU/mL, 50.0 mu IU/mL, 100.0 mu IU/mL, 250.0 mu IU/mL and 500.0 mu IU/mL by adopting healthy human serum without renin antigen, the renin detection reagent is adopted for detection, and a standard curve is drawn according to the renin WHO international standard concentration and the relative signal value detected by the renin international standard, the standard curve is shown in figure 1, and a mathematical formula of the standard curve obtained by adopting four-parameter fitting is as follows: y= (a-d)/[ 1+ (x/c) b ] +d, where a=3221.61, b=1.0881, c=1.10271e+07, d=4.66957e+10.
Example 4
In the embodiment, the minimum detection limit of the kit is detected and analyzed by adopting a blank detection limit method, and the method comprises the following steps:
detecting a zero concentration standard (S0) for 20 times, wherein the zero concentration standard is a blank sample without renin and renin, obtaining a signal value (RLU) of 20 times of measurement results, calculating an average value M and a standard deviation SD of the signal value (RLU), obtaining an RLU value corresponding to M+2SD, and carrying out two-point regression fitting according to the concentration between a zero concentration calibrator and an adjacent concentration standard and the result of the RLU value to obtain a primary equation, wherein the renin concentration of the adjacent concentration standard is 2 mu IU/mL, in the embodiment, the renin concentration of the adjacent concentration standard is 2 mu IU/mL, the standard with renin of 0.5 mu IU/mL is used as S1, the RLU value corresponding to the standard is detected, and the RLU value corresponding to M+2SD is carried into the primary equation, so that the corresponding concentration, namely the lowest detection Limit (LOB) is calculated.
The blank detection limit results are shown in Table 1, and it can be seen that the blank detection limit using the renin detection reagent of the present invention is only 0.07. Mu.IU/mL, and the confidence interval of the reference range of 2.5% -97.5% of the renin content distribution in the normal healthy human body is as follows: the vertical position is 4.3 mu IU/mL-48.3 mu IU/mL; the range of lying position is 2.2 mu IU/mL-38.9 mu IU/mL, so that the blank detection limit of the renin detection reagent is far lower than the renin content distribution in normal healthy human body, and the detection sensitivity of the kit meets the clinical detection requirement.
In the fourth embodiment of the patent CN108398423B 'renin chemiluminescence detection kit', the blank limit of the kit for detecting the renin sample of the similar product is only 0.2402 mu IU/mL and 0.2527 mu IU/mL, which are far higher than the detection method in the invention, therefore, the kit has lower detection limit compared with the prior art, and the kit has better detection sensitivity.
TABLE 1 blank detection limit results
Example 5
In the embodiment, the renin with an open active site and the renin with an open active site which are extracted naturally are treated by a renin inhibitor to be detected, and the correct identification capacity of the kit for the 'active' renin is analyzed, namely the detection specificity of the kit for the 'active' renin.
As shown in FIG. 2, the kit has good recognition capability on natural renin and active site-opened renin, and as the theoretical concentration of the natural renin and the active site-opened renin is increased, the tested concentration is increased, and a better dose-response curve relationship is presented, and after the renin inhibitor is added into the active site-opened renin, the active site-opened renin is converted into the active site-closed renin due to the fact that the active site is blocked, so that the content of the active site-opened renin cannot be detected by the kit. The kit and the detection method provided by the invention have obvious reactivity only to the 'active renin', can accurately detect the content of the 'active renin', can not detect inactive renin, ensure the reliability of a test result, and have higher detection specificity to the 'active' renin.
Example 6
In this embodiment, the detection accuracy of the assay kit is analyzed by adding a recovery test, and the method is as follows:
the human plasma without renin is used to dilute the international standard of renin WHO to final concentration of 0.0 mu IU/mL, 1.0 mu IU/mL, 50.0 mu IU/mL and 400.0 mu IU/mL, so as to form blank, low-concentration, medium-concentration and high-concentration plasma samples. The blank, low concentration, medium concentration, high concentration plasma samples were tested using the chemiluminescent immunoassay detection method of example 2 of the present invention and the standard curve of FIG. 1.
The results are shown in Table 2, and the recovery rate of the plasma samples of the international standard of the WHO of the renin with different concentrations measured by using the renin detection reagent of the invention is between 98.3% and 103.1%, which indicates that the renin detection reagent of the invention can be used for detecting the renin in the samples, and the accuracy of the results is high.
TABLE 2 preparation of renin test results in serum samples
Example 7
The detection precision of the kit of the invention is analyzed based on the following method:
the international standard of the renin WHO is diluted to a final concentration by adopting serum of healthy people without renin, and the final concentration is as follows: 5.0. Mu.IU/mL, 80.0. Mu.IU/mL, 250.0. Mu.IU/mL, to form reproducible samples of low, medium, and high concentrations. The repeated samples of low, medium and high concentrations were repeated 10 times by using the above-mentioned method for detecting renin by chemiluminescence immunoassay and the standard curve of FIG. 1, and the average value and the coefficient of variation were calculated.
The results are shown in Table 3, and the variation coefficient of renin in samples with different concentrations measured by using the renin detection reagent is less than 5%, which indicates that the kit has higher detection precision.
TABLE 3 precision test results
| Preparing a sample | Low concentration sample | Medium concentration sample | High concentration sample |
| Concentration (mu IU/mL) | 5.0 | 80.0 | 250.0 |
| Test 1 | 4.87 | 82.32 | 241.62 |
| Test 2 | 4.91 | 77.50 | 255.82 |
| Test 3 | 5.12 | 81.56 | 245.27 |
| Test 4 | 4.72 | 75.32 | 254.35 |
| Test 5 | 4.90 | 76.69 | 250.03 |
| Test 6 | 4.85 | 78.85 | 244.02 |
| Test 7 | 5.03 | 83.98 | 253.13 |
| Test 8 | 5.27 | 84.73 | 237.11 |
| Test 9 | 5.30 | 81.19 | 267.34 |
| Test 10 | 4.58 | 82.93 | 242.28 |
| Test mean (mu IU/mL) | 4.95 | 80.51 | 249.10 |
| Coefficient of variation (%) | 4.60％ | 4.01％ | 3.56％ |
Finally, it should be noted that the above embodiments are only for illustrating the technical solution of the present invention and not for limiting the scope of the present invention, and although the present invention has been described in detail with reference to the preferred embodiments, it should be understood by those skilled in the art that the technical solution of the present invention may be modified or substituted equally without departing from the spirit and scope of the technical solution of the present invention.
Claims (10)
1. An anti-renin active site antibody, wherein the anti-renin active site antibody specifically recognizes renin and active site-open renin, and wherein the anti-renin active site antibody specifically recognizes amino acid sequences from amino acid sequence 25 to amino acid sequence 50 in an amino acid sequence of a renin molecule and from amino acid sequence 91 to amino acid sequence 116 in an active site-open renin molecule amino acid sequence.
2. The anti-renin active site antibody of claim 1, wherein the amino acid sequence of the anti-renin active site antibody is set forth in SEQ ID No. 1.
3. Use of an anti-renin active site antibody according to claim 1 or 2 for renin and active site open renin detection.
4. A renin magnetic particle chemiluminescent detection kit for detection of renin and active site-opened renin, the kit comprising a biotin-labeled anti-renin active site antibody of claim 1 or 2.
5. The renin magnetic particle chemiluminescent assay kit of claim 4 further comprising a streptavidin-alkaline phosphatase conjugate, anti-renin monoclonal antibody coated magnetic particles, an enzymatic luminescent substrate, and a sample lysate.
6. A method for preparing a renin magnetic particle chemiluminescence detection kit according to claim 5, wherein the method comprises the steps of preparing a biotin-labeled anti-renin active site antibody, a streptavidin-alkaline phosphatase connector, anti-renin monoclonal antibody-coated magnetic particles and a sample lysate;
the preparation method of the biotin-labeled anti-renin active site antibody comprises the following steps:
(1) Dissolving biotin-N-succinimidyl ester in dimethyl sulfoxide to prepare biotin-N-succinimidyl ester solution;
(2) Adding the anti-renin active site antibody into biotin-N-succinimidyl ester solution for reaction to obtain a connection product, and purifying the connection product to obtain the biotin-marked anti-renin active site antibody.
7. The method for preparing a renin magnetic particle chemiluminescence detection kit of claim 6, wherein the method for preparing the anti-renin monoclonal antibody coated magnetic particles comprises the following steps:
(1) Mixing the anti-renin monoclonal antibody and magnetic beads in a buffer solution to obtain a mixed solution of the anti-renin monoclonal antibody and the magnetic beads;
(2) Adding (NH) to the mixture of anti-renin monoclonal antibody and magnetic beads 4 ) 2 SO 4 The solution is subjected to magnetic bead coating anti-renin monoclonal antibody reaction, after the reaction, a sealing liquid is added to seal the magnetic beads coated with the anti-renin monoclonal antibody, and then the anti-renin monoclonal antibody is prepared by cleaningMagnetic microparticles coated with a plain monoclonal antibody.
8. The method of preparing a renin magnetic particle chemiluminescent assay kit of claim 6 wherein the method of preparing a streptavidin-alkaline phosphatase conjugate comprises the steps of:
(1) Treating streptavidin by an activating agent to obtain activated streptavidin;
(2) Treating alkaline phosphatase with an activating agent to obtain activated alkaline phosphatase;
(3) Mixing activated or unactivated alkaline phosphatase with activated streptavidin to obtain streptavidin-alkaline phosphatase conjugate.
9. The method for preparing a renin magnetic particle chemiluminescence detection kit of claim 6, wherein the method for preparing the sample lysate comprises the following steps:
dispersing trometamol, a surfactant, tween-20 and a preservative in water to prepare a sample lysate.
10. Use of the renin magnetic particle chemiluminescence detection kit of claim 4 or 5 in primary aldosteronism index detection and kidney function evaluation index detection.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202310315221.7A CN116380882A (en) | 2023-03-21 | 2023-03-21 | Renin magnetic particle chemiluminescence detection kit, preparation method and application |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202310315221.7A CN116380882A (en) | 2023-03-21 | 2023-03-21 | Renin magnetic particle chemiluminescence detection kit, preparation method and application |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| CN116380882A true CN116380882A (en) | 2023-07-04 |
Family
ID=86976246
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN202310315221.7A Pending CN116380882A (en) | 2023-03-21 | 2023-03-21 | Renin magnetic particle chemiluminescence detection kit, preparation method and application |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN116380882A (en) |
Citations (6)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US5945512A (en) * | 1997-04-07 | 1999-08-31 | Tokiwa Chemical Industries Co., Ltd. | Prorenin antibody and renin-active substance containing the same |
| JP2000186100A (en) * | 1998-10-13 | 2000-07-04 | Tokiwa Kagaku Kogyo Kk | Substance for expressing activity of renin |
| US6159723A (en) * | 1998-10-13 | 2000-12-12 | Tokiwa Chemical Industries, Ltd. | Renin-active substance |
| US20100196367A1 (en) * | 2009-02-04 | 2010-08-05 | Molecular Innovations | Methods For Screening Candidate Agents For Modulating Prorenin And Renin, Assays for Detecting Prorenin And Antibodies |
| CN109187972A (en) * | 2018-08-17 | 2019-01-11 | 迪瑞医疗科技股份有限公司 | A kind of magnetic microparticle chemiluminescence kit of quantitative detection plasma renin content and preparation method thereof |
| US20200049712A1 (en) * | 2016-10-21 | 2020-02-13 | Fujirebio Inc. | Immunological measurement method for renin concentration |
-
2023
- 2023-03-21 CN CN202310315221.7A patent/CN116380882A/en active Pending
Patent Citations (6)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US5945512A (en) * | 1997-04-07 | 1999-08-31 | Tokiwa Chemical Industries Co., Ltd. | Prorenin antibody and renin-active substance containing the same |
| JP2000186100A (en) * | 1998-10-13 | 2000-07-04 | Tokiwa Kagaku Kogyo Kk | Substance for expressing activity of renin |
| US6159723A (en) * | 1998-10-13 | 2000-12-12 | Tokiwa Chemical Industries, Ltd. | Renin-active substance |
| US20100196367A1 (en) * | 2009-02-04 | 2010-08-05 | Molecular Innovations | Methods For Screening Candidate Agents For Modulating Prorenin And Renin, Assays for Detecting Prorenin And Antibodies |
| US20200049712A1 (en) * | 2016-10-21 | 2020-02-13 | Fujirebio Inc. | Immunological measurement method for renin concentration |
| CN109187972A (en) * | 2018-08-17 | 2019-01-11 | 迪瑞医疗科技股份有限公司 | A kind of magnetic microparticle chemiluminescence kit of quantitative detection plasma renin content and preparation method thereof |
Non-Patent Citations (1)
| Title |
|---|
| "PREDICTED: renin isoform X1[Rhinopithecus bieti]", NCBI * |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| CN102901810B (en) | Preparation method of latex particles coated with prostate specific antigen-antibody and PSA enhanced turbidimetric immunophelometry kit | |
| CN110862881B (en) | Special cleaning solution or diluent for full-automatic chemiluminescence determinator and preparation method thereof | |
| CN110726841B (en) | Method for detecting veterinary drug residues based on enzymatic click reaction signal amplification magnetic relaxation time immunosensor | |
| Johannsson | Heterogeneous enzyme immunoassay | |
| CN103018465A (en) | Human cystatin C chemiluminescence quantitative detection method | |
| JP5563470B2 (en) | Glycated hemoglobin detection method | |
| CN112816694A (en) | Abnormal prothrombin detection kit and preparation method thereof | |
| CN103063851A (en) | Free triiodothyronine nanometer magnetic particle chemiluminescence assay kit and preparation method thereof and detection method thereof | |
| Mao et al. | Simultaneous detection of carcinoembryonic antigen and neuron-specific enolase in human serum based on time-resolved chemiluminescence immunoassay | |
| Pollet et al. | Enzyme-antigen immunoassay for human placental alkaline phosphatase in serum and tissue extracts, and its application as a tumor marker. | |
| Millán et al. | Highly sensitive solid-phase immunoenzymometric assay for placental and placental-like alkaline phosphatases with a monoclonal antibody and monodisperse polymer particles. | |
| CN112067826A (en) | NT-proBNP detection kit constructed based on high-specific-activity alkaline phosphatase and application thereof | |
| CN111044724B (en) | Thymidine kinase 1 magnetic particle chemiluminescence assay kit and preparation method thereof | |
| CN107860914A (en) | A kind of anti-mitochondrial antibody IgG detection kit and its detection method | |
| CN113933521A (en) | Magnetic particle chemiluminescence detection kit and preparation method and application thereof | |
| Rogier et al. | Two-site immunoenzymometric assay for the 52-kDa cathepsin D in cytosols of breast cancer tissues. | |
| CN103048453A (en) | Nanometer magnetic particle chemiluminescence detection kit for carbohydrate antigen CA19-9 as well as preparation method thereof and detecting method thereof | |
| CN109142753A (en) | Squamous cell carcinoma-related antigen chemiluminescence immune detection reagent kit and preparation method thereof | |
| CN116380882A (en) | Renin magnetic particle chemiluminescence detection kit, preparation method and application | |
| CN102565037A (en) | Magnetic particle chemiluminescence kit for quantitatively detecting plasma rennin | |
| CN111693721A (en) | Preparation method and application of enzyme-linked immunosorbent assay based on prussian blue nano enzyme label | |
| Maiolini et al. | Study of an enzyme immunoassay kit for carcinoembryonic antigen. | |
| Lein et al. | Elimination of serum free and total prostate-specific antigen after radical retropubic prostatectomy | |
| CN103048461A (en) | Nanometer magnetic particle chemiluminescent assay kit for cancer antigen CA15-3, and preparation method and detection method thereof | |
| KR20010025027A (en) | Immunoassay reagents and immunoassay method |
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 |