CN106146651A - Women luteal function quick non-invasive monitoring technology - Google Patents
Women luteal function quick non-invasive monitoring technology Download PDFInfo
- Publication number
- CN106146651A CN106146651A CN201510185202.2A CN201510185202A CN106146651A CN 106146651 A CN106146651 A CN 106146651A CN 201510185202 A CN201510185202 A CN 201510185202A CN 106146651 A CN106146651 A CN 106146651A
- Authority
- CN
- China
- Prior art keywords
- couplet
- pregnanediol
- antibody
- glucuronate
- test 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
- 230000000938 luteal effect Effects 0.000 title claims abstract description 12
- 238000012544 monitoring process Methods 0.000 title claims abstract description 8
- 238000005516 engineering process Methods 0.000 title abstract description 11
- YWYQTGBBEZQBGO-BERLURQNSA-N Pregnanediol Chemical compound C([C@H]1CC2)[C@H](O)CC[C@]1(C)[C@@H]1[C@@H]2[C@@H]2CC[C@H]([C@@H](O)C)[C@@]2(C)CC1 YWYQTGBBEZQBGO-BERLURQNSA-N 0.000 claims abstract description 43
- YWYQTGBBEZQBGO-UHFFFAOYSA-N UC1011 Natural products C1CC2CC(O)CCC2(C)C2C1C1CCC(C(O)C)C1(C)CC2 YWYQTGBBEZQBGO-UHFFFAOYSA-N 0.000 claims abstract description 43
- 102000004169 proteins and genes Human genes 0.000 claims abstract description 32
- 108090000623 proteins and genes Proteins 0.000 claims abstract description 32
- 238000005859 coupling reaction Methods 0.000 claims abstract description 25
- AEMOLEFTQBMNLQ-AQKNRBDQSA-N D-glucopyranuronic acid Chemical compound OC1O[C@H](C(O)=O)[C@@H](O)[C@H](O)[C@H]1O AEMOLEFTQBMNLQ-AQKNRBDQSA-N 0.000 claims abstract description 23
- 238000010168 coupling process Methods 0.000 claims abstract description 23
- 229940097042 glucuronate Drugs 0.000 claims abstract description 23
- 230000008878 coupling Effects 0.000 claims abstract description 22
- 238000009396 hybridization Methods 0.000 claims abstract description 8
- 238000012360 testing method Methods 0.000 claims description 39
- 125000004429 atom Chemical group 0.000 claims description 21
- 229940092253 ovalbumin Drugs 0.000 claims description 20
- 229910052799 carbon Inorganic materials 0.000 claims description 14
- 125000004432 carbon atom Chemical group C* 0.000 claims description 14
- IJGRMHOSHXDMSA-UHFFFAOYSA-N Atomic nitrogen Chemical compound N#N IJGRMHOSHXDMSA-UHFFFAOYSA-N 0.000 claims description 8
- 108010058846 Ovalbumin Proteins 0.000 claims description 8
- 239000000084 colloidal system Substances 0.000 claims description 8
- 108091003079 Bovine Serum Albumin Proteins 0.000 claims description 6
- 229940098773 bovine serum albumin Drugs 0.000 claims description 6
- 239000002253 acid Substances 0.000 claims description 5
- 125000005842 heteroatom Chemical group 0.000 claims description 5
- NINIDFKCEFEMDL-UHFFFAOYSA-N Sulfur Chemical compound [S] NINIDFKCEFEMDL-UHFFFAOYSA-N 0.000 claims description 4
- QVGXLLKOCUKJST-UHFFFAOYSA-N atomic oxygen Chemical group [O] QVGXLLKOCUKJST-UHFFFAOYSA-N 0.000 claims description 4
- 229910052757 nitrogen Inorganic materials 0.000 claims description 4
- 229910052760 oxygen Inorganic materials 0.000 claims description 4
- 239000001301 oxygen Substances 0.000 claims description 4
- 229910052717 sulfur Inorganic materials 0.000 claims description 4
- 239000011593 sulfur Substances 0.000 claims description 4
- 102000004190 Enzymes Human genes 0.000 claims description 3
- 108090000790 Enzymes Proteins 0.000 claims description 3
- 108060003552 hemocyanin Proteins 0.000 claims description 3
- OKTJSMMVPCPJKN-UHFFFAOYSA-N Carbon Chemical compound [C] OKTJSMMVPCPJKN-UHFFFAOYSA-N 0.000 claims 1
- WQZGKKKJIJFFOK-GASJEMHNSA-N Glucose Natural products OC[C@H]1OC(O)[C@H](O)[C@@H](O)[C@@H]1O WQZGKKKJIJFFOK-GASJEMHNSA-N 0.000 claims 1
- 241000218636 Thuja Species 0.000 claims 1
- 239000008103 glucose Substances 0.000 claims 1
- 102000002710 Neurophysins Human genes 0.000 abstract description 22
- 108010018674 Neurophysins Proteins 0.000 abstract description 22
- 210000002700 urine Anatomy 0.000 abstract description 14
- 230000035945 sensitivity Effects 0.000 abstract description 12
- 230000035558 fertility Effects 0.000 abstract description 7
- 230000000624 ovulatory effect Effects 0.000 abstract description 4
- 150000003146 progesterones Chemical class 0.000 abstract description 2
- 230000016087 ovulation Effects 0.000 description 24
- 229940088597 hormone Drugs 0.000 description 21
- 239000005556 hormone Substances 0.000 description 21
- 238000001514 detection method Methods 0.000 description 17
- 239000003153 chemical reaction reagent Substances 0.000 description 13
- 239000000243 solution Substances 0.000 description 13
- 230000021615 conjugation Effects 0.000 description 11
- 208000000509 infertility Diseases 0.000 description 11
- 230000036512 infertility Effects 0.000 description 11
- 238000002372 labelling Methods 0.000 description 10
- 238000004458 analytical method Methods 0.000 description 9
- 230000001276 controlling effect Effects 0.000 description 9
- 108010000912 Egg Proteins Proteins 0.000 description 7
- 102000002322 Egg Proteins Human genes 0.000 description 7
- 229940011871 estrogen Drugs 0.000 description 7
- 239000000262 estrogen Substances 0.000 description 7
- ZBKIUFWVEIBQRT-UHFFFAOYSA-N gold(1+) Chemical compound [Au+] ZBKIUFWVEIBQRT-UHFFFAOYSA-N 0.000 description 7
- 231100000535 infertility Toxicity 0.000 description 7
- 210000004681 ovum Anatomy 0.000 description 7
- 238000004445 quantitative analysis Methods 0.000 description 7
- YMWUJEATGCHHMB-UHFFFAOYSA-N Dichloromethane Chemical compound ClCCl YMWUJEATGCHHMB-UHFFFAOYSA-N 0.000 description 6
- OKKJLVBELUTLKV-UHFFFAOYSA-N Methanol Chemical compound OC OKKJLVBELUTLKV-UHFFFAOYSA-N 0.000 description 6
- RJKFOVLPORLFTN-LEKSSAKUSA-N Progesterone Chemical compound C1CC2=CC(=O)CC[C@]2(C)[C@@H]2[C@@H]1[C@@H]1CC[C@H](C(=O)C)[C@@]1(C)CC2 RJKFOVLPORLFTN-LEKSSAKUSA-N 0.000 description 6
- ZMANZCXQSJIPKH-UHFFFAOYSA-N Triethylamine Chemical compound CCN(CC)CC ZMANZCXQSJIPKH-UHFFFAOYSA-N 0.000 description 6
- 230000002209 hydrophobic effect Effects 0.000 description 6
- 208000021267 infertility disease Diseases 0.000 description 6
- 238000000034 method Methods 0.000 description 6
- 239000008363 phosphate buffer Substances 0.000 description 6
- 229920001223 polyethylene glycol Polymers 0.000 description 6
- 238000002360 preparation method Methods 0.000 description 6
- 238000003756 stirring Methods 0.000 description 6
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 description 6
- 241000283973 Oryctolagus cuniculus Species 0.000 description 5
- 239000002202 Polyethylene glycol Substances 0.000 description 5
- PCHJSUWPFVWCPO-UHFFFAOYSA-N gold Chemical compound [Au] PCHJSUWPFVWCPO-UHFFFAOYSA-N 0.000 description 5
- HEDRZPFGACZZDS-UHFFFAOYSA-N Chloroform Chemical compound ClC(Cl)Cl HEDRZPFGACZZDS-UHFFFAOYSA-N 0.000 description 4
- XSQUKJJJFZCRTK-UHFFFAOYSA-N Urea Chemical compound NC(N)=O XSQUKJJJFZCRTK-UHFFFAOYSA-N 0.000 description 4
- 238000000502 dialysis Methods 0.000 description 4
- 230000036039 immunity Effects 0.000 description 4
- 238000003018 immunoassay Methods 0.000 description 4
- 238000011155 quantitative monitoring Methods 0.000 description 4
- 239000000020 Nitrocellulose Substances 0.000 description 3
- 230000015572 biosynthetic process Effects 0.000 description 3
- 125000003178 carboxy group Chemical group [H]OC(*)=O 0.000 description 3
- 238000010790 dilution Methods 0.000 description 3
- 239000012895 dilution Substances 0.000 description 3
- 239000010931 gold Substances 0.000 description 3
- 229910052737 gold Inorganic materials 0.000 description 3
- 239000000203 mixture Substances 0.000 description 3
- 229920001220 nitrocellulos Polymers 0.000 description 3
- 210000001672 ovary Anatomy 0.000 description 3
- 230000027758 ovulation cycle Effects 0.000 description 3
- 230000035935 pregnancy Effects 0.000 description 3
- 239000000047 product Substances 0.000 description 3
- 239000000186 progesterone Substances 0.000 description 3
- 229960003387 progesterone Drugs 0.000 description 3
- 238000003786 synthesis reaction Methods 0.000 description 3
- 201000005670 Anovulation Diseases 0.000 description 2
- 206010002659 Anovulatory cycle Diseases 0.000 description 2
- LFQSCWFLJHTTHZ-UHFFFAOYSA-N Ethanol Chemical compound CCO LFQSCWFLJHTTHZ-UHFFFAOYSA-N 0.000 description 2
- 241001529936 Murinae Species 0.000 description 2
- 229910019145 PO4.2H2O Inorganic materials 0.000 description 2
- 241001494479 Pecora Species 0.000 description 2
- NBIIXXVUZAFLBC-UHFFFAOYSA-N Phosphoric acid Chemical compound OP(O)(O)=O NBIIXXVUZAFLBC-UHFFFAOYSA-N 0.000 description 2
- 239000004698 Polyethylene Substances 0.000 description 2
- VYPSYNLAJGMNEJ-UHFFFAOYSA-N Silicium dioxide Chemical compound O=[Si]=O VYPSYNLAJGMNEJ-UHFFFAOYSA-N 0.000 description 2
- 230000000274 adsorptive effect Effects 0.000 description 2
- 238000013019 agitation Methods 0.000 description 2
- 231100000552 anovulation Toxicity 0.000 description 2
- 239000004202 carbamide Substances 0.000 description 2
- 235000013877 carbamide Nutrition 0.000 description 2
- 238000006243 chemical reaction Methods 0.000 description 2
- 210000004246 corpus luteum Anatomy 0.000 description 2
- 238000003745 diagnosis Methods 0.000 description 2
- 201000010099 disease Diseases 0.000 description 2
- 208000037265 diseases, disorders, signs and symptoms Diseases 0.000 description 2
- BNIILDVGGAEEIG-UHFFFAOYSA-L disodium hydrogen phosphate Chemical compound [Na+].[Na+].OP([O-])([O-])=O BNIILDVGGAEEIG-UHFFFAOYSA-L 0.000 description 2
- 229910000397 disodium phosphate Inorganic materials 0.000 description 2
- 238000001035 drying Methods 0.000 description 2
- 238000007689 inspection Methods 0.000 description 2
- 239000000463 material Substances 0.000 description 2
- 238000001840 matrix-assisted laser desorption--ionisation time-of-flight mass spectrometry Methods 0.000 description 2
- 230000009871 nonspecific binding Effects 0.000 description 2
- 229920000573 polyethylene Polymers 0.000 description 2
- 238000001556 precipitation Methods 0.000 description 2
- 239000000741 silica gel Substances 0.000 description 2
- 229910002027 silica gel Inorganic materials 0.000 description 2
- 239000002904 solvent Substances 0.000 description 2
- 238000002965 ELISA Methods 0.000 description 1
- 102000008394 Immunoglobulin Fragments Human genes 0.000 description 1
- 108010021625 Immunoglobulin Fragments Proteins 0.000 description 1
- ZFFFJLDTCLJDHL-JQYCEVDMSA-N Pregnanediol-3-glucuronide Chemical compound O([C@H]1C[C@H]2CC[C@H]3[C@@H]4CC[C@@H]([C@]4(CC[C@@H]3[C@@]2(C)CC1)C)[C@@H](O)C)[C@@H]1O[C@H](C(O)=O)[C@@H](O)[C@H](O)[C@H]1O ZFFFJLDTCLJDHL-JQYCEVDMSA-N 0.000 description 1
- 229910000147 aluminium phosphate Inorganic materials 0.000 description 1
- 239000000427 antigen Substances 0.000 description 1
- 102000036639 antigens Human genes 0.000 description 1
- 108091007433 antigens Proteins 0.000 description 1
- 238000013459 approach Methods 0.000 description 1
- 238000003149 assay kit Methods 0.000 description 1
- 230000009286 beneficial effect Effects 0.000 description 1
- 238000004159 blood analysis Methods 0.000 description 1
- 239000000872 buffer Substances 0.000 description 1
- 239000006227 byproduct Substances 0.000 description 1
- 238000005119 centrifugation Methods 0.000 description 1
- 239000003795 chemical substances by application Substances 0.000 description 1
- 238000004587 chromatography analysis Methods 0.000 description 1
- 238000004140 cleaning Methods 0.000 description 1
- 150000001875 compounds Chemical class 0.000 description 1
- 229940124558 contraceptive agent Drugs 0.000 description 1
- 239000003433 contraceptive agent Substances 0.000 description 1
- 230000002596 correlated effect Effects 0.000 description 1
- 238000007405 data analysis Methods 0.000 description 1
- 230000003111 delayed effect Effects 0.000 description 1
- 238000013461 design Methods 0.000 description 1
- 238000011161 development Methods 0.000 description 1
- 238000010586 diagram Methods 0.000 description 1
- 238000007865 diluting Methods 0.000 description 1
- 239000003085 diluting agent Substances 0.000 description 1
- 238000004090 dissolution Methods 0.000 description 1
- 230000000694 effects Effects 0.000 description 1
- 238000012921 fluorescence analysis Methods 0.000 description 1
- 239000012634 fragment Substances 0.000 description 1
- 210000004907 gland Anatomy 0.000 description 1
- 230000009027 insemination Effects 0.000 description 1
- 238000003780 insertion Methods 0.000 description 1
- 230000037431 insertion Effects 0.000 description 1
- 238000011835 investigation Methods 0.000 description 1
- 239000007788 liquid Substances 0.000 description 1
- 239000012160 loading buffer Substances 0.000 description 1
- 230000029849 luteinization Effects 0.000 description 1
- 238000000816 matrix-assisted laser desorption--ionisation Methods 0.000 description 1
- 230000035800 maturation Effects 0.000 description 1
- 238000005259 measurement Methods 0.000 description 1
- 238000002156 mixing Methods 0.000 description 1
- ZFFFJLDTCLJDHL-UHFFFAOYSA-N pregnanediol glucuronide Natural products C1CC2(C)C3CCC4(C)C(C(O)C)CCC4C3CCC2CC1OC1OC(C(O)=O)C(O)C(O)C1O ZFFFJLDTCLJDHL-UHFFFAOYSA-N 0.000 description 1
- 230000001737 promoting effect Effects 0.000 description 1
- 238000003908 quality control method Methods 0.000 description 1
- 239000000376 reactant Substances 0.000 description 1
- 239000011541 reaction mixture Substances 0.000 description 1
- 230000001850 reproductive effect Effects 0.000 description 1
- 230000001568 sexual effect Effects 0.000 description 1
- 230000009870 specific binding Effects 0.000 description 1
- 239000000126 substance Substances 0.000 description 1
- 239000006228 supernatant Substances 0.000 description 1
- 230000001225 therapeutic effect Effects 0.000 description 1
- 210000004885 white matter Anatomy 0.000 description 1
- 239000012224 working solution Substances 0.000 description 1
Classifications
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
- C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
- C07K14/76—Albumins
- C07K14/765—Serum albumin, e.g. HSA
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
- C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
- C07K14/76—Albumins
- C07K14/77—Ovalbumin
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
- C07K14/795—Porphyrin- or corrin-ring-containing peptides
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K19/00—Hybrid peptides, i.e. peptides covalently bound to nucleic acids, or non-covalently bound protein-protein complexes
-
- 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
-
- 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
- G01N33/581—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving labelled substances with enzyme label (including co-enzymes, co-factors, enzyme inhibitors or substrates)
-
- 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
Landscapes
- Health & Medical Sciences (AREA)
- Life Sciences & Earth Sciences (AREA)
- Chemical & Material Sciences (AREA)
- Molecular Biology (AREA)
- Engineering & Computer Science (AREA)
- Biochemistry (AREA)
- Immunology (AREA)
- Hematology (AREA)
- General Health & Medical Sciences (AREA)
- Biomedical Technology (AREA)
- Medicinal Chemistry (AREA)
- Urology & Nephrology (AREA)
- Organic Chemistry (AREA)
- Proteomics, Peptides & Aminoacids (AREA)
- Biotechnology (AREA)
- General Physics & Mathematics (AREA)
- Biophysics (AREA)
- Cell Biology (AREA)
- Genetics & Genomics (AREA)
- Microbiology (AREA)
- Pathology (AREA)
- Food Science & Technology (AREA)
- Physics & Mathematics (AREA)
- Analytical Chemistry (AREA)
- Gastroenterology & Hepatology (AREA)
- Zoology (AREA)
- Toxicology (AREA)
- Gynecology & Obstetrics (AREA)
- Pregnancy & Childbirth (AREA)
- Reproductive Health (AREA)
- Investigating Or Analysing Biological Materials (AREA)
- Peptides Or Proteins (AREA)
Abstract
The present invention relates to female fertility ability non-invasive monitoring technology, specifically, relate to a kind of complete quick noinvasive urine examination technology the most quantitatively or semi-quantitatively to measure the cycle growing amount of progesterone metabolite in female urine, with this monitor with assess women luteal function, judge different ovulatory cycle type, determine whether really to ovulate and ovulate after can become pregnant.First embodiment of the invention, relates to a kind of protein hybridization couplet for measuring pregnanediol glucuronate, comprises pregnanediol glucuronate and coupling protein, it is characterised in that also comprise the junctional complex of both connections.The neurophysin couplet of the present invention detects PdG with unsaturated traget antibody, and its sensitivity is up to 0.1ng/ml, and the range of linearity can be controlled between 0.1 50ng/ml.
Description
Technical field
The present invention relates to female fertility ability non-invasive monitoring technology, entirely fixed in particular to one
Amount or semiquantitative quick noinvasive urine examination technology measure the cycle generation of progesterone metabolite in female urine
Amount, monitors with this and assesses women luteal function, judges different ovulatory cycle type, determines whether
Can become pregnant after real ovulation and ovulation.
Background technology
According to " China's sterility and infertility Current Situation Investigation report " display in 2012, China's sterility and infertility rate was high
Reaching 12.5%, wife's side reason accounts for more than 50%.Number is more than 50,000,000.Sterility and infertility Endodontic failure
Patient accounts for 66%, wherein 98.9% causes because the most promptly and accurately detecting.Therefore, invention one
The women luteal function monitoring technology that noinvasive, inexpensive, hospital and family are easy-to-use, is particularly well-suited to China
The needs of prenatal and postnatal care.
When menstrual cycle starts, in each ovary, about 15-20 ovum starts maturation.Dress
The follicle having ovum produces estrogen.Finally, a follicle is in the ascendance and keeps ripe,
And remaining follicle starts to disintegrate.The estrogen level of this dominant follicle is a few days ago substantially increased in ovulation,
Until reaching critical level.This high estrogen level, triggers another kind of hormone referred to as rush corpus luteum and swashs
The surge of element (LH).Trigger ovulation, cause ovum to discharge from ovary.Indicate the knot of ovulatory cycle
Bundle.
The estrogen and the LH peak that successively occur in menstrual cycle have pass closely with ovary ovulation period
System.Once estrogen or the appearance at LH peak, ovulation typically occurs in following 12-48 hour.
Therefore, menstrual cycle is monitored estrogen or LH peak value, may be used to determine optimal Time to pregnancy,
Or need to take the time of contraceptives.
Before ovulation, the appearance at e surge and LH peak can not confirm that ovulation actually occurs, because
Estrogen increases sharply and LH release peak not always causes ovulation.Even with real ovulation, but by
After ovum, the body phase is not enough or luteal phase is short, and causes infertile.This have ovulation but the most conceptive existing
As, it is infertile modal reason in current women.
Commercialization inspection in the market is limited only to hormone blood examination or LH urine examination, due to hemocrinia
Inspection needs large-scale instrument, both inconvenient, and checks the result diagnosis for infertility, again without unified mark
Accurate.And LH urine examination (includes the CN00112779.9 patent of Kunming Yunnan University, Gansu Lanzhou University
The CN20110072240.9 patent of CN200610200333 patent and Wuhan deep all places hall) then
It is mainly used in the preliminary estimation of the onset of ovulation.Therefore, up to now, the most also do not have one convenient
Easy-to-use commercialization sterility and infertility external non-invasive diagnosis product, for Quantitative Monitoring women luteal function,
Determine the female fertility and hormone infertility can become pregnant after having anovulation, ovulation and be correlated with.
Summary of the invention
It is an object of the invention to, according to during ovulation in female urine hormone progesterone growing amount and ovulation and
The substantial connection become pregnant, measures progesterone urine metabolite-pregnanediol glucuronate after ovulation
The growing amount of (pregnanediol glucuronide PdG), thus provide a kind of hospital, family and
Individual carry out complete the most quantitatively or semi-quantitatively, inexpensive convenient immune analysis method and detection kit.With row
The ovum phase is reference point, and in being ovulated latter 6 days by quantitative determination, the concentration of PdG in urine, monitors corpus luteum
The change of function, thus examine and can become pregnant with or without after true ovulation and ovulation.(1) when pregnanediol value
< during 2.0mg/24h, show anovulation or elementization not ruptured follicle (luteinized unruptured
Follicle, LUF).(2) if pregnanediol value is between 2.0-3.0mg/24h, row is indicated
Ovum, but ILP or short after ovulation, it is impossible to become pregnant.(3) only when pregnanediol value > 3.0mg/24
During h, this cycle is only the ovulatory cycle with complete fertility.Therefore, in ovulating latter 6 days pregnant two
Alcohol number is that women has complete fertility and the criterion that can normally become pregnant more than 3.0mg/24h.
If by pregnanediol value in latter 6 days of ovulation whether more than 3.0mg/24h as standard, this
Bright also can analyze such as colloidal gold semi-quantitative immunoreagent bar with quantitative immune, determine whether women has
Completely fertility can normally be become pregnant (> 3.0mg/24h) or suffer from infertility (≤3.0mg/24h).
The present invention is by being connected pregnanediol glucuronate with coupling protein by junctional complex (linker)
Get up to be formed the protein hybridization couplet of pregnanediol glucuronate, and measure the egg of pregnanediol glucuronate
White hybridized coupling body, monitors the change of luteal function, thus after examining with or without true ovulation and ovulation
Can become pregnant.
Junctional complex of the present invention be divided into hydrophobicity (such as simple non-resilient carbochain) and hydrophilic (as oxygen-containing,
The heteroatomic elastic carbochain of sulfur, nitrogen etc.), its a length of 1-100 carbon atom, preferably 5-25
Carbon atom, more preferably 6-20 carbon atom, most preferably 6-18 carbon atom, wherein one or more
Carbon atom can be selected from the hetero atom of oxygen, sulfur, nitrogen etc. and replace.Although, the length of junctional complex and it
Hydrophilic or hydrophobic performance, affect the spirit of immunoassay by different way, to some extent simultaneously
Sensitivity and specificity.But relation between the two there is no so far and systematically studies, more do not report as
What effectively utilizes junctional complex length and hydrophilic/hydrophobic energy, and the business application for immunoassay is such as exempted from
The exploitation application of epidemic disease assay kit, carries out reasonable design.
The present invention, by changing hydrophilic and hydrophobic and the length of chain of junctional complex, can control pregnanediol Portugal
Glycuronide connection quantity on coupling protein, the most just have adjusted pregnanediol glucuronate and examines at reagent strip
Density on survey line, i.e. density are the highest, and the range of linearity is the widest, and density is the lowest, and sensitivity is the best.With
Time, the length of carbochain and type also affect pregnanediol glucuronate-coupling protein hybridized coupling body and antibody
Combination quality, can be prevented effectively from the non-specific binding with antibody, and suitably long carbochain is permissible
Minimizing is sterically hindered with antibodies.
First embodiment of the invention, relates to a kind of albumen for measuring pregnanediol glucuronate
Hybridized coupling body, comprises pregnanediol glucuronate and coupling protein, it is characterised in that also comprise connection
Both junctional complexs.
Second embodiment of the invention, relates to the couplet of the first embodiment, it is characterised in that
Described junctional complex is for comprising 1-100 carbon atom, and preferably 5-25 carbon atom, more preferably 6-20 is individual
The straight or branched group of carbon atom, most preferably 6-18 carbon atom.
Third embodiment of the invention, relates to the couplet of the second embodiment, it is characterised in that
One or more carbon atoms of described junctional complex are exchanged for heteroatoms.
Fourth embodiment of the invention, relates to the couplet of the 3rd embodiment, it is characterised in that
Described hetero atom is selected from oxygen, nitrogen and sulfur.
Fifth embodiment of the invention, relates to the coupling any one of the first to the 4th embodiment
Body, it is characterised in that described coupling protein selected from ovalbumin (OVA), bovine serum albumin (BSA),
Hemocyanin (KLH).
Sixth embodiment of the invention, relates to the coupling any one of the first to the 4th embodiment
Body, described couplet is selected from:
(1) pregnanediol glucuronate--6 atom carbochain--ovalbumin couplet
(2) pregnanediol glucuronate--13 atom carbochain--ovalbumin couplet
(3) pregnanediol glucuronate--20 atom carbochain--ovalbumin couplet
(4) pregnanediol glucuronate--18 atom carbochain--ovalbumin couplet.
Seventh embodiment of the invention, relates to the coupling any one of the first to the 4th embodiment
Body, it is characterised in that described pregnanediol glucuronate is 1:1 with junctional complex and coupling protein ratio
To 100:1.
Eigth embodiment of the invention, relates to a kind of reagent for detecting pregnanediol glucuronate
Box, the protein hybridization any one of the first to the 7th embodiment that described test kit comprises the present invention is even
Conjuncted, it is characterised in that also to comprise the antibody specific binding with described protein hybridization couplet.
Ninth embodiment of the invention, relates to the test kit of the 8th embodiment, it is characterised in that
Described antibody is monoclonal antibody or polyclonal antibody.
Tenth embodiment of the invention, relates to the test kit of the 9th embodiment, it is characterised in that
Possibly together with the detectable being connected with described antibody.
Eleventh embodiment of the invention, relates to the test kit of the 9th embodiment, and its feature exists
In, possibly together with the second antibody being combined with described antibody specificity.
Twelfth embodiment of the invention, relates to the test kit of the 11st embodiment, its feature
Being, described antibody is 1:2 to 2:1 with the ratio of second antibody.
Thirteenth embodiment of the invention, relates to the test kit of the 11st or the 12nd embodiment,
It is characterized in that, possibly together with the detectable being connected with described second antibody.
Fourteenth embodiment of the invention, relates to the tenth or the 13rd test kit of embodiment,
It is characterized in that, described detectable is selected from colloid, chromophore, chemiluminescent groups, fluorescence
Group, isotope or enzyme.
Fifteenth embodiment of the invention, relates to a kind of test kit for monitoring luteal function,
It is characterized in that, comprise the couplet any one of the first to the 14th embodiment or the examination of the present invention
Agent box.
The beneficial effects of the present invention is, for having the women of complete fertility, the present invention is undoubtedly
Information accurately is provided for the ideal time of natural conception and the time started of gestation.For suffering from not
Pregnant disease the women treated, the present invention provides one simultaneously and can judge to control also to doctor and patient
Therapeutic effect the most successfully ideal tools.At reproductive center, progesterone provided by the present invention measures undoubtedly
For determining the Best Times of artificial insemination, test-tube baby's (IVF) early stage of development induces for promoting sexual gland hormone
The monitoring of ovulation and determine ovulation and take time of ovum, thus improve Pregnancy Success rate, give huge
Help.
Should be understood that within the scope of the present invention, the above-mentioned various technical characteristics of the present invention and (real below
Execute example) in can be mutually combined between each technical characteristic of specifically describing, thus constitute new or preferably
Technical scheme.
Accompanying drawing explanation
Fig. 1 .PdG immunity test strip and quantitative fluorescence analysis schematic diagram
Fig. 2. the range of linearity analysis chart that albumen addition colloid gold immune is analyzed
Fig. 3. the range of linearity analysis chart of albumen addition fluorescence immunoassay
Detailed description of the invention
The present inventor, through extensively in-depth study, has been surprisingly found that, the pregnanediol of the present invention first
The protein hybridization couplet of glucosiduronic acid and detection method thereof and detection kit, by changing junctional complex
Hydrophilic and hydrophobic and the length of chain, pregnanediol glucuronate connection number on coupling protein can be controlled
Amount so that mensuration sensitivity is up to 1ng/ml, and the range of linearity can be controlled between 0.1-50ng/ml,
Thus complete the present invention.
Junctional complex
Herein described " junctional complex ", refers to by pregnanediol glucuronate with coupling protein even
The linker being connected together, includes but not limited to can use described in US7521425 and US8288349
Make the compound of linker.
Coupling protein
Herein described " coupling protein ", refers to by junctional complex of the present invention and pregnant two
The protein that alcohol glucosiduronic acid links together, include but not limited to ovalbumin, bovine serum albumin,
Hemocyanin.
Couplet
Herein described " couplet ", refers to that pregnanediol glucuronate and coupling protein are by this
The adduct that bright described junctional complex links together, includes but not limited to:
Pregnanediol glucuronate--6 atom carbochain--ovalbumin couplet (1)
Pregnanediol glucuronate--13 atom carbochain--ovalbumin couplet (2)
Pregnanediol glucuronate--20 atom carbochain--ovalbumin couplet (3)
Pregnanediol glucuronate--18 atom Polyethylene Glycol carbochain--ovalbumin couplet (4)
Antibody
Herein described " antibody ", refer to specifically with pregnanediol glucuronate and/or idol
The antibody that conjuncted middle pregnanediol glucuronate combines, in this application, term " antibody " and " special
Property antibody " it is used interchangeably.Present invention additionally comprises and pregnanediol glucuronate is had specific many grams
Grand antibody and monoclonal antibody, especially monoclonal antibody.Here, " specificity " refers to antibody energy
It is incorporated into pregnanediol glucuronate, but nonrecognition and be incorporated into other non-related molecules.The present invention is not only
Including complete monoclonal or polyclonal antibody, but also include that there is immunocompetent antibody fragment,
Such as Fab' or (Fab)2Fragment；Heavy chain of antibody；Light chain of antibody；Genetically engineered scFv divides
Son (Ladner etc., United States Patent (USP) No.4,946,778)；Or chimeric antibody, as having murine antibody knot
Close specificity but retain the antibody of the antibody moiety from people.The antibody of the present invention can pass through this area
Various technology known to the skilled person of in are prepared.
Detectable
Herein described " detectable ", refers to produce the material of detection signal, described
Detectable includes but not limited to colloid, chromophore, chemiluminescent groups, fluorogen, coordination
Element or enzyme.
As illustrated in fig. 1, the test strips of the present invention, can by nitrocellulose filter, sample pad,
Adsorptive pads and PVC or PS base plate etc. are constituted.Draw on nitrocellulose filter and have two lines: one is
PdG-protein conjugates coated detection line, another is independent two anti antibodys coated internal standard nature controlling line.
Gold colloidal (20nm-100nm) or various fluorometric reagent both can direct labelling PdG monoclonal antibodies, it is possible to
The first anti-polyclonal antibody of labelling two, is combined with PdG monoclonal antibody immunity the most again.As preferably side
Case, the first anti-polyclonal antibody of labelling two, then, then adjust and optimize two anti-labels and PdG Dan Ke
The optimal proportion of grand antibody, thus obtain maximally effective immunity and combine.It is applicable to the present invention's and colloid
Golden or that fluorescent quantitation test strip is general portable quantitative analysis instrument (TSR3000 Shanghai Jie Ningsheng
Thing Science and Technology Ltd.), the depth or the fluorescence intensity level of colloid golden red color on reagent strip can be carried out soon
Speed accurate quantitative analysis.The weight of its fluorescence intensity level or gold colloidal redness is dense with PdG in female urine
Degree is inversely.
The Cleaning Principle of the present invention: by the female urine drop after dilution by a certain percentage to sample pad
(a step dry method), or join in a test tube, drips in sample pad after fully mixing that (two steps are wet again
Method).Contain PdG monoclonal antibody murine antibody in sample pad or in test tube and use fluorescently-labeled sheep anti mouse respectively
Two anti-and goat-anti rabbits two anti-(as internal standard).In sample pad or in test tube, in urine sample PdG first with
Fluorescence PdG monoclonal antibody combines.By capillarity, in sample pad, remaining fluorescence PdG monoclonal antibody flows to again
Detection line on reagent strip, carries out immunity knot with the PdG-protein conjugates being coated in advance on detection line
Close, so that detection line display fluorescence.And the anti-fluorescence of goat-anti rabbit two in sample pad, Quality Control will be flowed to
With coated rabbit antibodies on line, nature controlling line is made to display that fluorescence.Fluorescent test paper strip is inserted portable
After formula quantitative analysis instrument, the fluorescence intensity on ELISA test strip line, nature controlling line is the most quantitatively divided
In analysis, and applying detection line and nature controlling line, the ratio of fluorescence intensity, determines the concentration of PdG in urine.
No matter whether containing hormone PdG in urine sample, nature controlling line shows fluorescence all the time, if nature controlling line does not show fluorescence,
Then show that test strips is invalid.It will be understood by those skilled in the art that and carry out labelling two when gold colloidal replaces fluorescence
During anti antibody, above-mentioned quantitative immunoassay principle is equally applicable to quantitatively divide with colloid gold immune test paper bar
Analysis.
As in figure 2 it is shown, sensitivity low can reach 1ng/ml, the range of linearity can be controlled in
Between 5-100ng/ml (neurophysin couplet 1,2) or 1-100ng/ml (neurophysin couplet 4)
(see Fig. 2), gold colloidal quantitative testing test paper bar can meet hospital and household person to PdG (i.e. completely
Luteal function) Quantitative Monitoring.
As it is shown on figure 3, with unsaturated traget antibody detection PdG, its sensitivity up to 0.1ng/ml,
The range of linearity can be controlled in 0.5-20ng/ml (neurophysin couplet 1,2) or 0.1-50ng/ml it
Between (neurophysin couplet 4), fluorescent quantitation test strip can meet hospital and family completely
People's Quantitative Monitoring to PdG (i.e. luteal function).
The foregoing invention content of the present invention be not intended as describe the present invention each disclosed embodiment or
Every kind of embodiment.Hereinafter describe and more particularly exemplify exemplary embodiment.Throughout the specification,
Being thered is provided by example and instruct, these examples can be used in various combinations.In either case,
Exemplified content is as just representative illustration, and should not be construed as exclusiveness example.
Below in conjunction with specific embodiment, the present invention is expanded on further.Only should be understood that these embodiments
For the present invention being described rather than limiting the scope of the present invention.
Embodiment
It will be understood by those skilled in the art that following example are the most by way of example to couplet of the present invention
Synthesis illustrate, any can be used to be connected to junctional complex pregnanediol glucuronate and coupling protein
The method coupled together can use, teaching according to embodiments of the present invention, those skilled in the art
Can be controlled both by the reaction ratio of regulation pregnanediol glucuronate with junctional complex and coupling protein
Ratio.
Embodiment 1:PdG-Linker-CO2The preparation of H (1)
PdG (0.1mmol) is dissolved in DMF (0.5mL), add DCC (0.143mmol) and
The DMF solution (0.8ml) of NHS (0.143mmol).Reactant mixture is stirred 7 hours, is subsequently added
(0.119mmol) chloroform (1mL) of 6-aminocaprolc acid and triethylamine (0.5mL) solution.Stirring was reacted
At night, after removing solvent, residue is with dichloromethane: methanol (8:2) chromatographs on silica gel: obtain product
PdG-Linker-CO2H(1；0.025mmol).Productivity: 25%.
The preparation of embodiment 2:PdG-Linker-OVA couplet (1)
PdG-linker-CO2H(1；0.05mmol) in small test tube, the lower addition of stirring is above-mentioned
DCC/DMF solution 0.2ml dissolves, and then adds 0.2ml, puts and reacts 3 hours at room temperature directly
To there being a large amount of Precipitation (carbamide is its by-product).NaH2PO4.2H2O (0.1186g) and anhydrous
Na2HPO4(0.46g) it is dissolved in 20ml water and is prepared as 0.2M phosphate buffer (pH7.4).?
At 4 DEG C, OVA (44.34mg, 1 μm ol) stirring and dissolving is delayed in above-mentioned 4ml, 0.2M phosphoric acid
Rush in liquid；Above-mentioned DCC/NHS PdG solution (DMF solution of 0.4ml) is under agitation instilled to OVA
In solution.The coupling reaction of PdG-linker-OVA is stirred overnight in 4 DEG C.PdG-Linker-OVA
Couplet with water dialyse 4 times (each 1.2L), 48 hours, then with phosphate buffer (1.2L,
PH7.4) dialysis 24 hours.Collect after purified PdG-Linker-OVA couplet (1) dialysis,
Lyophilizing is stored.
The preparation of embodiment 3:PdG-Linker-OVA couplet (2-4)
With the similar approach of above-described embodiment 1, take three parts of PdG (0.1mmol) and be dissolved in DMF (0.5mL)
In, add DCC (0.143mmol) and the DMF solution (0.8ml) of NHS (0.143mmol).Will reaction
Mixture stir 7 hours, be separately added into subsequently 7-azepine-8-oxo-13-amino-tridecanoic acid,
7,14-diaza-8,15-dioxo-20-amino-arachic acid and 4-oxo-5-azepine
(0.119mmol) chloroform (1mL) of-9,12,15-trioxa-18-amino-octadecanoid acid and triethylamine
(0.5mL) solution.Overnight, after removing solvent, residue is with dichloromethane: methanol (8:2) in stirring reaction
Silica gel chromatographs: obtain product PdG-Linker-CO2H(2-4；0.023-0.026mmol).Produce
Rate is respectively as follows: 23-26%.
With the method for above-described embodiment 2, take above-mentioned PdG-linker-CO respectively2H(2-4；0.05
Mmol) in small test tube, the lower above-mentioned DCC/DMF solution 0.2ml of addition of stirring dissolves, and then adds
0.2ml, puts and reacts 3 hours at room temperature until there being a large amount of Precipitation (carbamide is its accessory substance).
NaH2PO4.2H2O (0.1186g) and anhydrous Na2HPO4(0.46g) preparation it is dissolved in 20ml water
Become 0.2M phosphate buffer (pH7.4).At 4 DEG C, OVA (44.34mg, 1 μm ol) is stirred
Mix and be dissolved in above-mentioned 4ml, in 0.2M phosphate buffer；By above-mentioned DCC/NHS PdG solution (0.4ml
DMF solution) under agitation instill to OVA solution.The coupling reaction of PdG-linker-OVA is in 4
DEG C it is stirred overnight.The couplet of PdG-Linker-OVA is with water 4 times (each 1.2L) of dialysis, and 48 is little
Time, then dialyse 24 hours with phosphate buffer (1.2L, pH7.4).Purified
Collect after PdG-Linker-OVA couplet (2) dialysis, lyophilizing is stored.
Embodiment 4: the preparation of colloid gold reagent bar
The labelling of hormone pregnanediol glucuronate monoclonal antibody (mAb): by colloidal gold solution
After pH value is adjusted to 7.5-8.5, mAb labelling uses the half of saturation flags amount, has met sensitivity
Needs.If with the range of linearity as target, then mAb uses saturation flags.After mAb labelling is complete,
Carry out the closing of 1%BSA.Finally, implement centrifugation, after the careful removing supernatant, often add
Rule buffer (10mM PBS, pH7.4,1%BSA) resuspended mAb-colloidal gold conjugate, and under 4 °
Preserve.By adsorptive pads, 8964 materials (sample pad and couplet pad) respectively with nitrocellulose filter
After the most overlapping coincidence 1-2mm, carry out a film or draw film.Two anti-loading buffer are diluted to
After 1mg/ml, nature controlling line carries out a film (0.5-1 μ l) or draws film (1 μ l/cm, 0.8mg/ml).
Application same principle, carries out PdG-protein conjugates a film (1 μ l) on detection line or draws film
(1μl/cm).After natural drying 1 hour, shred (4mm width) by test strips.
Test (two step wet methods): by PdG antigen standard specimen with phosphate buffer (PB, 10mM, pH7.2)
Dilution, standard specimen blank is diluent.With sensitivity as target, use unsaturated labelling gold colloidal (the fullest
With labelling one incomplete antibody mAb consumption), after diluting with working solution 1:1, point sample (10 μ l) is in sample
On pad.With the range of linearity as target, then use saturation flags gold colloidal (the whole antibody of saturation flags
mAb).After same 1:1 dilution, on point sample (10-12 every sample pad of μ l/).Controlling the time (7 points
Clock) on the premise of, test strips is installed, inserts quantitative analysis instrument (TSR3000, the prompt peaceful biology in Shanghai
Science and Technology Ltd.) carry out card reader reading, it is thus achieved that quantitative analysis results.
Low can reach 1ng/ml with unsaturated traget antibody detection PdG: sensitivity, the range of linearity is controlled
(neurophysin is even between 5-100ng/ml (neurophysin couplet 1,2) or 1-100ng/ml for system
Conjuncted 4).From Fig. 2 result: above-mentioned gold colloidal quantitative testing test paper bar can meet hospital completely
With the household person Quantitative Monitoring to PdG (i.e. luteal function).
Embodiment 5: the preparation of fluorometric reagent bar
PdG-linker-OVA (1-4) is carried out on detection line a film (1 μ l) or draws film
(1μl/cm).After natural drying 1 hour, shred (4mm width) by test strips, at control line
Upper goat-anti rabbit two is anti-to carry out a film or draws film.Containing PdG sample 0.1ml and 0.05ml PdG monoclonal antibody mouse-anti
Body, two anti-mixture (sheep anti mouse and the goat-anti rabbit two resist) quantitative point of 0.05ml fluorescence (such as Cy3) labelling
Carry out lateral chromatography after in sample pad, on the premise of the time of control (7 minutes), test strips is filled
Card, insertion quantitative analysis instrument carry out card reader reading (ESE-Quant FLUO reader, DCN Inc),
Obtain quantitative analysis results.
Embodiment 6: data analysis is tested
Neurophysin couplet is for the analysis of immune reagent kit
[note]: neurophysin conjugation (hapten density), the most each protein molecular is connected and is swashed
The average number of element molecule, is to pass through Matrix-assisted laser desorption ionization
(Matrix-Assisted Laser Desorption/Ionization Time of Flight Mass
Spectrometry, MALDI-TOF-MS, Bruker Daltonics AutodlexTM spectrometer)
Mensuration and obtain.
Present inventors have surprisingly discovered that, the height of immune reagent kit sensitivity for analysis and linear model
The size enclosed, will depend on " neurophysin matter conjugation " and " water dissolution of neurophysin matter simultaneously
Property " two factors, the present inventor without being bound by existing principle, propose the most in a creative way following the most absolutely
It is different from the brand-new mentality of designing of routine, and achieves excellent technique effect:
(1) in the synthesis of neurophysin couplet, neurophysin matter conjugation (i.e. hapten density)
Simultaneously tight with the molecule molar ratio of albumen with hormone molecule and the hydrophilicity of junctional complex and hormone
Close relevant；In general, both hydrophilicities are the best, hormone molecule and the mol ratio of protein molecular
The highest, then hormone is the highest with the conjugation of albumen；Vice versa.Due to the hydrophobicity of hormone molecule,
The hydrophilicity of junctional complex just seems particular importance.Before controlling identical hormone/protein molecular mol ratio
Put, when hydrophobic junctional complex is risen to 13 atoms (2) or 20 atoms (3) by 6 atoms (1),
Its neurophysin conjugation also drops to 2.4 from 4.1 accordingly.From its absolutely unlike, work as hydrophilic
18 atom Polyethylene Glycol as (4) during junctional complex, its neurophysin matter conjugation is up to 11, almost
It is 5 times with similar chain length hydrophobicity junctional complex (3).
(2) between hormone and protein molecular introduce different performance (hydrophilic or hydrophobicity) and
The junctional complex of different length, does not only result in the huge difference beyond routine anticipation of neurophysin matter conjugation
Not, the water-soluble of final products-neurophysin matter is also directly affected；And its water-soluble is determined
Determine this neurophysin matter quantity on immunoreagent bar detection line.Therefore, hormone egg on detection line
The hormone number connected in the quantity of white matter and each protein molecular, just determines hormone on detection line
Molecular density.Thus affect the sensitivity and linear measurement range that immune reagent kit is analyzed.When hydrophobic
When junctional complex is risen to 20 atoms (3) by 6 atoms (1), not only its neurophysin conjugation is from 4.1
Drop to 2.4, and the water-soluble of the neurophysin couplet after its synthesis also drastically reduces, this
Due to ultralow neurophysin conjugation and water-soluble, cause detecting hormone molecule density on line the lowest,
Cannot be carried out immune reagent kit analysis effectively.And when hydrophilic 18 atom Polyethylene Glycol are as connection
Thing, provide not only very highland neurophysin conjugation (11), is additionally, since longer chain polyethylene glycols even
Connect the excellent hydrophilic of thing so that hormone-Polyethylene Glycol-protein couplet has good water-soluble,
Thus neurophysin conjugate point sample in high concentration can be allowed to detecting on line.This by high protein concentration,
High hormone conjugation and the hormone molecule highdensity detection line that causes, be immune reagent kit analysis undoubtedly
Provide the wider range of linearity.Further, since the long-chain of Polyethylene Glycol junctional complex (18 atom) improves
Effective combination of antibody/hormone, adds Polyethylene Glycol carbochain and has and avoid antibody non-specific binding
Premium properties, therefore, hormone-Polyethylene Glycol-protein couplet (4) immune reagent kit analyze in
Sensitivity the most fitst water.
When introducing element or the preferred embodiment of the present invention of the present invention, article " ", " being somebody's turn to do "
" described " means and there is one or more element.Term " comprises ", " including " and " having "
Comprise, mean the extra element that can exist in addition to listed element.
Owing to can hereinbefore carry out various change, without deviating from the scope of the present invention, this mean be included in
All the elements in upper description should be read as illustrative, does not has restrictive implication.The present invention's
Scope is limited by appending claims, and embodiment above can be carried out without departing from
The amendment of the scope of the present invention.
Claims (15)
1., for measuring a protein hybridization couplet for pregnanediol glucuronate, comprise pregnanediol Portugal
Glycuronide and coupling protein, it is characterised in that also comprise the junctional complex of both connections.
2. the couplet of claim 1, it is characterised in that described junctional complex is for comprising 1-100
Individual carbon atom, preferably 6-25 carbon atom, more preferably 6-20 carbon atom, most preferably 6-18 carbon
The straight or branched group of atom.
3. the couplet of claim 2, it is characterised in that one or more in described junctional complex
Carbon atom is exchanged for heteroatoms.
4. the couplet of claim 3, it is characterised in that described hetero atom is selected from oxygen, nitrogen and sulfur.
5. the couplet any one of claim 1-4, it is characterised in that described coupling protein selects
From ovalbumin, bovine serum albumin, hemocyanin.
6. the couplet any one of claim 1-4, is selected from:
(1) pregnanediol glucuronate--6 atom carbochain--ovalbumin couplet
(2) pregnanediol glucuronate--13 atom carbochain--ovalbumin couplet
(3) pregnanediol glucuronate--20 atom carbochain--ovalbumin couplet
(4) pregnanediol glucuronate--18 atom carbochain--ovalbumin couplet.
7. the couplet any one of claim 1-4, it is characterised in that described pregnanediol glucose
Thuja acid is 1:1 to 100:1 with junctional complex and coupling protein ratio.
8., for detecting a test kit for pregnanediol glucuronate, comprise claim 1-7 arbitrary
The protein hybridization couplet of item, it is characterised in that also comprise and described protein hybridization couplet specificity
In conjunction with antibody.
9. the test kit of claim 8, it is characterised in that described antibody is monoclonal antibody or many
Clonal antibody.
10. the test kit of claim 9, it is characterised in that possibly together be connected with described antibody
Detectable.
The test kit of 11. claim 9, it is characterised in that possibly together with described antibody specificity
In conjunction with second antibody.
The test kit of 12. claim 11, it is characterised in that described antibody and the ratio of second antibody
Example is 1:2 to 2:1.
The test kit of 13. claim 11 or 12, it is characterised in that possibly together with described second
The detectable that antibody connects.
The test kit of 14. claim 10 or 13, it is characterised in that described detectable
Selected from colloid, chromophore, chemiluminescent groups, fluorogen, isotope or enzyme.
15. 1 kinds for monitoring the test kit of luteal function, it is characterised in that comprises claim
Couplet any one of 1-14 or test kit.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201910771749.9A CN110526964B (en) | 2015-04-17 | 2015-04-17 | Rapid noninvasive female corpus luteum function monitoring technology |
Applications Claiming Priority (2)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN2015076841 | 2015-04-17 | ||
| CNPCT/CN2015/076841 | 2015-04-17 |
Related Child Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN201910771749.9A Division CN110526964B (en) | 2015-04-17 | 2015-04-17 | Rapid noninvasive female corpus luteum function monitoring technology |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| CN106146651A true CN106146651A (en) | 2016-11-23 |
| CN106146651B CN106146651B (en) | 2019-08-23 |
Family
ID=58058149
Family Applications (2)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN201510185202.2A Active CN106146651B (en) | 2015-04-17 | 2015-04-17 | The quick non-invasive monitoring technology of women luteal function |
| CN201910771749.9A Active CN110526964B (en) | 2015-04-17 | 2015-04-17 | Rapid noninvasive female corpus luteum function monitoring technology |
Family Applications After (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN201910771749.9A Active CN110526964B (en) | 2015-04-17 | 2015-04-17 | Rapid noninvasive female corpus luteum function monitoring technology |
Country Status (1)
| Country | Link |
|---|---|
| CN (2) | CN106146651B (en) |
Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN108459167A (en) * | 2018-03-16 | 2018-08-28 | 江苏维尔生物科技有限公司 | A kind of kit and preparation method thereof quantitatively detected for β-HCG, PDG in human urine |
| EP4017375A4 (en) * | 2019-08-19 | 2023-12-06 | MFB Fertility, Inc. | Systems and methods for menstrual cycle testing |
Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| EP0075193A1 (en) * | 1981-09-23 | 1983-03-30 | Northwestern University | Method of pregnanediol glucuronide detection and indicator strip for use therein |
| CN1314593A (en) * | 2000-03-16 | 2001-09-26 | 昆明云大生物技术有限公司 | Test paper for detecting sterility and its test method |
| CN101389964A (en) * | 2005-10-24 | 2009-03-18 | 马纳瓦图生物技术投资有限公司（Mbil） | Ovulation cycle monitoring and management |
| CN1892223B (en) * | 2005-04-11 | 2012-08-22 | 兰州大学 | Colloidal gold semi-quantitative quick immunity diagnosis test-paper stripe |
Family Cites Families (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101058584B (en) * | 2007-05-23 | 2010-05-26 | 上海骏捷生化科技有限公司 | Method of preparing cefprozil parent nucleus 7-amino-3-propenylcephalosporanic acid |
| CN101289457A (en) * | 2008-06-13 | 2008-10-22 | 河源市制药工程技术研究开发中心 | Novel process for synthesizing 3-deacetyl cefuroxime sodium (DCCF) |
-
2015
- 2015-04-17 CN CN201510185202.2A patent/CN106146651B/en active Active
- 2015-04-17 CN CN201910771749.9A patent/CN110526964B/en active Active
Patent Citations (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| EP0075193A1 (en) * | 1981-09-23 | 1983-03-30 | Northwestern University | Method of pregnanediol glucuronide detection and indicator strip for use therein |
| IE53755B1 (en) * | 1981-09-23 | 1989-02-01 | Univ Northwestern | Method of determining the concentration of pregnanediol glucuronide in a woman's urine and test device for use therein |
| CN1314593A (en) * | 2000-03-16 | 2001-09-26 | 昆明云大生物技术有限公司 | Test paper for detecting sterility and its test method |
| CN1892223B (en) * | 2005-04-11 | 2012-08-22 | 兰州大学 | Colloidal gold semi-quantitative quick immunity diagnosis test-paper stripe |
| CN101389964A (en) * | 2005-10-24 | 2009-03-18 | 马纳瓦图生物技术投资有限公司（Mbil） | Ovulation cycle monitoring and management |
Non-Patent Citations (2)
| Title |
|---|
| C. MARK SMALES等: "Purification and characterization of lysozyme-pregnanediol glucuronide conjugates: the effect of the hapten and coupling reagent on the substitution level, sites of acylation and the consequences for the development of future immunoassays", 《BIOTECHNOLOGY AND APPLIED BIOCHEMISTRY》 * |
| 刘文泰: "《医学免疫学》", 28 February 2009, 中国中医药出版社 * |
Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN108459167A (en) * | 2018-03-16 | 2018-08-28 | 江苏维尔生物科技有限公司 | A kind of kit and preparation method thereof quantitatively detected for β-HCG, PDG in human urine |
| EP4017375A4 (en) * | 2019-08-19 | 2023-12-06 | MFB Fertility, Inc. | Systems and methods for menstrual cycle testing |
Also Published As
| Publication number | Publication date |
|---|---|
| CN106146651B (en) | 2019-08-23 |
| CN110526964A (en) | 2019-12-03 |
| CN110526964B (en) | 2021-03-23 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| US8278109B2 (en) | Hyperglycosylated hCG detection device | |
| EP0327843B1 (en) | Positive step immunoassay | |
| EP0075193B1 (en) | Method of pregnanediol glucuronide detection and indicator strip for use therein | |
| CN116754756A (en) | Methotrexate detection kit | |
| ES2349649T3 (en) | PROBE COMPLEX. | |
| US20110020847A1 (en) | Direct Enzyme Immunoassay and Kit for Measurement of Serum Progesterone Levels | |
| CN108982860A (en) | A kind of reagent using latex immunoturbidimetry technology measurement human serum content of glycocholic acid | |
| CN109116038A (en) | Unbonded estriol chemiluminescence immune detection reagent kit and preparation method thereof | |
| US5514598A (en) | Prenatal detection of meconium | |
| CN106146651A (en) | Women luteal function quick non-invasive monitoring technology | |
| EP1301790B1 (en) | Direct assessment of analyte to reference molecule ratios | |
| CN109212196A (en) | A kind of kit of quick detection free estriol | |
| CN113358864A (en) | Detection method of anti-phospholipid syndrome related antibody | |
| CN106706931A (en) | Progesterone measurement kit and preparation method | |
| CN110196336A (en) | Hematopoietin chemiluminescence immune detection reagent kit and preparation method thereof | |
| CN108982859A (en) | A kind of reagent measuring human serum content of glycocholic acid | |
| KR20020078032A (en) | Kit for diagnosing non-pregnancy, and method for diagnosing non-pregnancy using the same | |
| CN103229057B (en) | The method amplified for signal and reagent | |
| CN112326976A (en) | Fluorescence quantitative detection kit for progesterone, estradiol and beta-human chorionic gonadotropin | |
| CN201021925Y (en) | Colloid gold immune test paper for detecting progestin | |
| US4255412A (en) | T3 Uptake test employing a precipitated nylon separating agent | |
| KHATKHATAY et al. | Application of penicillinase linked Elisa of pregnanediol glucuronide for detection of ovulation and assessment of corpus luteal function | |
| EP0114614A2 (en) | Determination of steroid hormone glucuronides | |
| Hsu | Immunoperoxidase techniques using the avidin-biotin system | |
| KR102594836B1 (en) | Reagents for immunochromatography including latex beads, linkers and antibodies, and rapid kits comprising the same |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| C06 | Publication | ||
| PB01 | Publication | ||
| C10 | Entry into substantive examination | ||
| SE01 | Entry into force of request for substantive examination | ||
| GR01 | Patent grant | ||
| GR01 | Patent grant |