CN105807071A - E3G and LH colloidal gold detection kit - Google Patents
E3G and LH colloidal gold detection kit Download PDFInfo
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- CN105807071A CN105807071A CN201610297896.3A CN201610297896A CN105807071A CN 105807071 A CN105807071 A CN 105807071A CN 201610297896 A CN201610297896 A CN 201610297896A CN 105807071 A CN105807071 A CN 105807071A
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- monoclonal antibody
- gold
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- colloid gold
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- PCHJSUWPFVWCPO-UHFFFAOYSA-N gold Chemical compound [Au] PCHJSUWPFVWCPO-UHFFFAOYSA-N 0.000 title claims abstract description 85
- 238000001514 detection method Methods 0.000 title claims abstract description 41
- 238000002360 preparation method Methods 0.000 claims abstract description 27
- 239000000020 Nitrocellulose Substances 0.000 claims abstract description 21
- 229920001220 nitrocellulos Polymers 0.000 claims abstract description 21
- 239000000427 antigen Substances 0.000 claims abstract description 11
- 102000036639 antigens Human genes 0.000 claims abstract description 11
- 108091007433 antigens Proteins 0.000 claims abstract description 11
- 239000010931 gold Substances 0.000 claims description 52
- 229910052737 gold Inorganic materials 0.000 claims description 50
- 239000000084 colloidal system Substances 0.000 claims description 45
- 239000000243 solution Substances 0.000 claims description 45
- 239000003153 chemical reaction reagent Substances 0.000 claims description 27
- 238000000034 method Methods 0.000 claims description 27
- 238000003556 assay Methods 0.000 claims description 19
- ZBKIUFWVEIBQRT-UHFFFAOYSA-N gold(1+) Chemical compound [Au+] ZBKIUFWVEIBQRT-UHFFFAOYSA-N 0.000 claims description 16
- 239000003085 diluting agent Substances 0.000 claims description 14
- 230000036039 immunity Effects 0.000 claims description 9
- 239000002250 absorbent Substances 0.000 claims description 8
- 230000002745 absorbent Effects 0.000 claims description 8
- 239000007788 liquid Substances 0.000 claims description 8
- 239000001509 sodium citrate Substances 0.000 claims description 8
- HRXKRNGNAMMEHJ-UHFFFAOYSA-K trisodium citrate Chemical compound [Na+].[Na+].[Na+].[O-]C(=O)CC(O)(CC([O-])=O)C([O-])=O HRXKRNGNAMMEHJ-UHFFFAOYSA-K 0.000 claims description 8
- 229940038773 trisodium citrate Drugs 0.000 claims description 8
- 239000007921 spray Substances 0.000 claims description 6
- 238000005520 cutting process Methods 0.000 claims description 5
- 239000012530 fluid Substances 0.000 claims description 5
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- 238000002372 labelling Methods 0.000 claims description 4
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- FJWGYAHXMCUOOM-QHOUIDNNSA-N [(2s,3r,4s,5r,6r)-2-[(2r,3r,4s,5r,6s)-4,5-dinitrooxy-2-(nitrooxymethyl)-6-[(2r,3r,4s,5r,6s)-4,5,6-trinitrooxy-2-(nitrooxymethyl)oxan-3-yl]oxyoxan-3-yl]oxy-3,5-dinitrooxy-6-(nitrooxymethyl)oxan-4-yl] nitrate Chemical compound O([C@@H]1O[C@@H]([C@H]([C@H](O[N+]([O-])=O)[C@H]1O[N+]([O-])=O)O[C@H]1[C@@H]([C@@H](O[N+]([O-])=O)[C@H](O[N+]([O-])=O)[C@@H](CO[N+]([O-])=O)O1)O[N+]([O-])=O)CO[N+](=O)[O-])[C@@H]1[C@@H](CO[N+]([O-])=O)O[C@@H](O[N+]([O-])=O)[C@H](O[N+]([O-])=O)[C@H]1O[N+]([O-])=O FJWGYAHXMCUOOM-QHOUIDNNSA-N 0.000 description 13
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- 239000000463 material Substances 0.000 description 6
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- HDTRYLNUVZCQOY-UHFFFAOYSA-N α-D-glucopyranosyl-α-D-glucopyranoside Natural products OC1C(O)C(O)C(CO)OC1OC1C(O)C(O)C(O)C(CO)O1 HDTRYLNUVZCQOY-UHFFFAOYSA-N 0.000 description 5
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- HDTRYLNUVZCQOY-LIZSDCNHSA-N alpha,alpha-trehalose Chemical compound O[C@@H]1[C@@H](O)[C@H](O)[C@@H](CO)O[C@@H]1O[C@@H]1[C@H](O)[C@@H](O)[C@H](O)[C@@H](CO)O1 HDTRYLNUVZCQOY-LIZSDCNHSA-N 0.000 description 5
- 230000005906 menstruation Effects 0.000 description 5
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- BTBUEUYNUDRHOZ-UHFFFAOYSA-N Borate Chemical compound [O-]B([O-])[O-] BTBUEUYNUDRHOZ-UHFFFAOYSA-N 0.000 description 4
- 108010077544 Chromatin Proteins 0.000 description 4
- 241000699666 Mus <mouse, genus> Species 0.000 description 4
- FAPWRFPIFSIZLT-UHFFFAOYSA-M Sodium chloride Chemical compound [Na+].[Cl-] FAPWRFPIFSIZLT-UHFFFAOYSA-M 0.000 description 4
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- IAZDPXIOMUYVGZ-UHFFFAOYSA-N Dimethylsulphoxide Chemical compound CS(C)=O IAZDPXIOMUYVGZ-UHFFFAOYSA-N 0.000 description 3
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- DGAQECJNVWCQMB-PUAWFVPOSA-M Ilexoside XXIX Chemical compound C[C@@H]1CC[C@@]2(CC[C@@]3(C(=CC[C@H]4[C@]3(CC[C@@H]5[C@@]4(CC[C@@H](C5(C)C)OS(=O)(=O)[O-])C)C)[C@@H]2[C@]1(C)O)C)C(=O)O[C@H]6[C@@H]([C@H]([C@@H]([C@H](O6)CO)O)O)O.[Na+] DGAQECJNVWCQMB-PUAWFVPOSA-M 0.000 description 3
- 102000009151 Luteinizing Hormone Human genes 0.000 description 3
- 108010073521 Luteinizing Hormone Proteins 0.000 description 3
- SJUCACGNNJFHLB-UHFFFAOYSA-N O=C1N[ClH](=O)NC2=C1NC(=O)N2 Chemical compound O=C1N[ClH](=O)NC2=C1NC(=O)N2 SJUCACGNNJFHLB-UHFFFAOYSA-N 0.000 description 3
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- HOGDNTQCSIKEEV-UHFFFAOYSA-N n'-hydroxybutanediamide Chemical compound NC(=O)CCC(=O)NO HOGDNTQCSIKEEV-UHFFFAOYSA-N 0.000 description 3
- 108090000623 proteins and genes Proteins 0.000 description 3
- 102000004169 proteins and genes Human genes 0.000 description 3
- 229910052708 sodium Inorganic materials 0.000 description 3
- 239000011734 sodium Substances 0.000 description 3
- PROQIPRRNZUXQM-UHFFFAOYSA-N (16alpha,17betaOH)-Estra-1,3,5(10)-triene-3,16,17-triol Natural products OC1=CC=C2C3CCC(C)(C(C(O)C4)O)C4C3CCC2=C1 PROQIPRRNZUXQM-UHFFFAOYSA-N 0.000 description 2
- 206010003445 Ascites Diseases 0.000 description 2
- 102000012673 Follicle Stimulating Hormone Human genes 0.000 description 2
- 108010079345 Follicle Stimulating Hormone Proteins 0.000 description 2
- 229930006000 Sucrose Natural products 0.000 description 2
- CZMRCDWAGMRECN-UGDNZRGBSA-N Sucrose Chemical compound O[C@H]1[C@H](O)[C@@H](CO)O[C@@]1(CO)O[C@@H]1[C@H](O)[C@@H](O)[C@H](O)[C@@H](CO)O1 CZMRCDWAGMRECN-UGDNZRGBSA-N 0.000 description 2
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- 210000004246 corpus luteum Anatomy 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
- 235000019800 disodium phosphate Nutrition 0.000 description 2
- PROQIPRRNZUXQM-ZXXIGWHRSA-N estriol Chemical compound OC1=CC=C2[C@H]3CC[C@](C)([C@H]([C@H](O)C4)O)[C@@H]4[C@@H]3CCC2=C1 PROQIPRRNZUXQM-ZXXIGWHRSA-N 0.000 description 2
- 229960001348 estriol Drugs 0.000 description 2
- 229940028334 follicle stimulating hormone Drugs 0.000 description 2
- 210000004408 hybridoma Anatomy 0.000 description 2
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- 230000027758 ovulation cycle Effects 0.000 description 2
- 239000004033 plastic Substances 0.000 description 2
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- 239000011780 sodium chloride Substances 0.000 description 2
- 239000001488 sodium phosphate Substances 0.000 description 2
- 239000000126 substance Substances 0.000 description 2
- 239000005720 sucrose Substances 0.000 description 2
- PXFBZOLANLWPMH-UHFFFAOYSA-N 16-Epiaffinine Natural products C1C(C2=CC=CC=C2N2)=C2C(=O)CC2C(=CC)CN(C)C1C2CO PXFBZOLANLWPMH-UHFFFAOYSA-N 0.000 description 1
- 239000000579 Gonadotropin-Releasing Hormone Substances 0.000 description 1
- 229910004042 HAuCl4 Inorganic materials 0.000 description 1
- 241001529936 Murinae Species 0.000 description 1
- 241000699670 Mus sp. Species 0.000 description 1
- 108010058846 Ovalbumin Proteins 0.000 description 1
- 206010035226 Plasma cell myeloma Diseases 0.000 description 1
- 108020004511 Recombinant DNA Proteins 0.000 description 1
- 101000857870 Squalus acanthias Gonadoliberin Proteins 0.000 description 1
- 101710120037 Toxin CcdB Proteins 0.000 description 1
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- 239000005557 antagonist Substances 0.000 description 1
- 230000003466 anti-cipated effect Effects 0.000 description 1
- 210000003651 basophil Anatomy 0.000 description 1
- 210000000481 breast Anatomy 0.000 description 1
- 150000001718 carbodiimides Chemical class 0.000 description 1
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- 239000003292 glue Substances 0.000 description 1
- FDWREHZXQUYJFJ-UHFFFAOYSA-M gold monochloride Chemical compound [Cl-].[Au+] FDWREHZXQUYJFJ-UHFFFAOYSA-M 0.000 description 1
- XLXSAKCOAKORKW-AQJXLSMYSA-N gonadorelin Chemical compound C([C@@H](C(=O)NCC(=O)N[C@@H](CC(C)C)C(=O)N[C@@H](CCCNC(N)=N)C(=O)N1[C@@H](CCC1)C(=O)NCC(N)=O)NC(=O)[C@H](CO)NC(=O)[C@H](CC=1C2=CC=CC=C2NC=1)NC(=O)[C@H](CC=1N=CNC=1)NC(=O)[C@H]1NC(=O)CC1)C1=CC=C(O)C=C1 XLXSAKCOAKORKW-AQJXLSMYSA-N 0.000 description 1
- 229940035638 gonadotropin-releasing hormone Drugs 0.000 description 1
- 210000003016 hypothalamus Anatomy 0.000 description 1
- 238000007654 immersion Methods 0.000 description 1
- 238000001727 in vivo Methods 0.000 description 1
- 230000005764 inhibitory process Effects 0.000 description 1
- 238000011081 inoculation Methods 0.000 description 1
- 238000009434 installation Methods 0.000 description 1
- 230000029849 luteinization Effects 0.000 description 1
- 229940040129 luteinizing hormone Drugs 0.000 description 1
- 238000004519 manufacturing process Methods 0.000 description 1
- 230000009245 menopause Effects 0.000 description 1
- 229910052751 metal Inorganic materials 0.000 description 1
- 239000002184 metal Substances 0.000 description 1
- 238000012986 modification Methods 0.000 description 1
- 230000004048 modification Effects 0.000 description 1
- 238000010369 molecular cloning Methods 0.000 description 1
- 239000002808 molecular sieve Substances 0.000 description 1
- 201000000050 myeloid neoplasm Diseases 0.000 description 1
- 229940092253 ovalbumin Drugs 0.000 description 1
- 230000011599 ovarian follicle development Effects 0.000 description 1
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- 238000003908 quality control method Methods 0.000 description 1
- 230000000630 rising effect Effects 0.000 description 1
- 238000012216 screening Methods 0.000 description 1
- URGAHOPLAPQHLN-UHFFFAOYSA-N sodium aluminosilicate Chemical compound [Na+].[Al+3].[O-][Si]([O-])=O.[O-][Si]([O-])=O URGAHOPLAPQHLN-UHFFFAOYSA-N 0.000 description 1
- 210000004989 spleen cell Anatomy 0.000 description 1
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Classifications
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/74—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving hormones or other non-cytokine intercellular protein regulatory factors such as growth factors, including receptors to hormones and growth factors
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
- G01N33/577—Immunoassay; Biospecific binding assay; Materials therefor involving monoclonal antibodies binding reaction mechanisms characterised by the use of monoclonal antibodies; monoclonal antibodies per se are classified with their corresponding antigens
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N2333/00—Assays involving biological materials from specific organisms or of a specific nature
- G01N2333/435—Assays involving biological materials from specific organisms or of a specific nature from animals; from humans
- G01N2333/575—Hormones
- G01N2333/59—Follicle-stimulating hormone [FSH]; Chorionic gonadotropins, e.g. HCG; Luteinising hormone [LH]; Thyroid-stimulating hormone [TSH]
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- Health & Medical Sciences (AREA)
- Life Sciences & Earth Sciences (AREA)
- Immunology (AREA)
- Engineering & Computer Science (AREA)
- Chemical & Material Sciences (AREA)
- Molecular Biology (AREA)
- Biomedical Technology (AREA)
- Urology & Nephrology (AREA)
- Hematology (AREA)
- Microbiology (AREA)
- Analytical Chemistry (AREA)
- Biotechnology (AREA)
- Pathology (AREA)
- Food Science & Technology (AREA)
- Medicinal Chemistry (AREA)
- Physics & Mathematics (AREA)
- Cell Biology (AREA)
- Biochemistry (AREA)
- General Health & Medical Sciences (AREA)
- General Physics & Mathematics (AREA)
- Endocrinology (AREA)
- Chemical Kinetics & Catalysis (AREA)
- Investigating Or Analysing Biological Materials (AREA)
Abstract
The invention relates to the field of biological detection and especially relates to an E3G and LH colloidal gold detection kit, and a preparation method and application thereof. The E3G and LH colloidal gold detection kit comprises a base plate, wherein a sample pad, a colloidal gold pad, an immune nitrocellulose membrane and water-absorbing paper are successively arranged on the base plate from a sample adding end; a colloidal gold marked anti-E3G monoclonal antibody and a colloidal gold marked anti-LH monoclonal antibody 1 envelop the colloidal gold pad; a detection line and a control line are arranged on the immune nitrocellulose membrane; an anti-LH monoclonal antibody 2 envelops the detection line; an E3G-OVA antigen envelops the control line. The E3G and LH colloidal gold detection kit provided by the invention combines E3G and LH hormone indexes with each other and adopts a colloidal gold quick chromatography detection method for judging if a human body is at the optimal pregnancy time according to the content of LH and E3G.
Description
Technical field
The present invention relates to field of biological detection, particularly relate to a kind of E3G and LH gold-immunochromatographyreagent reagent for assay box and preparation method thereof
And purposes.
Background technology
Interstitialcellstimulating hormone (ICSH) (LH, luteinizing hormone) is secreted by adenohypophysis basophil.Promote corpus luteum in women to generate
Element (LH) collaborative follicle stimulating hormone (FSH, follicle-stimulating hormone) effect jointly maintains the menstruation week of ovary
Phase, ovulation is caused to be formed with corpus luteum.The generation of LH is controlled by hypothalamus gonadotropin releasing hormone, simultaneously positive and negative by ovary
Feedback regulation.Once appearance, 24-36 is indicated with ovary ovulation close relation, LH peak on the release peak of menstrual cycle LH
The ovulation of hour ovary, therefore can monitor serum Lh peak value, to determine optimal Time to pregnancy in menstrual cycle.For menstruation
Women in cycle, LH value term of reference is as follows: follicular phase 1.9-12.5IU/L；The onset of ovulation 8.7-76.3IU/L；Luteal phase 0.5-16.9
IU/L；Menopause 15.9-54IU/L；Trimester of pregnancy 0-1.5IU/L.
Although to the monitoring of LH peak value can predicting ovulation time to a certain extent, but use owing to existing product is the most single
LH is as the index of Monitoring Ovulation, about the time of its prediction Women of Childbearing Age ovulation only has 2 day time, for wanting child's
Personnel have bigger probability can miss the optimal conceived time.
Summary of the invention
The shortcoming of prior art in view of the above, it is an object of the invention to provide a kind of E3G and LH gold-immunochromatographyreagent reagent for assay
Box and its production and use, is used for solving the problems of the prior art.
For achieving the above object and other relevant purposes, first aspect present invention provides a kind of E3G and LH gold-immunochromatographyreagent reagent for assay
Box, including base plate, on described base plate from sample-adding end start to be sequentially provided with sample pad, colloidal gold pad, immunity nitrocellulose filter and
Absorbent paper, wherein, described colloidal gold pad is coated with the anti-E3G monoclonal antibody of colloid gold label and the anti-of colloid gold label
LH monoclonal antibody 1, described immunity nitrocellulose filter is provided with detection line (T line) and control line (C line), on detection line
It is coated with anti-LH monoclonal antibody 2, control line is coated with E3G-BSA antigen.
The anti-E3G monoclonal antibody of described colloid gold label is anti-E3G monoclonal antibody-colloidal gold conjugate, described colloid
The anti-LH monoclonal antibody 1 of gold labelling is anti-LH monoclonal antibody 1-colloidal gold conjugate, is coated above described colloidal gold pad
There are anti-E3G monoclonal antibody-colloidal gold conjugate and the complex of anti-LH monoclonal antibody 1-colloidal gold conjugate.
Preferably, in the anti-E3G monoclonal antibody of colloid gold label and the anti-LH monoclonal antibody 1 of colloid gold label, colloid
Gold is the granule of particle diameter 30-50nm, more preferably spheroidal particle.
Described anti-LH monoclonal antibody 1 and anti-LH monoclonal antibody 2 can be identical anti-LH monoclonal antibodies, it is also possible to
It it is different anti-LH monoclonal antibodies.
Preferably, described anti-LH monoclonal antibody 1 is anti-β-LH monoclonal antibody, and described anti-LH monoclonal antibody 2 is anti-
α-LH monoclonal antibody.
Preferably, described detection line is also coated with trehalose and Tween-20.
When described E3G and LH gold-immunochromatographyreagent reagent for assay box uses, as on the LH antigen in the urine of tested material and test kit
The anti-LH monoclonal antibody 1 of prior coated colloid gold label is combined into conjugate, and this conjugate is along with the capillarity of film
Migrate up to reach detection zone, react with the anti-LH monoclonal antibody 2 on detection line, a red stripes occurs；And urinate
The anti-E3G monoclonal antibody of the E3G antigen in liquid and the prior coated colloid gold label on test pen is combined into conjugate,
And fail anti-E3G monoclonal antibody-colloidal gold conjugate of combining and migrate up to reach detection zone along with the capillarity of film,
React with the E3G-BSA antigen on control line, it may appear that a red stripes.Therefore line (T line) color is detected the deepest,
Then represent that in urine, LH content is the highest, and control line (C line) color is the deepest, then it represents that in urine, E3G content is the lowest.
Second aspect present invention provides the preparation method of described E3G and LH gold-immunochromatographyreagent reagent for assay box, comprises the steps:
1) by anti-for colloid gold label LH monoclonal antibody 1, the anti-LH i.e. obtaining colloid gold label after closing after centrifugal concentrating is mono-
Clonal antibody 1；By anti-for colloid gold label E3G monoclonal antibody, after closing, after centrifugal concentrating, i.e. obtain the anti-E3G of colloid gold label
Monoclonal antibody；The anti-LH monoclonal antibody 1 of the colloid gold label of gained and the anti-E3G monoclonal of colloid gold label will be prepared
Mix after antibody dilution.
Preferably, in described step 1, described gold colloidal uses reducing process to prepare.
It is furthermore preferred that in described step 1, reducing process is specially trisodium citrate reduction method, and described trisodium citrate reduction method can
Comprise the steps: chlorauric acid solution is heated to boiling, be subsequently adding trisodium citrate or citric acid three sodium solution, continue to boil
Boil 20 minutes, then cooling constant volume.Described trisodium citrate reduction method is the conventional method that existing gold colloidal produces, ability
Field technique personnel can adjust the ratio of gold chloride and trisodium citrate as required, to obtain the gold colloidal of different-grain diameter size.
It is furthermore preferred that in described step 1, colloidal gold solution OD > 2.9OD (light path 1cm, the 533nm that reducing process prepares
Place), pH scope is 3.5～4.5.
Those skilled in the art can select suitably according to anti-LH monoclonal antibody 1 and the kind of anti-LH monoclonal antibody 2
Condition is by colloidal gold labeled monoclonal antibody.
Preferably, in described step 1, anti-LH monoclonal antibody 1 is anti-β-LH monoclonal antibody, by colloid gold label anti-β-LH
Monoclonal antibody method particularly includes: by anti-β-LH monoclonal antibody with 0.9-1.1mg/ml antibody correspondence 150OD (533nm)
Gold colloidal (concrete as when the absorbance of colloidal gold solution is 150OD, add anti-β-LH with the final concentration of 0.9-1.1mg/ml
Monoclonal antibody, when the absorbance of colloidal gold solution is 300OD, adds anti-β-LH with the final concentration of 1.8-2.2mg/ml single
Clonal antibody) ratio under conditions of pH=6.95-7.05, by colloid gold label anti-β-LH monoclonal antibody, after 30 minutes,
Close with BSA under conditions of pH=7.35-7.45, after centrifugal concentrating, i.e. obtain the anti-β-LH monoclonal of colloid gold label
Antibody.
Preferably, in described step 1, by anti-for colloid gold label E3G monoclonal antibody method particularly includes: by mono-for anti-E3G
Clonal antibody with the ratio of 0.9-1.1mg/ml antibody correspondence 150OD gold colloidal under conditions of pH=7.95-8.05, by colloid
Gold labelling anti-E3G monoclonal antibody, after 30 minutes, closes with BSA under conditions of pH=7.35-7.45, centrifugal dense
The anti-E3G monoclonal antibody of colloid gold label is i.e. obtained after contracting.
Preferably, also dialyse before described anti-LH monoclonal antibody 1 and anti-E3G labeling of monoclonal antibody.
Those skilled in the art can select suitable dialysis process antagonist to dialyse, at this according to the kind of monoclonal antibody
In a bright embodiment, the actual conditions of dialysis is: antibody is placed in 0.9-1.1mM disodium phosphate soln dialysis more than 16h.
Preferably, in described step 1, during two kinds of antibody mixing, colloidal gold film diluent is used to be diluted.
In an embodiment of the present invention, the anti-LH monoclonal antibody 1 (anti-β-LH monoclonal antibody) of colloid gold label is dissolved in
Colloidal gold film diluent, the OD=3.8-4.2 of gained diluent, the anti-E3G monoclonal antibody of colloid gold label is dissolved in gold colloidal
Film diluent, the OD=2.8-3.2 of gained diluent, then both diluents are mixed (volume according to the ratio of 1:0.95-1.05
Than).
Those skilled in the art may select suitable colloidal gold film diluent and are diluted the antibody of colloid gold label, in the present invention
In one embodiment, described colloidal gold film diluent is 1.1%NaCl, 3.75%BSA, 20% sucrose, 1.25%Tween-20
With 50mM borate buffer, pH 9.0.
2) after glass fibre fully being soaked in step 1 gained mixed liquor, take out and squeeze and remove surplus liquid, tile, after drying
Obtain colloidal gold pad, standby.
3) the specking detection on nitrocellulose filter respectively of anti-LH monoclonal antibody 2 solution and E3G-OVA antigenic solution is used
Line and control line, and nitrocellulose filter that preparation is completed dried immune nitrocellulose filter, standby.
Preferably, in described step 3, described anti-LH monoclonal antibody 2 solution is anti alpha-LH monoclonal antibody solution, solution
Concentration is 0.9-1.1mg/ml, and specking amount is 0.9-1.1ul/cm, also containing the trehalose of 5wt% and 1wt% in the solution of specking
Tween-20.Described solution the most also filters before specking, in an embodiment of the present invention, and specially 0.22 μm mistake
Filter.
Preferably, in described step 3, the solution concentration of described E3G-OVA antigenic solution is 1.8-2.2mg/ml, and specking amount is
0.9-1.1ul/cm.Described solution the most also filters before specking, in an embodiment of the present invention, and specially 0.22 μm
Filter.
4) untreated filter sample paper is fully soaked in sample pad treatment fluid, be dried after taking out and extract surplus liquid, it is thus achieved that sample
Product pad, standby.
Preferably, in described step 4, sample pad treatment fluid can be selected at the various applicable colloidal gold kit sample pad in this area
Reason liquid, in an embodiment of the present invention, described sample pad treatment fluid is the 50mM containing 1%TWEEN-20 (percent by volume)
Borate buffer, pH=9.0.
5) prepared by immune nitrocellulose filter, step 4 prepared by the colloidal gold pad absorbent paper, step 2 prepared, step 3
Sample pad is pasted onto on base plate, and cutting, encapsulation prepare detection kit.
Third aspect present invention provides a kind of method detecting the human ovulation phase, by described E3G and LH gold-immunochromatographyreagent reagent for assay box
Sample-adding end immerse in urine the 3-7 second, keep flat after taking-up 5-10 minute, it is determined that result.
This product is continuously monitoring hormone in vivo level, determines whether ovulation according to the rising of internal LH and E3G.Judge E3G
During numerical value, if less than threshold value (color the most shallow expression E3G concentration is the highest), then assert and will be in the onset of ovulation, at this
In a bright embodiment, i.e. think less than threshold value less than reference value more than 15%；When judging LH numerical value, if it exceeds thresholding
Value (color the deepest LH concentration is the highest), then regard as being in peak period of ovulation, in an embodiment of the present invention, exceed benchmark
Value more than 15% is i.e. thought and is exceeded threshold value.These two kinds of situations, all represent that this period of time period is for the suitable pregnancy period.
In an embodiment of the present invention, start the 7th day to detect from menstruation, it is determined that during result, by test bar (containing test strips),
Inserting test pen and carry out interpretation, test pen light path detection module uses RGB color sensor, reads T line and C line respectively
Color, is converted to R, and G, B color frequency signal exports, and test pen acquisition module frequency acquisition signal is also converted into digital value,
Then the measured value of the 7th day is compared with the threshold value of machine set, as the 7th day measured value is higher than threshold value, then by this
Measured value is set to reference value, and the most still using the threshold value of machine set as reference value, the hereafter detection of every day is entered with this reference value
Row compares, and carries out result judgement.When judging E3G numerical value, if less than reference value more than 15% (the most shallow expression of color
E3G concentration is the highest), then assert and will be in the onset of ovulation；Judge LH magnitude value, if it exceeds determinating reference value 15% with
Upper (color the deepest LH concentration is the highest), then regard as being in peak period of ovulation.These two kinds of situations, all represent this period of time period
For the suitable pregnancy period.
E3G and the LH gold-immunochromatographyreagent reagent for assay box that the present invention provides is by E3G (estriol) and LH (induced ovulation generates element) 2
Individual Hormone traits combines, and uses gold colloidal flash chromatography detection method, according to the content height of LH content and E3G, permissible
Judge whether human body is in the optimal conceived time, thus reach conceived purpose.Specifically, described E3G and LH colloid
The time of anticipated ovulation can be expanded to 4 days by gold detection kit, can preferably indicate the optimal conceived time, thus improve bosom
Pregnant success rate.In the solution of detection line, add trehalose and Tween-20 simultaneously, the color of detection lines can be made to keep homogeneous,
Improve the sensitivity of reagent and attractive in appearance.
Accompanying drawing explanation
Fig. 1 is shown as the structural representation of the present invention.
Fig. 2 is shown as the embodiment of the present invention 4 testing result schematic diagram.
Element numbers explanation
1 sample pad
2 colloidal gold pad
3 detections line (T line)
4 control lines (C line)
5 immunity nitrocellulose filter (NC film)
6 absorbent paper
Detailed description of the invention
Below by way of specific instantiation, embodiments of the present invention being described, those skilled in the art can be by disclosed by this specification
Content understand other advantages and effect of the present invention easily.The present invention can also be added by the most different detailed description of the invention
To implement or application, the every details in this specification can also be based on different viewpoints and application, in the essence without departing from the present invention
Various modification or change is carried out under god.
Before further describing the specific embodiment of the invention, it should be appreciated that protection scope of the present invention is not limited to following specific
Specific embodiments；It is also understood that the term used in the embodiment of the present invention is to describe specific specific embodiments,
Rather than in order to limit the scope of the invention；In description of the invention and claims, unless literary composition additionally clearly refers to
Going out, singulative " ", " one " and " this " include plural form.
When embodiment provides numerical range, it should be appreciated that unless the present invention is otherwise noted, two end points of each numerical range with
And any one numerical value all can be selected between two end points.Unless otherwise defined, all technology used in the present invention and section are academic
The same meaning that language and those skilled in the art of the present technique are generally understood that.In addition to the concrete grammar used in embodiment, equipment, material,
According to those skilled in the art's grasp to prior art and the record of the present invention, it is also possible to use and the embodiment of the present invention
Described in method, any method, equipment and the material of the similar or equivalent prior art of equipment, material realize the present invention.
Unless otherwise indicated, the experimental technique that disclosed in this invention, detection method, preparation method all use the art normal
Rule molecular biology, biochemistry, chromatin Structure and analysis, analytical chemistry, cell cultivate, recombinant DNA technology and
The routine techniques of association area.These technology have improved explanation in existing document, specifically can be found in Sambrook etc.
MOLECULAR CLONING:A LABORATORY MANUAL, Second edition, Cold Spring Harbor
Laboratory Press, 1989and Third edition, 2001；Ausubel etc., CURRENT PROTOCOLS IN
MOLECULAR BIOLOGY, John Wiley&Sons, New York, 1987and periodic updates；the series
METHODS IN ENZYMOLOGY, Academic Press, San Diego；Wolffe, CHROMATIN
STRUCTURE AND FUNCTION, Third edition, Academic Press, San Diego, 1998；METHODS
IN ENZYMOLOGY, Vol.304, Chromatin (P.M.Wassarman and A.P.Wolffe, eds.), Academic
Press, San Diego, 1999；With METHODS IN MOLECULAR BIOLOGY, Vol.119, Chromatin
Protocols (P.B.Becker, ed.) Humana Press, Totowa, 1999 etc..
Each raw material information used in embodiment is as follows:
Anti alpha-LH monoclonal antibody and anti-β-LH Antibodies Monoclonal antibodies: purchased from Arista company of the U.S., protein concentration
≥10mg/ml。
Anti-E3G monoclonal antibody: self-control (detailed in Example 1), for affinity purification, purity > 90%, protein concentration >=2mg/ml.
E3G-OVA antigen: self-control (detailed in Example 2), protein concentration >=4mg/ml.
Nitrocellulose membrane (NC film): purchased from Sartorius company of the U.S., length standard: 100 ± 2m, width criteria: 2.54 ± 0.05cm,
Metal run out time standard: 107～133 seconds.
Glass fibre (Glass Fiber Conjugate Pad): purchased from Millipore company of the U.S., length standard: 31 ± 0.2cm,
Width criteria: 8.0 ± 0.5mm.
Sample pad and absorbent filter: purchased from Shanghai Jie Ning Bioisystech Co., Ltd, the wide 11 ± 0.5mm of absorbent paper, long 31 ± 0.2cm,
Wide 22 ± the 0.5mm of sample pad, long 31 ± 0.2cm.
PVC base plate: purchased from Shanghai Jie Ning Bioisystech Co., Ltd, length standard: 31 ± 0.2cm, width criteria:
60.4～61.4mm.
HAuCl4Purchased from Sigma or Amersco company
Trisodium citrate, E3G antigen, ovalbumin OVA: purchased from Sigma company.
Other raw materials not be given are marketable material, and concentration is not less than chemical pure, and should meet existing " Chinese Pharmacopoeia " or " in
State's biological product main raw and auxiliary material quality control standard " requirement.
Embodiment 1
The preparation of anti-E3G monoclonal antibody and qualification:
1. by E3G antigen 6 mices of immunity respectively, ELISA method detection serum titer, until titre > 100,000.
2. immune mouse spleen cell and the SP2/0 murine myeloma cell of step 1 gained merges, and uses ELISA competing
Strive method screening stable cell line 5-10 strain.
3. select the highest strain cell strain of affinity costant and optimize Inhibition ELISA to reach optimal sensitivity, special
Property, the range of linearity, repeatability.
4. select, pass on and 2 hybridoma cell strains of low temperature storage.
5. the hybridoma cell strain of inoculation step 4 gained in mouse peritoneal, after obtaining mouse ascites, affine with Protein G post
Purified mouse ascites, it is thus achieved that required anti-E3G monoclonal antibody.
Embodiment 2
The preparation of E3G-OVA antigen:
1. prepare the ovalbumin solution of 5mg/ml as solvent with the PBS of 0.2M.
2. prepare E3G (estriol) solution of 10mg/ml as solvent with DMF (DMSO).
3. prepare NHS (N-hydroxy-succinamide) solution of 20mg/ml as solvent with DMF.
4. prepare EDC (carbodiimide) solution of 18mg/ml as solvent with DMF.
5. E3G solution, EDC solution and the NHS solution of above-mentioned configuration gained are mixed 2 hours with the ratio of 6:2:1.
6. after, above-mentioned mixed liquor is slowly added in the OVA solution of 4.5 times of volumes, ambient temperature overnight.
7. the E3G-OVA solution molecular sieve purification column purification prepared is obtained E3G-OVA antigen.
Embodiment 3
The preparation of test kit:
1. the preparation of gold colloidal:
Trisodium citrate reduction method is used to prepare gold colloidal: 0.03% (mass percent) chlorauric acid solution is heated to boiling,
The citric acid three sodium solution of rear addition 3% (mass percent) (1 liter of chlorauric acid solution adds 12.202ml 3% Fructus Citri Limoniae
Acid three sodium solutions), continue to boil 20 minutes, rear cooling constant volume.Colloid gold particle size is at about 40nm.The glue prepared
Body gold solution OD answers > 2.9, pH scope is 3.5～4.5.
2. the preparation of colloidal gold pad:
A) anti-β-LH monoclonal antibody and the dialysis of anti-E3G monoclonal antibody: by mono-to anti-β-LH monoclonal antibody and anti-E3G
Clonal antibody is placed in dialysed overnight in 1mM disodium phosphate soln.
B) anti-β-LH monoclonal antibody and the preparation of anti-E3G monoclonal antibody Au probe: by anti-β-LH monoclonal antibody with
The ratio of 1mg/mL antibody correspondence 150OD gold colloidal under conditions of pH7.0, anti-by colloid gold label step a gained
β-LH monoclonal antibody, after 30 minutes, closes with BSA under conditions of pH7.4, i.e. obtains anti-LH after centrifugal concentrating
Antibody-gold probe；By anti-E3G monoclonal antibody with the ratio of 1mg/mL antibody correspondence 150OD gold colloidal at the bar of pH8.0
Under part, by the anti-E3G monoclonal antibody of colloid gold label step a gained, after 30 minutes, with BSA under conditions of pH7.4
Close, after centrifugal concentrating, i.e. obtain anti-E3G antibody-gold probe.
C) both Au probes are with colloidal gold film diluent (1.1%NaCl, 3.75%BSA, 20% sucrose, 1.25%Tween-20
With 50mM borate buffer, pH 9.0) it is diluted to OD4.0 (anti-LH antibody-gold probe) and OD3.0 (anti-E3G respectively
Antibody-gold probe) after, then 1:1 mixing for standby use in proportion.
D) colloidal gold pad preparation, be dried: after glass fibre is fully soaked in colloidal gold film solution, take out and squeeze go unnecessary
Liquid, is laid on special rack；In the environment of relative humidity≤20%, dry overnight at room temperature obtains colloidal gold pad, standby.
3. the preparation of immunity nitrocellulose membrane (NC film)
A) preparation of detection line (T line) spray membrane antibody solution: with pH7.4 and containing 5wt% trehalose and 1wt%Tween-20
10mM PBS solution anti alpha-LH antibody is diluted to 1mg/ml, 0.22 μm filter.
B) preparation of control line (C line) spray membrane antibody solution: E3G-OVA is diluted with the 10mM PBS solution of pH7.4
To 2.0mg/ml, 0.22 μm filters.
C) immunity nitrocellulose membrane (NC film) preparation, be dried: respectively with detection line spray membrane antibody solution and control line spray film
Antibody-solutions is specking detection line and control line (1.0 ± 0.1ul/cm) on nitrocellulose filter, and cellulose nitrate preparation completed
Element film dry overnight at room temperature in the environment of relative humidity≤20%, standby.
4. the process of sample pad:
Untreated filter sample paper is abundant in filtering in sample paper treatment fluid (50mM borate buffer pH9.0 and 1%TWEEN-20)
Soak, be laid on special rack after taking out and extract surplus liquid, be dried overnight in the environment of relative humidity≤20%, standby.
5. the preparation of mucosa kilocalorie:
During mucosa, shopwork envionmental humidity requires less than 30%.
Take 60mm × 300mm offset plate (end liner) a piece, paste the immune nitrocellulose membrane (NC after being coated at the over center position
Film), the detection line position of NC film is down；NC film detection line one side lower part patch one colloidal gold pad, it is ensured that both have 1～
The overlap of 2mm；Pasting a sample pad processed on the downside of colloidal gold pad, it must overlap with colloidal gold pad；?
PVC offset plate does not pastes one end of sample pad and pastes absorbent paper one, partly overlaps with NC film；NC film is pasted transparent preventing
Spatter adhesive tape；Will paste after snap fits into greatly plastic bag, relative humidity less than 20% low humidity store between preserve (the base of test kit
This structure is as shown in Figure 1).
6. test strips cutting, assemble and pack
Shopwork envionmental humidity when cutting, assembling requires less than 30%.
Kilocalorie cutting machine is cut into the test strips of 6mm width, 6mm test strips is sequentially loaded by test pen installation step
Working of plastics is assembled into test pen, test pen In Aluminium Foil Packing is sealed, and is packaged into test kit by reagent constituents requirement.
Embodiment 4
Ovulation period prognostic experiment:
1. sample canonical: collect 18～45 one full year of life women and start to every day when menstruation starts next time for the 7th day from what menstruation started
Urina sanguinis sample, totally 114 of the right age women.
2. packet: totally 114 groups of samples, produces with this product (using embodiment 3 gained reagent strip to detect) and contrast respectively
Product (the hormone test bar onset of ovulation (emulsion technique) of Britain Unipath) compare, to determine both coincidence rates.
3. detection method: collect experimenter's urine every day, prepares gained test kit (ovulation colloidal gold method immunity layer by embodiment 3
Analysis test pen) and contrast agents detect simultaneously, by sample-adding end immersion urine in 5 seconds, keep flat after taking-up, after 5 minutes use
Readout instrument detects, it is determined that result, and concrete outcome is as shown in table 1.
Table 1
4. result: find when the sample of 97 normal cycles is detected, the tested of LH and E3G increasing state occurs
Person's natural law (503 days) accounts for the 17.4% of the whole cycle (503+1806+97 × 6=2891 days), then for one a length of 28 days just
In the often cycle, what this reagent place can point out has the natural law of higher probability pregnancy is 28 × 17.4%=4.87 days, removes the LH of 2 days
Can pregnant state, the extra date proposing to be easier to pregnancy for 2.87 days.
Test kit sensitivity and the experiment of line color homogeneity:
Detection method:
The gold-immunochromatographyreagent reagent for assay box that Example 3 prepares, is placed on test kit on level table, takes and does not use new detection
The strip of line formula is as comparison (not adding trehalose and Tween-20 when i.e. detecting the preparation of line spray membrane antibody solution), simultaneously
The E3G standard substance of the detection LH and 10ng/ml containing 25mIU/ml, with sample-adding suction pipe pipette samples, then drip 4 (about
150ul) sample is in the well of test kit.
Observed result:
5 minutes sentence read result after dropping sample.
Testing result (the left side: the testing result figure of reagent strip before adjustment formula as shown in Figure 2；The right: adjust reagent after formula
The testing result figure of bar).
Under same standard product concentration, when using the reagent strip detection of new formula, line color is substantially deep, before detection lines
Tailing edge color is basically identical, observes attractive in appearance, therefore, when reading, uses the reagent strip lines detection sensitivity of new formula to improve.
In sum, the present invention effectively overcomes various shortcoming of the prior art and has high industrial utilization.
The principle of above-described embodiment only illustrative present invention and effect thereof, not for limiting the present invention.Any it is familiar with this skill
Above-described embodiment all can be modified under the spirit and the scope of the present invention or change by the personage of art.Therefore, such as
All that in art, tool usually intellectual is completed under without departing from disclosed spirit and technological thought etc.
Effect is modified or changes, and must be contained by the claim of the present invention.
Claims (10)
1. an E3G and LH gold-immunochromatographyreagent reagent for assay box, including base plate, described base plate starts to be sequentially provided with sample from sample-adding end
Pad, colloidal gold pad, immunity nitrocellulose filter and absorbent paper, wherein, described colloidal gold pad is coated with colloid gold label
Anti-E3G monoclonal antibody and the anti-LH monoclonal antibody 1 of colloid gold label, described immunity nitrocellulose filter is provided with inspection
Survey line and control line, detection line is coated with anti-LH monoclonal antibody 2, control line is coated with E3G-OVA antigen.
2. a kind of E3G and LH gold-immunochromatographyreagent reagent for assay box as claimed in claim 1, it is characterised in that resisting of colloid gold label
In the anti-LH monoclonal antibody 1 of E3G monoclonal antibody and colloid gold label, gold colloidal is the granule of particle diameter 30-50nm.
3. a kind of E3G and LH gold-immunochromatographyreagent reagent for assay box as claimed in claim 1, it is characterised in that described anti-LH monoclonal
Antibody 1 is anti-β-LH monoclonal antibody, and described anti-LH monoclonal antibody 2 is anti alpha-LH monoclonal antibody.
4. the preparation method of E3G and the LH gold-immunochromatographyreagent reagent for assay box as described in claim 1-3 any claim, including as follows
Step:
1) by anti-for colloid gold label LH monoclonal antibody 1, the anti-LH i.e. obtaining colloid gold label after closing after centrifugal concentrating is mono-
Clonal antibody 1；By anti-for colloid gold label E3G monoclonal antibody, after closing, after centrifugal concentrating, i.e. obtain the anti-E3G of colloid gold label
Monoclonal antibody；The anti-LH monoclonal antibody 1 of the colloid gold label of gained and the anti-E3G monoclonal of colloid gold label will be prepared
Mix after antibody dilution；
2) after glass fibre fully being soaked in step 1 gained mixed liquor, take out and squeeze and remove surplus liquid, tile, after drying
Obtain colloidal gold pad, standby；
3) the specking detection on nitrocellulose filter respectively of anti-LH monoclonal antibody 2 solution and E3G-OVA antigenic solution is used
Line and control line, and nitrocellulose filter that preparation is completed dried immune nitrocellulose filter, standby；
4) untreated filter sample paper is fully soaked in sample pad treatment fluid, be dried after taking out and extract surplus liquid, it is thus achieved that sample
Product pad, standby；
5) prepared by immune nitrocellulose filter, step 4 prepared by the colloidal gold pad absorbent paper, step 2 prepared, step 3
Sample pad is pasted onto on base plate, and cutting, encapsulation prepare detection kit.
5. the preparation method of E3G and LH gold-immunochromatographyreagent reagent for assay box as claimed in claim 4, it is characterised in that described step 1
In, described gold colloidal uses trisodium citrate reduction method to prepare.
6. the preparation method of E3G and LH gold-immunochromatographyreagent reagent for assay box as claimed in claim 4, it is characterised in that described step 1
In, anti-LH monoclonal antibody 1 is anti-β-LH monoclonal antibody, by the tool of colloid gold label anti-β-LH monoclonal antibody
Body method is: anti-β-LH monoclonal antibody existed with the ratio of 0.9-1.1mg/ml antibody correspondence 150OD gold colloidal
Under conditions of pH=6.95-7.05, by colloid gold label anti-β-LH monoclonal antibody, after 30 minutes, at pH=7.35-7.45
Under conditions of close with BSA, i.e. obtain the anti-β-LH monoclonal antibody of colloid gold label after centrifugal concentrating.
7. the preparation method of E3G and LH gold-immunochromatographyreagent reagent for assay box as claimed in claim 4, it is characterised in that described step 1
In, by anti-for colloid gold label E3G monoclonal antibody method particularly includes: by anti-E3G monoclonal antibody with 0.9-1.1mg/ml
The ratio of antibody correspondence 150OD gold colloidal is under conditions of pH=7.95-8.05, by anti-for colloid gold label E3G monoclonal anti
Body, after 30 minutes, closes with BSA under conditions of pH=7.35-7.45, i.e. obtains gold colloidal mark after centrifugal concentrating
The anti-E3G monoclonal antibody of note.
8. the preparation method of E3G and LH gold-immunochromatographyreagent reagent for assay box as claimed in claim 4, it is characterised in that described anti-LH
Also dialyse before monoclonal antibody 1 and anti-E3G labeling of monoclonal antibody.
9. the preparation method of E3G and LH gold-immunochromatographyreagent reagent for assay box as claimed in claim 4, it is characterised in that described step 1
In, the anti-LH monoclonal antibody 1 of colloid gold label is dissolved in colloidal gold film diluent, the OD=3.8-4.2 of gained diluent,
The anti-E3G monoclonal antibody of colloid gold label is dissolved in colloidal gold film diluent, the OD=2.8-3.2 of gained diluent, then
Both diluents are mixed according to the ratio of 1:0.95-1.05.
10. the preparation method of E3G and LH gold-immunochromatographyreagent reagent for assay box as claimed in claim 4, it is characterised in that described
In step 3, described anti-LH monoclonal antibody 2 solution is anti alpha-LH monoclonal antibody solution, and solution concentration is 0.9-1.1
Mg/ml, specking amount is 0.9-1.1ul/cm；The solution concentration of described E3G-OVA antigenic solution is 1.8-2.2mg/ml, spray
Point amount is 0.9-1.1ul/cm.
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| CN107132356A (en) * | 2017-04-27 | 2017-09-05 | 北京航空航天大学 | A kind of colloidal gold test paper card for detecting carcinoma of urinary bladder and preparation method thereof |
| CN110346554A (en) * | 2018-04-02 | 2019-10-18 | 洛阳普莱柯万泰生物技术有限公司 | A kind of double-antibody method enzyme immunochromatographytest test kit and preparation method and application |
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Application publication date: 20160727 |