CN109613252A - Echinococcus granulosus hyperimmune yolk antibody and test strip and preparation method and application - Google Patents
Echinococcus granulosus hyperimmune yolk antibody and test strip and preparation method and application Download PDFInfo
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- CN109613252A CN109613252A CN201811230812.XA CN201811230812A CN109613252A CN 109613252 A CN109613252 A CN 109613252A CN 201811230812 A CN201811230812 A CN 201811230812A CN 109613252 A CN109613252 A CN 109613252A
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- echinococcus
- yolk antibody
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- 201000003808 Cystic echinococcosis Diseases 0.000 title claims abstract description 113
- 241000244170 Echinococcus granulosus Species 0.000 title claims abstract description 93
- 238000012360 testing method Methods 0.000 title claims abstract description 66
- 238000002360 preparation method Methods 0.000 title claims abstract description 35
- 230000000521 hyperimmunizing effect Effects 0.000 title claims 24
- PCHJSUWPFVWCPO-UHFFFAOYSA-N gold Chemical compound [Au] PCHJSUWPFVWCPO-UHFFFAOYSA-N 0.000 claims abstract description 73
- 241000244160 Echinococcus Species 0.000 claims abstract description 66
- 102000036639 antigens Human genes 0.000 claims abstract description 51
- 108091007433 antigens Proteins 0.000 claims abstract description 51
- 239000010931 gold Substances 0.000 claims abstract description 41
- 208000009366 Echinococcosis Diseases 0.000 claims abstract description 36
- 229910052737 gold Inorganic materials 0.000 claims abstract description 27
- 239000000084 colloidal system Substances 0.000 claims abstract description 26
- 238000001514 detection method Methods 0.000 claims abstract description 24
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- UQLDLKMNUJERMK-UHFFFAOYSA-L di(octadecanoyloxy)lead Chemical compound [Pb+2].CCCCCCCCCCCCCCCCCC([O-])=O.CCCCCCCCCCCCCCCCCC([O-])=O UQLDLKMNUJERMK-UHFFFAOYSA-L 0.000 claims description 20
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- 229910021538 borax Inorganic materials 0.000 claims description 6
- UQGFMSUEHSUPRD-UHFFFAOYSA-N disodium;3,7-dioxido-2,4,6,8,9-pentaoxa-1,3,5,7-tetraborabicyclo[3.3.1]nonane Chemical compound [Na+].[Na+].O1B([O-])OB2OB([O-])OB1O2 UQGFMSUEHSUPRD-UHFFFAOYSA-N 0.000 claims description 6
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- IVLXQGJVBGMLRR-UHFFFAOYSA-N 2-aminoacetic acid;hydron;chloride Chemical compound Cl.NCC(O)=O IVLXQGJVBGMLRR-UHFFFAOYSA-N 0.000 claims description 4
- FPQQSJJWHUJYPU-UHFFFAOYSA-N 3-(dimethylamino)propyliminomethylidene-ethylazanium;chloride Chemical compound Cl.CCN=C=NCCCN(C)C FPQQSJJWHUJYPU-UHFFFAOYSA-N 0.000 claims description 4
- NQTADLQHYWFPDB-UHFFFAOYSA-N N-Hydroxysuccinimide Chemical compound ON1C(=O)CCC1=O NQTADLQHYWFPDB-UHFFFAOYSA-N 0.000 claims description 4
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- LOKCTEFSRHRXRJ-UHFFFAOYSA-I dipotassium trisodium dihydrogen phosphate hydrogen phosphate dichloride Chemical compound P(=O)(O)(O)[O-].[K+].P(=O)(O)([O-])[O-].[Na+].[Na+].[Cl-].[K+].[Cl-].[Na+] LOKCTEFSRHRXRJ-UHFFFAOYSA-I 0.000 claims 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
- G01N33/68—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids
- G01N33/6854—Immunoglobulins
-
- 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
- G01N33/531—Production of immunochemical test materials
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
- G01N33/543—Immunoassay; Biospecific binding assay; Materials therefor with an insoluble carrier for immobilising immunochemicals
- G01N33/54313—Immunoassay; Biospecific binding assay; Materials therefor with an insoluble carrier for immobilising immunochemicals the carrier being characterised by its particulate form
- G01N33/54326—Magnetic particles
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
- G01N33/558—Immunoassay; Biospecific binding assay; Materials therefor using diffusion or migration of antigen or antibody
-
- 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
- G01N2333/43504—Assays involving biological materials from specific organisms or of a specific nature from animals; from humans from invertebrates
- G01N2333/43526—Assays involving biological materials from specific organisms or of a specific nature from animals; from humans from invertebrates from worms
- G01N2333/43539—Assays involving biological materials from specific organisms or of a specific nature from animals; from humans from invertebrates from worms from cestodes
- G01N2333/43543—Assays involving biological materials from specific organisms or of a specific nature from animals; from humans from invertebrates from worms from cestodes from Taenia
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- Health & Medical Sciences (AREA)
- Life Sciences & Earth Sciences (AREA)
- Immunology (AREA)
- Engineering & Computer Science (AREA)
- Molecular Biology (AREA)
- Biomedical Technology (AREA)
- Chemical & Material Sciences (AREA)
- Hematology (AREA)
- Urology & Nephrology (AREA)
- Biotechnology (AREA)
- Microbiology (AREA)
- Cell Biology (AREA)
- Food Science & Technology (AREA)
- Medicinal Chemistry (AREA)
- Physics & Mathematics (AREA)
- Analytical Chemistry (AREA)
- Biochemistry (AREA)
- General Health & Medical Sciences (AREA)
- General Physics & Mathematics (AREA)
- Pathology (AREA)
- Proteomics, Peptides & Aminoacids (AREA)
- Peptides Or Proteins (AREA)
Abstract
The present invention relates to Yolk antibody and its preparation method and application technical fields, it is a kind of Echinococcus granulosus high immunity yolk antibody and test strips and preparation method and application, the Echinococcus granulosus high immunity yolk antibody, it carries out as follows: the preparation of echinococcus antigen protein, the preparation of immunogene, layer breeding and immune, the preparation of Echinococcus granulosus high immunity yolk antibody.The first public Echinococcus granulosus high immunity yolk antibody of the present invention, freeze-drying high immunity yolk antibody and its application in terms of Diagnosis of Human Hydatidosis, when using colloid gold chromatographic test paper strip Diagnosis of Human Hydatidosis made of Echinococcus granulosus high immunity yolk antibody of the present invention, freeze-drying high immunity yolk antibody, the colloid gold chromatographic test paper strip has good sensitivity, specificity, Stability and veracity, provides a kind of simpler, lower detection means of cost for the diagnosis of echinococcosis.
Description
Technical field
It is that a kind of Echinococcus granulosus height exempts from ovum the present invention relates to Yolk antibody and its preparation method and application technical field
Yellow antibody and test strips and preparation method and application comprising a kind of Echinococcus granulosus high immunity yolk antibody and preparation method thereof
With its application in the reagent or reagent strip or drug for preparing Diagnosis of Human Hydatidosis；Further include a kind of freeze-drying high immunity yolk antibody and
Preparation method and its application in the reagent or reagent strip or drug for preparing Diagnosis of Human Hydatidosis；It further include a kind of colloid layer gold
Analyse test strips and preparation method and its application in the kit for preparing Diagnosis of Human Hydatidosis.Test strips in denomination of invention represent
Colloid gold chromatographic test paper strip.
Background technique
Echinococcosis is also known as hydatidosis, is infecting both domestic animals and human parasitic disease, population health even life is seriously endangered, due to passing
Dye source is difficult to control and lack efficient prevention and control measure, China, the seven provinces and regions Endemic Areas Reng Weigao, western part, some areas crowd's echinococcosis
Illness rate is up to 8.93% to 12.38%, and farming and animal husbandry area is presented and spreads to city, and the Northwest is spread to east and south
Trend is classified as one of the infectious disease of priority control by country at present.Echinococcosis is often by parasitizing the particulate of canid small intestine
Echinococcus worm's ovum or gravid proglottid are discharged with excrement pollutes grass, drinking-water etc., is eaten by mistake and causes by people and domestic animal.It is advised as echinococcosis prevents and treats
Draw implementation, diagnostic monitoring dog excrement Echinococcus granulosus, be control its propagated between people and animals and immune expelling parasite after it is antipollution must
Want means.Existing cause of disease checks that rate of missed diagnosis constantly increases, and etiological examination is time-consuming, laborious, and operation is loaded down with trivial details, and discomfort is on site
Extensive generaI investigation and epidemic monitoring.Therefore, it is necessary to establish a kind of simple and quick, high sensitivity, economy immunology diagnosis side
Method provides important tool for Synthetical prevention echinococcosis, also provides a new technological means and approach for hydatidosis diagnosis.
Immune colloidal gold technique is one of the main Types of in-vitro diagnosis in immunodiagnosis, mutual using antigen and antibody
In conjunction with specific reaction carry out qualitative or quantitative diagnosis, be widely used to clinical diagnosis at present, especially medicine,
It is had been widely used in veterinary science clinical examination, has that expense is low, high sensitivity, speed are fast, easy to use, easy to operate and be easy to
The features such as universal.
In field of immunology bird inlay (birds) are immunized by using specific antigen, make the immune thorn of its lymphocyte receptor
Antibody IgY is generated after swashing, and antibody IgY is selectively transferred in yolk and constantly accumulate through blood, referred to as ball is immunized in yolk
Albumen (Immunoglobulinof Yolk, IgY) is unique existing antibody in yolk, also anti-for a species specificity for birds
Body obtains specific polyclonal antibody eventually by yolk liquid extraction purification.It is mainly used in the diagnosis detection of disease and prevents
It controls, veterinary clinic, food additives and cosmetic field etc..However, about Yolk immunoglobulin, (yolk is not anti-at present
Body) for the report in terms of hydatidosis diagnosis.
Summary of the invention
The present invention provides a kind of Echinococcus granulosus high immunity yolk antibodies and test strips and preparation method and application, overcome
The deficiency of the above-mentioned prior art, the first public Echinococcus granulosus high immunity yolk antibody of the present invention are preparing Diagnosis of Human Hydatidosis
Application in reagent or reagent strip or drug, and Echinococcus granulosus high immunity yolk antibody of the invention is used for Diagnosis of Human Hydatidosis
When, there is good sensitivity, specificity and stability.
Technical solution of the present invention first is that being realized by following measures: a kind of Echinococcus granulosus high-immunity yolk is anti-
The preparation method of body carries out as follows: the preparation of echinococcus antigen protein: the first step uses 0.9% sterile physiological salt
The sterilizing PBS that water or concentration are 0.01mol/L, pH value is 7.2 to 7.4 washs echinococcus polypide, by the spine ball silk ribbon after washing
After worm polypide crushes the echinococcus polypide after freeze thawing multigelation 3 times to 5 times at liquid nitrogen and 37 DEG C, then every 1mg is crushed
Echinococcus polypide add 50 μ L protein lysates crack after obtain lysate, lysate is centrifuged, supernatant is taken after centrifugation, will
Supernatant liquid filtering degerming obtains echinococcus antigen egg after adjusting the protein concentration in the supernatant after degerming to 1mg/mL
It is white；
Second step, the preparation of immunogene: by echinococcus antigen protein that the first step obtains with concentration be 0.01mol/L,
The sterilizing PBS that pH value is 7.2 to 7.4 obtains antigen protein dilution after being diluted, and antigen protein dilution and Freund is complete
Full adjuvant obtains Water-In-Oil Freund's complete adjuvant echinococcus protein immunogen after mixing and emulsify in equal volume, and antigen protein is dilute
It releases and obtains Water-In-Oil incomplete Freund's adjuvant echinococcus protein immunogen after liquid is mixed and emulsified with incomplete Freund's adjuvant；
Third step, layer breeding and immune: the selection 15 weeks laying hens to 20 week old health, hello avermectin for the first time, by
Avermectin is admixed in feed, and hello avermectin is primary again after 7 days to 10 days, and it is 0.2mg/ that laying hen feeds avermectin amount every time
Kilogram, avermectin after feeding, after 7 days to 10 days, carries out first immunisation to the laying hen for being no different paradoxical reaction, for the first time twice
It after immune, be spaced and carry out within 10 days to 14 days being immunized for second, after second immune, the interval third time of progress in 10 days to 14 days is exempted from
Epidemic disease, first immunisation use Water-In-Oil Freund's complete adjuvant echinococcus protein immunogen 1mL, and non-first immunisation uses Water-In-Oil not
Family name Freund's incomplete adjuvant echinococcus protein immunogen 1mL after third time is immune, collects gained egg；
4th step, the preparation of Echinococcus granulosus high immunity yolk antibody:
4.1 sterilize egg obtained by third step, then break eggshell, separate egg white and yolk, yolk is inclined on filter paper, and
Yolk is obtained after absorbing the egg white on yolk completely by filter paper, during obtaining yolk, vitellinae membrana does not rupture, then
Vitellinae membrana is punctured, the yolk stoste in yolk is poured into container and obtains yolk stoste, obtains the operation of yolk stoste from egg
For sterile working；
The PBS that yolk stoste is 0.01mol/L with sterile concentration by 4.2, pH value is 7.4 with the ratio of 1:5 to 10 into
Row dilution, is then stirred at room temperature mixing, then is centrifuged layering after standing, and takes the first supernatant, and 1% is being added into the first supernatant just
Freezing 16h to obtaining IgY first extract for 24 hours, after suction filtration after the stirring of sad solution；
4.3 are added ammonium sulfate into IgY first extract, make the protein content 20% in IgY first extract, then extremely at -5 DEG C
50min to 80min is stirred at 0 DEG C, then is centrifuged at 4 DEG C to 5 DEG C with the revolving speed of 8000rpm/min to 12000rpm/min
It is layered to obtain supernatant one and precipitating one again after 10min to 20min, ammonium sulfate is added into supernatant one, makes in supernatant one
Protein content be 50%, 30min to 60min is then stirred at -5 DEG C to 0 DEG C, then with 8000rpm/ at 4 DEG C to 5 DEG C
It is layered to obtain supernatant two and precipitating two again after the revolving speed centrifugation 10min to 20min of min to 12000rpm/min, to supernatant
Ammonium sulfate is added in two, makes the protein content 80% in supernatant two, then stirs 20min extremely at -5 DEG C to 0 DEG C
40min, then be layered again after being centrifuged 10min to 20min at 4 DEG C to 5 DEG C with the revolving speed of 8000rpm/min to 12000rpm/min
Obtain supernatant three and precipitating three；
Precipitating one, precipitating two obtained in 4.3 steps and precipitating three are mixed to get primary sedimentation by 4.4, by primary sedimentation
With obtaining lysate after the PBS dissolution that the sterile concentration isometric with yolk is 0.01mol/L, pH value is 7.4, then with poly- second
Glycol 6000 precipitates the Yolk antibody in lysate, then 10min to 30min is stirred at -5 DEG C to 0 DEG C, then at 4 DEG C to 5 DEG C
Under 10min to 20min is centrifuged with the revolving speed of 8000rpm/min to 12000rpm/min after centrifugation obtain secondary precipitation；
4.5 dissolution is resuspended with PBS again in secondary precipitation after obtain that lysate one is resuspended, be then added under the conditions of 4 DEG C poly-
The Yolk antibody in lysate one is resuspended in the precipitating of ethylene glycol 6000, then 10min to 30min is stirred at -5 DEG C to 0 DEG C, then exists
It is centrifuged to be centrifuged after 10min to 20min with the revolving speed of 8000rpm/min to 12000rpm/min at 4 DEG C to 5 DEG C and be sunk three times
It forms sediment；
4.6 will precipitate three times again with PBS be resuspended dissolution obtain be resuspended lysate two, will be resuspended lysate two using PBS as
Dialyzate dialysis is for 24 hours to obtaining Echinococcus granulosus high immunity yolk antibody after 48h, wherein preceding 10 hours every 2h to 3h replacement one
Secondary dialyzate, every 5h to 6h replaces a dialyzate later.
Technical solution of the present invention second is that being realized by following measures: a kind of Echinococcus granulosus high-immunity yolk is anti-
Body obtains as follows: the first step, the preparation of echinococcus antigen protein: use 0.9% sterile saline or concentration for
0.01mol/L, the sterilizing PBS that pH value is 7.2 to 7.4 wash echinococcus polypide, by the echinococcus polypide after washing in liquid
Multigelation 3 times to 5 times at nitrogen and 37 DEG C, the echinococcus polypide after freeze thawing is crushed, then the smashed echinococcus of every 1mg
Polypide obtains lysate after adding 50 μ L protein lysates to crack, and lysate is centrifuged, supernatant is taken after centrifugation, by supernatant liquid filtering
Degerming obtains echinococcus antigen protein after adjusting the protein concentration in the supernatant after degerming to 1mg/mL；
Second step, the preparation of immunogene: by echinococcus antigen protein that the first step obtains with concentration be 0.01mol/L,
The sterilizing PBS that pH value is 7.2 to 7.4 obtains antigen protein dilution after being diluted, and antigen protein dilution and Freund is complete
Full adjuvant obtains Water-In-Oil Freund's complete adjuvant echinococcus protein immunogen after mixing and emulsify in equal volume, and antigen protein is dilute
It releases and obtains Water-In-Oil incomplete Freund's adjuvant echinococcus protein immunogen after liquid is mixed and emulsified with incomplete Freund's adjuvant；
Third step, layer breeding and immune: the selection 15 weeks laying hens to 20 week old health, hello avermectin for the first time, by
Avermectin is admixed in feed, and hello avermectin is primary again after 7 days to 10 days, and it is 0.2mg/ that laying hen feeds avermectin amount every time
Kilogram, avermectin after feeding, after 7 days to 10 days, carries out first immunisation to the laying hen for being no different paradoxical reaction, for the first time twice
It after immune, be spaced and carry out within 10 days to 14 days being immunized for second, after second immune, the interval third time of progress in 10 days to 14 days is exempted from
Epidemic disease, first immunisation use Water-In-Oil Freund's complete adjuvant echinococcus protein immunogen 1mL, and non-first immunisation uses Water-In-Oil not
Family name Freund's incomplete adjuvant echinococcus protein immunogen 1mL after third time is immune, collects gained egg；
4th step, the preparation of Echinococcus granulosus high immunity yolk antibody:
4.1 sterilize egg obtained by third step, then break eggshell, separate egg white and yolk, yolk is inclined on filter paper, and
Yolk is obtained after absorbing the egg white on yolk completely by filter paper, during obtaining yolk, vitellinae membrana does not rupture, then
Vitellinae membrana is punctured, the yolk stoste in yolk is poured into container and obtains yolk stoste, obtains the operation of yolk stoste from egg
For sterile working；
The PBS that yolk stoste is 0.01mol/L with sterile concentration by 4.2, pH value is 7.4 with the ratio of 1:5 to 10 into
Row dilution, is then stirred at room temperature mixing, then is centrifuged layering after standing, and takes the first supernatant, and 1% is being added into the first supernatant just
Freezing 16h to obtaining IgY first extract for 24 hours, after suction filtration after the stirring of sad solution；
4.3 are added ammonium sulfate into IgY first extract, make the protein content 20% in IgY first extract, then extremely at -5 DEG C
50min to 80min is stirred at 0 DEG C, then is centrifuged at 4 DEG C to 5 DEG C with the revolving speed of 8000rpm/min to 12000rpm/min
It is layered to obtain supernatant one and precipitating one again after 10min to 20min, ammonium sulfate is added into supernatant one, makes in supernatant one
Protein content be 50%, 30min to 60min is then stirred at -5 DEG C to 0 DEG C, then with 8000rpm/ at 4 DEG C to 5 DEG C
It is layered to obtain supernatant two and precipitating two again after the revolving speed centrifugation 10min to 20min of min to 12000rpm/min, to supernatant
Ammonium sulfate is added in two, makes the protein content 80% in supernatant two, then stirs 20min extremely at -5 DEG C to 0 DEG C
40min, then be layered again after being centrifuged 10min to 20min at 4 DEG C to 5 DEG C with the revolving speed of 8000rpm/min to 12000rpm/min
Obtain supernatant three and precipitating three；
Precipitating one, precipitating two obtained in 4.3 steps and precipitating three are mixed to get primary sedimentation by 4.4, by primary sedimentation
With obtaining lysate after the PBS dissolution that the sterile concentration isometric with yolk is 0.01mol/L, pH value is 7.4, then with poly- second
Glycol 6000 precipitates the Yolk antibody in lysate, then 10min to 30min is stirred at -5 DEG C to 0 DEG C, then at 4 DEG C to 5 DEG C
Under 10min to 20min is centrifuged with the revolving speed of 8000rpm/min to 12000rpm/min after centrifugation obtain secondary precipitation；
4.5 dissolution is resuspended with PBS again in secondary precipitation after obtain that lysate one is resuspended, be then added under the conditions of 4 DEG C poly-
The Yolk antibody in lysate one is resuspended in the precipitating of ethylene glycol 6000, then 10min to 30min is stirred at -5 DEG C to 0 DEG C, then exists
It is centrifuged to be centrifuged after 10min to 20min with the revolving speed of 8000rpm/min to 12000rpm/min at 4 DEG C to 5 DEG C and be sunk three times
It forms sediment；
4.6 will precipitate three times again with PBS be resuspended dissolution obtain be resuspended lysate two, will be resuspended lysate two using PBS as
Dialyzate dialysis is for 24 hours to obtaining Echinococcus granulosus high immunity yolk antibody after 48h, wherein preceding 10 hours every 2h to 3h replacement one
Secondary dialyzate, every 5h to 6h replaces a dialyzate later.
Technical solution of the present invention third is that being realized by following measures: a kind of Echinococcus granulosus high-immunity yolk is anti-
Application of the body in the reagent or reagent strip or drug for preparing Diagnosis of Human Hydatidosis.
Technical solution of the present invention fourth is that being realized by following measures: a kind of preparation that high immunity yolk antibody is lyophilized
Method carries out as follows:
Then with MEST buffer solution for cleaning magnetic bead 3 times 1ml is added into 2ml activator in 1ml to 2ml magnetic bead by step 1
Concussion be uniformly mixed, activator include 10mg/mL 1- (3- dimethylamino-propyl) -3- ethyl-carbodiimide hydrochloride solution and
The n-hydroxysuccinimide solution of 10mg/mL, then be protected from light at room temperature, revolving speed be 150r/min under conditions of react
15min to 1h stands 5min to 10min after reaction, comes out the Beads enrichment after reaction with magnetic frame and obtains activation magnetic
Pearl, and the supernatant of reaction solution is discarded, retain lower layer's object of reaction solution,
Step 2 obtains lower layer's object of reaction solution with 0.01mol/L, the PBST buffer solution that pH value is 5.0 to 7.2
Lower layer's object lysate, at least once using the magnetic bead after the cleaning activation of lower layer's object lysate, then by the activated magnetic beads after cleaning
Be dispersed in 500 μ l to 1000 μ l l0.01mol/L, in the PBST buffer that pH value is 5.0 to 7.2 after the magnetic bead that is activated it is outstanding
Supernatant liquid；
Step 3, the solution by 100 μ l to 200 μ l containing 500 μ g to 1mg echinococcus antigen protein are added to the magnetic of activation
It being mixed in pearl suspension, the oscillation incubation 1h to 2h at 37 DEG C is then allowed to stand Magneto separate after 5min to 10min, and abandon supernatant,
It removes the reaction solution on magnetic bead and obtains the magnetic bead of coupled antigen after not being coupled echinococcus granulosus antigen albumen；
Step 4, closing: wash the magnetic bead 1mg of coupled antigen with PBST buffer, then be added contain 2% fish glue albumen,
The 0.01mol/L of 1% bovine serum albumin(BSA), the PBS1ml that pH value is 7.2 to 7.4 are closed, then the oscillation incubation at 37 DEG C
2h to 2.5h, after standing 5min to 10min, Magneto separate simultaneously abandons supernatant, then washs the magnetic bead after closing with PBST buffer, obtains
Magnetic bead is incubated for closing；
Step 5 is coupled antibody purification: closing incubation magnetic bead being added to 80 μ g to 110 μ g Echinococcus granulosus height and exempts from ovum
In yellow antibody, 37 DEG C of oscillation incubation 1h；Make closing be incubated for magnetic bead to be evenly distributed state, during incubation, closing is incubated for magnetic
Pearl in conjunction with Echinococcus granulosus high immunity yolk antibody, magnetic frame absorption closing be incubated for magnetic bead, suck not with echinococcus antigen
Closing after incubation is incubated for magnetic bead and is washed 3 times with PBST buffer by protein bound Echinococcus granulosus high immunity yolk antibody,
Then it is incubated in magnetic bead to the closing after washing and glycine-HCl eluent is added, blow and beat 1min, immune magnetic is obtained after Magneto separate
The Echinococcus granulosus high immunity yolk antibody of pearl purifying；
The Echinococcus granulosus high immunity yolk antibody of immunomagnetic beads purifying is passed through filtration sterilization and freeze-drying by step 6
After obtain freeze-drying high immunity yolk antibody.
Technical solution of the present invention fifth is that being realized by following measures: a kind of freeze-drying high immunity yolk antibody presses
The method of stating obtains:
Then with MEST buffer solution for cleaning magnetic bead 3 times 1ml is added into 2ml activator in 1ml to 2ml magnetic bead by step 1
Concussion be uniformly mixed, activator include 10mg/mL 1- (3- dimethylamino-propyl) -3- ethyl-carbodiimide hydrochloride solution and
The n-hydroxysuccinimide solution of 10mg/mL, then be protected from light at room temperature, revolving speed be 150r/min under conditions of react
15min to 1h stands 5min to 10min after reaction, comes out the Beads enrichment after reaction with magnetic frame and obtains activation magnetic
Pearl, and the supernatant of reaction solution is discarded, retain lower layer's object of reaction solution,
Step 2 obtains lower layer's object of reaction solution with 0.01mol/L, the PBST buffer solution that pH value is 5.0 to 7.2
Lower layer's object lysate, at least once using the magnetic bead after the cleaning activation of lower layer's object lysate, then by the activated magnetic beads after cleaning
Be dispersed in 500 μ l to 1000 μ l l0.01mol/L, in the PBST buffer that pH value is 5.0 to 7.2 after the magnetic bead that is activated it is outstanding
Supernatant liquid；
Step 3, the solution by 100 μ l to 200 μ l containing 500 μ g to 1mg echinococcus antigen protein are added to the magnetic of activation
It being mixed in pearl suspension, the oscillation incubation 1h to 2h at 37 DEG C is then allowed to stand Magneto separate after 5min to 10min, and abandon supernatant,
It removes the reaction solution on magnetic bead and obtains the magnetic bead of coupled antigen after not being coupled echinococcus granulosus antigen albumen；
Step 4, closing: wash the magnetic bead 1mg of coupled antigen with PBST buffer, then be added contain 2% fish glue albumen,
The 0.01mol/L of 1% bovine serum albumin(BSA), the PBS1ml that pH value is 7.2 to 7.4 are closed, then the oscillation incubation at 37 DEG C
2h to 2.5h, after standing 5min to 10min, Magneto separate simultaneously abandons supernatant, then washs the magnetic bead after closing with PBST buffer, obtains
Magnetic bead is incubated for closing；
Step 5 is coupled antibody purification: closing incubation magnetic bead being added to 80 μ g to 110 μ g Echinococcus granulosus height and exempts from ovum
In yellow antibody, 37 DEG C of oscillation incubation 1h；Make closing be incubated for magnetic bead to be evenly distributed state, during incubation, closing is incubated for magnetic
Pearl in conjunction with Echinococcus granulosus high immunity yolk antibody, magnetic frame absorption closing be incubated for magnetic bead, suck not with echinococcus antigen
Closing after incubation is incubated for magnetic bead and is washed 3 times with PBST buffer by protein bound Echinococcus granulosus high immunity yolk antibody,
Then it is incubated in magnetic bead to the closing after washing and glycine-HCl eluent is added, blow and beat 1min, immune magnetic is obtained after Magneto separate
The Echinococcus granulosus high immunity yolk antibody of pearl purifying；
The Echinococcus granulosus high immunity yolk antibody of immunomagnetic beads purifying is passed through filtration sterilization and freeze-drying by step 6
After obtain freeze-drying high immunity yolk antibody.
Technical solution of the present invention sixth is that being realized by following measures: it is prepared by a kind of freeze-drying high immunity yolk antibody
Application in the reagent or reagent strip or drug of Diagnosis of Human Hydatidosis.
Technical solution of the present invention seventh is that being realized by following measures: a kind of colloid gold chromatographic test paper strip, including
PVC liner plate, nitrocellulose filter, colloidal gold conjugate pad, sample pad and water absorption pad, the top surface of PVC liner plate from a left side it is right according to
Sequence is fixed with sample pad, colloidal gold conjugate pad and nitrocellulose filter, and water absorption pad is fixed on the right part top of nitrocellulose filter
Face, nitrocellulose filter top surface between colloidal gold conjugate pad and water absorption pad from a left side and right septum be provided with test strip and
Quality Control band；Colloidal gold conjugate pad is coated with Echinococcus granulosus high immunity yolk antibody, and test strip endoperidium has spine ball silk ribbon
Worm antigen protein, Quality Control band endoperidium have goat-anti chicken antibody IgG.
Here is seven further optimization and/or improvements to invention technology described above scheme:
Above-mentioned colloidal gold conjugate pad is coated with the Echinococcus granulosus high immunity yolk antibody of Au colloidal nanoparticles label,
The Echinococcus granulosus high immunity yolk antibody of Au colloidal nanoparticles label obtains as follows: will be added in colloidal gold solution
Sodium tetraborate solution be uniformly mixed, by 0.3ml to 0.5ml concentration be 1mg/ml Echinococcus granulosus high immunity yolk antibody dropwise
It is added in 100ml colloidal gold solution and mixes and react, then the avoid light place 45min to 1h at 4 DEG C, then be added dropwise containing 30%
The Tris- that concentration to 50% sucrose, 5% to 10% bovine serum albumin(BSA) or fish glue albumen is 0.05mol/L to 0.2mol/L
HCL buffer is closed, and is mixed, and the membrane filtration for being 0.22 μm with aperture, filtrate is what Au colloidal nanoparticles marked
Echinococcus granulosus high immunity yolk antibody；Or/and test strip endoperidium echinococcus antigen protein obtains as follows:
The Tris-HCL that the phosphate buffer or pH value for being 7.2 to 7.4 with pH value by echinococcus antigen protein are 7.2 to 7.4 delays
Fliud flushing dilutes to obtain 1.0mg/ml test strip coating buffer, is centrifuged at 4 DEG C with 8000r/min to 12000r/min speed
After 60min, the supernatant after the centrifugation of test strip coating buffer is test strip antibody, will test band antibody and is sprayed on nitric acid fibre
It ties up the corresponding test strip position of plain film and dries；Or/and Quality Control band endoperidium goat-anti chicken antibody IgG is obtained as follows
To: the Tris-HCL that the phosphate buffer or pH value for being 7.2 to 7.4 with pH value by goat-anti chicken antibody IgG are 8.0 to 8.2 delays
Fliud flushing dilutes to obtain 1.0mg/ml Quality Control band coating buffer, is centrifuged at 4 DEG C with 8000r/min to 12000r/min speed
After 60min, the supernatant after the centrifugation of Quality Control band coating buffer is Quality Control band antibody, and Quality Control band antibody is sprayed on nitric acid fibre
It ties up the corresponding Quality Control pillar location of plain film and dries；Or/and sample pad, colloidal gold conjugate pad be by pH value be 7.2 to
7.4 confining liquid Seal treatment, the confining liquid are the phosphoric acid containing 1.0% tween, 0.5% to 5.0% bovine serum albumin(BSA)
Salt buffer；Or/and further include shell, shell is wrapped in PVC liner plate, nitrocellulose filter, colloidal gold conjugate pad, sample
The outside of pad and water absorption pad is provided with sample-adding window and detection window on shell, and sample-adding window is located at the sample pad, detection
Window is located at test strip and Quality Control band.
Above-mentioned colloidal gold conjugate pad is coated with the particulate spine ball silk ribbon of the immunomagnetic beads purifying of Au colloidal nanoparticles label
The Echinococcus granulosus high immunity yolk antibody of worm high immunity yolk antibody, the immunomagnetic beads purifying of Au colloidal nanoparticles label is pressed
The method of stating obtains: being uniformly mixed sodium tetraborate solution is added in colloidal gold solution, is 1mg/ml by 0.3ml to 0.5ml concentration
The Echinococcus granulosus high immunity yolk antibody of immunomagnetic beads purifying be added dropwise in 100ml colloidal gold solution and mix and react,
Then the avoid light place 45min to 1h at 4 DEG C, then be added dropwise containing 30% to 50% sucrose, 5% to 10% bovine serum albumin(BSA)
Or the Tris-HCL buffer that the concentration of fish glue albumen is 0.05mol/L to 0.2mol/L is closed, and is mixed, is with aperture
0.22 μm of membrane filtration, filtrate are that the Echinococcus granulosus height of the immunomagnetic beads purifying of Au colloidal nanoparticles label exempts from ovum
Yellow antibody.
Technical solution of the present invention eighth is that being realized by following measures: it is prepared by a kind of colloid gold chromatographic test paper strip
Application in the kit of Diagnosis of Human Hydatidosis.
High immunity yolk antibody is lyophilized and its in diagnosis Echinococcus hydatid cyst in the first public Echinococcus granulosus high immunity yolk antibody of the present invention
The application of sick aspect, using glue made of Echinococcus granulosus high immunity yolk antibody of the present invention, freeze-drying high immunity yolk antibody
When body gold chromatograph test strip Diagnosis of Human Hydatidosis, the colloid gold chromatographic test paper strip has good sensitivity, specificity, accuracy
And stability, a kind of simpler, lower detection means of cost is provided for the diagnosis of echinococcosis.
Detailed description of the invention
Attached drawing 1 is the schematic perspective view of colloid gold chromatographic test paper strip of the present invention.
Coding in attached drawing is respectively as follows: 1 for PVC liner plate, and 2 be nitrocellulose filter, and 3 be colloidal gold conjugate pad, and 4 be sample
Product pad, 5 be water absorption pad, and 6 be test strip, and 7 be Quality Control band.
Specific embodiment
The present invention is not limited by the following examples, can determine according to the technique and scheme of the present invention with actual conditions specific
Embodiment.It is previously mentioned various chemical reagent and chemical article in the present invention unless otherwise specified, is public in the prior art
Know common chemical reagent and chemical article；Percentage in the present invention does not have specified otherwise such as, is mass percent；This hair
It is the aqueous solution that solvent is water, for example, hydrochloric acid solution is aqueous hydrochloric acid solution if the solution in bright is without specified otherwise；This
Room temperature, room temperature in invention refer generally to 15 DEG C to 25 DEG C of temperature, are commonly defined as 25 DEG C；PBS in the present invention is slow for phosphoric acid
Rush salting liquid.
However, the present invention is not limited to exemplary embodiment as disclosed below；It can be by different form come to it
It is realized.The essence of specification is only to aid in those skilled in the relevant arts' Integrated Understanding detail of the invention.
The present invention will be further described below with reference to examples:
Embodiment 1: a kind of Echinococcus granulosus high immunity yolk antibody is carried out by following preparation methods: the first step, spine ball silk ribbon
The preparation of worm antigen protein: the sterilizing that 0.9% sterile saline or concentration are 7.2 to 7.4 for 0.01mol/L, pH value is used
PBS washing echinococcus polypide will be frozen by the echinococcus polypide after washing multigelation 3 times to 5 times at liquid nitrogen and 37 DEG C
Echinococcus polypide after melting crushes, then the smashed echinococcus polypide of every 1mg add 50 μ L protein lysates to crack after obtain
Lysate is centrifuged by lysate, and supernatant is taken after centrifugation, by supernatant liquid filtering degerming, by the albumen in the supernatant after degerming
Concentration obtains echinococcus antigen protein after adjusting to 1mg/mL；
Second step, the preparation of immunogene: by echinococcus antigen protein that the first step obtains with concentration be 0.01mol/L,
The sterilizing PBS that pH value is 7.2 to 7.4 obtains antigen protein dilution after being diluted, and antigen protein dilution and Freund is complete
Full adjuvant obtains Water-In-Oil Freund's complete adjuvant echinococcus protein immunogen after mixing and emulsify in equal volume, and antigen protein is dilute
It releases and obtains Water-In-Oil incomplete Freund's adjuvant echinococcus protein immunogen after liquid is mixed and emulsified with incomplete Freund's adjuvant；
Third step, layer breeding and immune: the selection 15 weeks laying hens to 20 week old health, hello avermectin for the first time, by
Avermectin is admixed in feed, and hello avermectin is primary again after 7 days to 10 days, and it is 0.2mg/ that laying hen feeds avermectin amount every time
Kilogram, avermectin after feeding, after 7 days to 10 days, carries out first immunisation to the laying hen for being no different paradoxical reaction, for the first time twice
It after immune, be spaced and carry out within 10 days to 14 days being immunized for second, after second immune, the interval third time of progress in 10 days to 14 days is exempted from
Epidemic disease, first immunisation use Water-In-Oil Freund's complete adjuvant echinococcus protein immunogen 1mL, and non-first immunisation uses Water-In-Oil not
Family name Freund's incomplete adjuvant echinococcus protein immunogen 1mL after third time is immune, collects gained egg；
4th step, the preparation of Echinococcus granulosus high immunity yolk antibody:
4.1 sterilize egg obtained by third step, then break eggshell, separate egg white and yolk, yolk is inclined on filter paper, and
Yolk is obtained after absorbing the egg white on yolk completely by filter paper, during obtaining yolk, vitellinae membrana does not rupture, then
Vitellinae membrana is punctured, the yolk stoste in yolk is poured into container and obtains yolk stoste, obtains the operation of yolk stoste from egg
For sterile working；
The PBS that yolk stoste is 0.01mol/L with sterile concentration by 4.2, pH value is 7.4 with the ratio of 1:5 to 10 into
Row dilution, is then stirred at room temperature mixing, then is centrifuged layering after standing, and takes the first supernatant, and 1% is being added into the first supernatant just
Freezing 16h to obtaining IgY first extract for 24 hours, after suction filtration after the stirring of sad solution；
4.3 are added ammonium sulfate into IgY first extract, make the protein content 20% in IgY first extract, then extremely at -5 DEG C
50min to 80min is stirred at 0 DEG C, then is centrifuged at 4 DEG C to 5 DEG C with the revolving speed of 8000rpm/min to 12000rpm/min
It is layered to obtain supernatant one and precipitating one again after 10min to 20min, ammonium sulfate is added into supernatant one, makes in supernatant one
Protein content be 50%, 30min to 60min is then stirred at -5 DEG C to 0 DEG C, then with 8000rpm/ at 4 DEG C to 5 DEG C
It is layered to obtain supernatant two and precipitating two again after the revolving speed centrifugation 10min to 20min of min to 12000rpm/min, to supernatant
Ammonium sulfate is added in two, makes the protein content 80% in supernatant two, then stirs 20min extremely at -5 DEG C to 0 DEG C
40min, then be layered again after being centrifuged 10min to 20min at 4 DEG C to 5 DEG C with the revolving speed of 8000rpm/min to 12000rpm/min
Obtain supernatant three and precipitating three；
Precipitating one, precipitating two obtained in 4.3 steps and precipitating three are mixed to get primary sedimentation by 4.4, by primary sedimentation
With obtaining lysate after the PBS dissolution that the sterile concentration isometric with yolk is 0.01mol/L, pH value is 7.4, then with poly- second
Glycol 6000 precipitates the Yolk antibody in lysate, then 10min to 30min is stirred at -5 DEG C to 0 DEG C, then at 4 DEG C to 5 DEG C
Under 10min to 20min is centrifuged with the revolving speed of 8000rpm/min to 12000rpm/min after centrifugation obtain secondary precipitation；
4.5 dissolution is resuspended with PBS again in secondary precipitation after obtain that lysate one is resuspended, be then added under the conditions of 4 DEG C poly-
The Yolk antibody in lysate one is resuspended in the precipitating of ethylene glycol 6000, then 10min to 30min is stirred at -5 DEG C to 0 DEG C, then exists
It is centrifuged to be centrifuged after 10min to 20min with the revolving speed of 8000rpm/min to 12000rpm/min at 4 DEG C to 5 DEG C and be sunk three times
It forms sediment；
4.6 will precipitate three times again with PBS be resuspended dissolution obtain be resuspended lysate two, will be resuspended lysate two using PBS as
Dialyzate dialysis is for 24 hours to obtaining Echinococcus granulosus high immunity yolk antibody after 48h, wherein preceding 10 hours every 2h to 3h replacement one
Secondary dialyzate, every 5h to 6h replaces a dialyzate later.
During obtaining Echinococcus granulosus high immunity yolk antibody according to the present embodiment 1, pass through the egg white and yolk
Separate mode, can make that egg white and yolk separates more thoroughly, then make the extraction of Echinococcus granulosus high immunity yolk antibody
Purity is higher, and by its subsequent third step and the 4th step, can further mention Echinococcus granulosus high immunity yolk antibody
DNA purity.
And the Echinococcus granulosus high immunity yolk antibody specificity obtained according to the present embodiment 1 is high, not activating complement: passing
The antibody Fc section of system can specifically bind Rheumatoid factors, in immune detection, increase the reaction of false positive, and of the invention
Echinococcus granulosus high immunity yolk antibody (IgY) not in conjunction with rheumatism sex factor, as detection reagent, have unique excellent
Gesture.
Due to containing complement in the serum of mammal, to be inactivated when correlation test, and the IgY invented is free of
Complement system can avoid the interference of complement system, reduce the generation of false negative.The present invention is using hen as generation IgY antibody
Parent, so that its generation is resisted the stabilization of antibodies (IgY) of a greater variety of antigenic determinant antibody and more efficient valence.
In addition, having fine when using obtained Echinococcus granulosus high immunity yolk antibody Diagnosis of Human Hydatidosis of the invention
Sensitivity, specificity, Stability and veracity.
Embodiment 2: a kind of Echinococcus granulosus high immunity yolk antibody in the reagent or reagent strip for preparing Diagnosis of Human Hydatidosis or
Application in drug.
Embodiment 3: a kind of freeze-drying high immunity yolk antibody is carried out by following preparation methods:
Then with MEST buffer solution for cleaning magnetic bead 3 times 1ml is added into 2ml activator in 1ml to 2ml magnetic bead by step 1
Concussion be uniformly mixed, activator include 10mg/mL 1- (3- dimethylamino-propyl) -3- ethyl-carbodiimide hydrochloride solution and
The n-hydroxysuccinimide solution of 10mg/mL, then be protected from light at room temperature, revolving speed be 150r/min under conditions of react
15min to 1h stands 5min to 10min after reaction, comes out the Beads enrichment after reaction with magnetic frame and obtains activation magnetic
Pearl, and the supernatant of reaction solution is discarded, retain lower layer's object of reaction solution,
Step 2 obtains lower layer's object of reaction solution with 0.01mol/L, the PBST buffer solution that pH value is 5.0 to 7.2
Lower layer's object lysate, at least once using the magnetic bead after the cleaning activation of lower layer's object lysate, then by the activated magnetic beads after cleaning
Be dispersed in 500 μ l to 1000 μ l l0.01mol/L, in the PBST buffer that pH value is 5.0 to 7.2 after the magnetic bead that is activated it is outstanding
Supernatant liquid；
Step 3, the solution by 100 μ l to 200 μ l containing 500 μ g to 1mg echinococcus antigen protein are added to the magnetic of activation
It being mixed in pearl suspension, the oscillation incubation 1h to 2h at 37 DEG C is then allowed to stand Magneto separate after 5min to 10min, and abandon supernatant,
It removes the reaction solution on magnetic bead and obtains the magnetic bead of coupled antigen after not being coupled echinococcus granulosus antigen albumen；
Step 4, closing: wash the magnetic bead 1mg of coupled antigen with PBST buffer, then be added contain 2% fish glue albumen,
The 0.01mol/L of 1% bovine serum albumin(BSA), the PBS1ml that pH value is 7.2 to 7.4 are closed, then the oscillation incubation at 37 DEG C
2h to 2.5h, after standing 5min to 10min, Magneto separate simultaneously abandons supernatant, then washs the magnetic bead after closing with PBST buffer, obtains
Magnetic bead is incubated for closing；
Step 5 is coupled antibody purification: closing incubation magnetic bead being added to 80 μ g to 110 μ g Echinococcus granulosus height and exempts from ovum
In yellow antibody, 37 DEG C of oscillation incubation 1h；Make closing be incubated for magnetic bead to be evenly distributed state, during incubation, closing is incubated for magnetic
Pearl in conjunction with Echinococcus granulosus high immunity yolk antibody, magnetic frame absorption closing be incubated for magnetic bead, suck not with echinococcus antigen
Closing after incubation is incubated for magnetic bead and is washed 3 times with PBST buffer by protein bound Echinococcus granulosus high immunity yolk antibody,
Then it is incubated in magnetic bead to the closing after washing and glycine-HCl eluent is added, blow and beat 1min, immune magnetic is obtained after Magneto separate
The Echinococcus granulosus high immunity yolk antibody of pearl purifying；
The Echinococcus granulosus high immunity yolk antibody of immunomagnetic beads purifying is passed through filtration sterilization and freeze-drying by step 6
After obtain freeze-drying high immunity yolk antibody.
When using high immunity yolk antibody Diagnosis of Human Hydatidosis being lyophilized described in the present embodiment 3, with better sensitivity, specifically
Property, Stability and veracity.
Embodiment 4: a kind of freeze-drying high immunity yolk antibody is in the reagent or reagent strip or drug for preparing Diagnosis of Human Hydatidosis
Using.
Embodiment 5: as shown in Fig. 1, a kind of colloid gold chromatographic test paper strip, including PVC liner plate 1, nitrocellulose filter 2,
Colloidal gold conjugate pad 3, sample pad 4 and water absorption pad 5 are sequentially fixed with sample pad 4, glue from a left side is right in the top surface of PVC liner plate 1
Body gold conjugate pad 3 and nitrocellulose filter 2, water absorption pad 5 is fixed on the right part top surface of nitrocellulose filter 2, in colloidal gold knot
2 top surface of nitrocellulose filter between object pad 3 and water absorption pad 5 is closed from a left side and right septum is provided with test strip 6 and Quality Control band
7；Colloidal gold conjugate pad 3 is coated with Echinococcus granulosus high immunity yolk antibody, and 6 endoperidium of test strip has echinococcus antigen
Albumen, 7 endoperidium of Quality Control band have goat-anti chicken antibody IgG.
Embodiment 6: as the optimization of above-described embodiment 5, colloidal gold conjugate pad 3 is coated with Au colloidal nanoparticles label
Echinococcus granulosus high immunity yolk antibody, the Echinococcus granulosus high immunity yolk antibody of Au colloidal nanoparticles label is by following
Method obtains: being uniformly mixed sodium tetraborate solution is added in colloidal gold solution, is 1mg/ml's by 0.3ml to 0.5ml concentration
Echinococcus granulosus high immunity yolk antibody is added dropwise in 100ml colloidal gold solution and mixes and react, and is then protected from light and puts at 4 DEG C
45min to 1h is set, then the concentration containing 30% to 50% sucrose, 5% to 10% bovine serum albumin(BSA) or fish glue albumen is added dropwise
It is closed, is mixed for the Tris-HCL buffer of 0.05mol/L to 0.2mol/L, the membrane filtration for being 0.22 μm with aperture,
Filtrate is the Echinococcus granulosus high immunity yolk antibody of Au colloidal nanoparticles label；Or/and 6 endoperidium spine of test strip
Ball tapeworm antigen protein obtains as follows: the phosphate buffer for being 7.2 to 7.4 by echinococcus antigen protein pH value
Or pH value be 7.2 to 7.4 Tris-HCL buffer dilute to obtain 6 coating buffer of 1.0mg/ml test strip, at 4 DEG C with
After 8000r/min is centrifuged 60min to 12000r/min speed, the supernatant after the centrifugation of 6 coating buffer of test strip is test strip 6
Antibody will test 6 antibody of band and be sprayed on corresponding 6 position of test strip of nitrocellulose filter 2 and dry；Or/and Quality Control item
7 endoperidium goat-anti chicken antibody IgG of band is obtained as follows: the phosphate for being 7.2 to 7.4 by goat-anti chicken antibody IgG pH value
The Tris-HCL buffer that buffer or pH value are 8.0 to 8.2 dilutes to obtain 7 coating buffer of 1.0mg/ml Quality Control band, at 4 DEG C
After being centrifuged 60min with 8000r/min to 12000r/min speed, the supernatant after the centrifugation of 7 coating buffer of Quality Control band is Quality Control item
7 antibody of Quality Control band is sprayed on corresponding 7 position of Quality Control band of nitrocellulose filter 2 and dried by 7 antibody of band；Or/and sample
This pad, colloidal gold conjugate pad 3 be by pH value be 7.2 to 7.4 confining liquid Seal treatment, the confining liquid be containing
The phosphate buffer of 1.0% tween, 0.5% to 5.0% bovine serum albumin(BSA)；Or/and further include shell, shell is wrapped in
The outside of PVC liner plate 1, nitrocellulose filter 2, colloidal gold conjugate pad 3, sample pad 4 and water absorption pad 5, be provided on shell plus
Sample window and detection window, sample-adding window are located at the sample pad, and detection window is located at test strip 6 and Quality Control band 7.
Embodiment 7: as the optimization of above-described embodiment 5 to 6, colloidal gold conjugate pad 3 is coated with Au colloidal nanoparticles
The Echinococcus granulosus high immunity yolk antibody of the immunomagnetic beads purifying of label, the immunomagnetic beads purifying of Au colloidal nanoparticles label
Echinococcus granulosus high immunity yolk antibody obtain as follows: will be added in colloidal gold solution sodium tetraborate solution mix it is equal
It is even, the Echinococcus granulosus high immunity yolk antibody that the immunomagnetic beads that 0.3ml to 0.5ml concentration is 1mg/ml purify is added dropwise
Mix and react in 100ml colloidal gold solution, then the avoid light place 45min to 1h at 4 DEG C, then be added dropwise containing 30% to
The concentration of 50% sucrose, 5% to 10% bovine serum albumin(BSA) or fish glue albumen is the Tris-HCL of 0.05mol/L to 0.2mol/L
Buffer is closed, and is mixed, the membrane filtration for being 0.22 μm with aperture, and filtrate is the immune of Au colloidal nanoparticles label
The Echinococcus granulosus high immunity yolk antibody of magnetic beads for purifying.
A kind of embodiment 8: the application of colloid gold chromatographic test paper strip in the kit for preparing Diagnosis of Human Hydatidosis.
Embodiment 9: the application method of colloid gold chromatographic test paper strip includes the following steps: to acquire dog excrement sample 2ml to 3ml, room
Temperature places 15min to 120min, is centrifuged 10min to 30min with 3000r/min speed, Aspirate supernatant is into Sample dilution
It mixes to get sample to be tested；100 μ L samples to be tested are accurately drawn using pipettor, and the sample in colloid gold chromatographic test paper strip is added dropwise
On this gasket；Protein antibodies flow under siphonage to 5 one end of water absorption pad with sample liquid, and reaction can determine whether to examine in 5min
Survey as a result, and in 30min, colour developing result will not change.After sample to be tested chromatography is complete, observing colloid gold chromatograph test strip
Test strip 6 and Quality Control band 7；
When there is tested antigenic substance (i.e. echinococcus) in determinand, the antigenic substance in determinand will be with colloid
The Echinococcus granulosus high immunity yolk antibody IgY of colloid gold label on golden conjugate pad 3 forms Ag-Ab-Au compound, compound
Object can only occur a colour band (C line) not in conjunction with the echinococcus antigen protein on test strip 6 at Quality Control band 7, knot
Fruit is the positive, illustrates that dog excrement sample is by Echinococcus infections in detected sample；
When the antigenic substance not being tested in test substance, the Echinococcus granulosus high immunity yolk antibody of colloid gold label
IgY will form Ag-Ab-Au compound, at this time at test strip 6 in conjunction with the echinococcus antigen protein on test strip 6
An obvious colour band (T line) can be seen, therefore when two lines (C line and T line) can be seen on Test paper, result is feminine gender,
Illustrate that middle dog excrement sample is not by Echinococcus infections in detected sample.
Embodiment 10: colloid gold chromatographic test paper strip performance detection of the present invention
1, sensitivity
Echinococcus adult albumen is extracted, after measuring echinococcus adult protein concentration, is diluted to 1mg/mL solution for standby,
It is diluted to 0,0.001,0.01,0.1,1,10,20 μ g/L respectively with 0.01mol/L, the PBS buffer solution that pH value is 7.2 to 7.4 again
A series of positive quality control solution, take 90 μ L above-mentioned positive quality control solution drop in the sample pad 4 of test strips respectively, seen after 3min
Examine the colour developing situation of test strips.
When echinococcus adult protein concentration is 0.01 μ g/L, T line when T line is relative to 0 μ g/L shows slightly light color, and works as
When echinococcus adult protein concentration is 1 μ g/L, T line shows claret, illustrates that the detection of colloidal gold strip is limited to 0.01 μ g/L,
Illustrate that colloid gold chromatographic test paper strip of the present invention has good sensitivity.
2, specific
It chooses 3 kinds of common dog enteron aisles that parasitize and obtains helminth (band tapeworm, roundworm, coccidia), extract total egg of each polypide
White is albumen mother liquor, and albumen mother liquor is prepared as 1 μ g/L with 0.01mol/L, the PBS dilution that pH value is 7.4 respectively, takes 90 respectively
The sample well that μ L is added drop-wise to test strips is tested, and test strips color developing effect is observed.
Testing result shows, colloid gold chromatographic test paper strip of the present invention be 33% and ascarid with tapeworm cross reacting rate
The cross reacting rate of worm is 26%, cross reaction is not present with coccidia, illustrates that colloid gold chromatographic test paper strip of the present invention has
Specificity well.
3, accuracy
Randomly select 22 parts of negative dog excrement samples after the positive dog excrement sample of artificial challenge echinococcus and 22 parts of expelling parasites
Product, as sample of having a try.Take out sample be added sample diluting liquid be placed at room temperature for 15min to 120min, with 3000r/min speed from
Heart 10min to 30min, Aspirate supernatant are mixed into Sample dilution, using colloid gold chromatographic test paper strip of the present invention into
Row detection, compares testing result and is shown in Table 1.It can be seen that colloid gold chromatographic test paper strip accuracy of the present invention by 1 data of table
It is high.
4, stability
Accelerated ageing and refrigeration test are done simultaneously, colloid gold chromatographic test paper strip of the present invention is placed in 37 DEG C, -20 DEG C
7 days, measurement result also indicated that the indices of kit are normal.This kit can save 12 months at 2 DEG C to 8 DEG C.
In conclusion the first public Echinococcus granulosus high immunity yolk antibody of the present invention, freeze-drying high immunity yolk antibody and its
Application in terms of Diagnosis of Human Hydatidosis, it is anti-using Echinococcus granulosus high immunity yolk antibody of the present invention, freeze-drying high-immunity yolk
Made of body when colloid gold chromatographic test paper strip Diagnosis of Human Hydatidosis, the colloid gold chromatographic test paper strip has good sensitivity, spy
Anisotropic, Stability and veracity provides a kind of simpler, lower detection means of cost for the diagnosis of echinococcosis.
The above technical features constitute embodiments of the present invention, can basis with stronger adaptability and implementation result
Actual needs increases and decreases non-essential technical characteristic, to meet the needs of different situations.
1 accuracy measurement result of table
| Sample | Quantity | It is positive | It is negative | Repetitive rate | Imprecision |
| It is positive | 22 | 22 | 0 | 100% | 0% |
| It is negative | 22 | 0 | 22 | 100% | 0% |
Claims (10)
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| CN113016713A (en) * | 2021-03-03 | 2021-06-25 | 新疆医科大学第一附属医院 | Method for separating echinococcus granulosus eggs from dog feces and hatching oncosphere in vitro to carry out primary infection animal molding |
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