CN107402298A - A kind of dog excrement echinococcosis antigen quick detection kit and its application method - Google Patents
A kind of dog excrement echinococcosis antigen quick detection kit and its application method Download PDFInfo
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Abstract
The present invention relates to a kind of dog excrement echinococcosis antigen quick detection kit and its application method, the dog excrement echinococcosis antigen quick detection kit, including sample pad, gold standard pad, nitrocellulose filter, adsorptive pads and bottom plate, sample pad is fixed with the left part top of bottom plate, gold standard pad is fixed between the right part and bottom plate of sample pad, adsorptive pads are fixed with the right part top of bottom plate, nitrocellulose filter is fixed between gold standard pad and adsorptive pads.The dog excrement echinococcosis antigen quick detection kit (colloidal gold method) of the present invention has higher sensitiveness, specificity and stability, it can carry with, immediately the test at the scene of carrying out, special test accessory is not needed, shortens the testing time, improves testing efficiency, in addition, the dog excrement echinococcosis antigen quick detection kit of the present invention is tested dog excrement, it is therefore prevented that the injury to dog so that detection work is more easy.
Description
Technical field
The present invention relates to canine Echinococcus granulosus detection technique technical field, in particular to a kind of dog excrement Echinococcus hydatid cyst
Sick rapid antigen detection kit (colloidal gold method) and its application method.
Background technology
Echinococcus granulosus is the mankind and domestic animal（Sheep, ox and camel etc.）The cause of disease of cystic echinococcosis.Particulate spine ball silk ribbon
Worm has two life stages, and one of life stage is adult stage, adult stage hermaphroditic, colonizes in domesticated dog small intestine
In, it is fixed on by the head hook on cephalomere and sucker on mucous membrane of small intestine, fertilization process is completed by autologous or allogamy.Adult
It is made up of four sections：Cephalomere, neck section, Cheng Jie and gravid proglottid.Embryonated egg is after gravid proglottid intrauterine growth maturation, and gravid proglottid comes off disintegration, worm
Ovum is discharged with excrement, pollutes environment, and livestock swallows worm's ovum during food grass, after then worm's ovum enters alimentary canal, is digested liquid
Activation, hexacanth embryo is hatched, hexacanth embryo is through intestinal mucosa and enters blood flow, and it can be echinococcus in each portion's implantation development of body
（Hydatid）, cause echinococcosis.Common echinococcosis is hepatic echinococcosis, echinococcosis pulmonum and abdominal cavity echinococcosis.Hexacanth embryo can also invade
Violate other organs and cenuriasis, osseous hydatid disease and renal hydatid disease etc. are caused in position.Germinal cell in hydatid on cyst wall
Develop into protoscolex, domesticated dog or other Carnivoras eaten Echinococcus hydatid cyst sick sheep internal organ after, protoscolex is fixed on intestinal mucosa,
Develop for adult, start the life circulation of a new round, thus dog and other Carnivoras are the definitive hosts of Echinococcus granulosus；
People and domestic animal are in-between hosts.
The communication process of cystic echinococcosis can be carried out by following processes：After slaughtering the sheep with echinococcosis, parasitism is had
The internal organ of hydatid organize the parasitism after hello dog, or discarding to have the internal organ of hydatid or tissue to be eaten by dog, in echinococcus
Protoscolex is developed in dog enteron aisle for after adult, worm's ovum is discharged with excrement, pollutes environment, livestock by pasture, drink water or other
Approach is eaten worm's ovum and is infected.And the infection of people is except eating by the vegetables of worm's ovum pollution, in addition to fruit, due to infecting the body of dog
Table has usually been infected with worm's ovum, and when touching domesticated dog, the worm's ovum being infected with hand enters oral cavity and is infected people particularly children.Cause
This, in addition to ensureing environmental sanitation and personal hygiene, crucial effective measures are to prevent house for the prevention and control of livestock echinococcosis
Dog infect and in time find infection domesticated dog simultaneously carry out expelling parasite, and domesticated dog infection timely discovery and anthelminthic effect monitoring, examine
Core is required for sensitiveness high and the diagnostic techniques of high specificity.It is to ensure the pre- prevention and control of echinococcosis thus to provide effective diagnostic tool
The mission critical of carrying out measures processed.
The diagnostic techniques current situation of echinococcus granulosus infection is as follows,
1st, cut open inspection method：After domesticated dog is put to death, cut open the belly and take out small intestine, after collecting intestinal contents and scraping intestinal mucosa, rushed repeatedly with water
After washing, precipitating, adult is collected from sediment, by Morphological Identification, determines that domesticated dog infects, this method be only used in domesticated dog into
The investigation of insect infection situation；
2nd, floating method checks the worm's ovum in dog faecal specimen：The problem of this method avoids having to put to death domesticated dog, but due to Taeniidae
Worm's ovum form be identical, detect worm's ovum after, cannot be distinguished by under the microscope its category and kind；
3rd, arecaline katharsis：Arecaline can make echinococcus polypide benumb and be split away off from intestinal mucosa, while have and lead
The effect of rushing down, causes domesticated dog to be suffered from diarrhoea, and domesticated dog produces diarrhoea in 1 hour or so after Arecoline hydrobromide solution is gavaged, and discharges simultaneously
A large amount of adults, although this method wastes time and energy and had by the risk of dog bite, but effect is reliable, is still used as diagnosing house so far
The goldstandard of dog Echinococcus infections；
4th, immunological method-EUSA（ELISA）：In the early 1990s, European scholar uses ELISA skills
Art, establishes the method for detection dog stool antigen diagnosis echinococcus granulosus infection, and is promoted in Australia and New Zealand rapidly
Using the Chinese patent literature of Publication No. 1299972 discloses to be exempted from using Echinococcus granulosus adult polypide soluble antigen
Epidemic disease rabbit anteserum IgG antibody, establishing double-antibodies sandwich ELISA has high susceptibility and specificity, detects corresponding antigens
Lower limit reach 2.5 ng/ml, technique is in western four provinces and regions on a large scale for the investigation of domesticated dog infection level and to domesticated dog
The detection of anthelminthic effect, great function is played in echinococcosis prevention and control, although ELISA technologies have enough sensitivities
Property and specificity but need special instrument and laboratory equipment, can not be carried out at farming and pastoral area scene, and the testing time is longer,
Limit its extensive use in prevention and control and monitoring echinococcus granulosus infection；
5th, Protocols in Molecular Biology --- polymerase chain amplification technique（PCR）：Specific DNA primer is designed, expands dog faecal specimen
Middle echinococcus DNA fragment specific, for diagnosing dog Echinococcus infections, there is high susceptibility and specificity, but due to
It needs specialized equipment and laboratory condition and can not be in farming and pastoral area field application.
The content of the invention
The invention provides a kind of dog excrement echinococcosis antigen quick detection kit (colloidal gold method) and its application method, gram
The deficiency of above-mentioned prior art is taken, it can effectively solve the existing existing detection during dog Echinococcus infections are detected
Time is long, wastes time and energy, needs the problem of special test accessory.
An object of the present invention, there is provided a kind of dog echinococcosis antigen quick detection kit, dog Echinococcus hydatid cyst of the invention
Sick rapid antigen detection kit is achieved through the following technical solutions：
A kind of dog excrement echinococcosis antigen quick detection kit (colloidal gold method), including sample pad, gold standard pad, nitrocellulose
Film, adsorptive pads and bottom plate, sample pad is fixed with the left part top of bottom plate, gold is fixed between the right part and bottom plate of sample pad
Mark pad, is fixed with adsorptive pads on the right part top of bottom plate, nitrocellulose filter, nitric acid is fixed between gold standard pad and adsorptive pads
The height of cellulose membrane is below gold standard pad right part height and the height of adsorptive pads left part, is indulged in the upper surface of nitrocellulose filter
There is detection line to drawing, longitudinally being drawn in the upper surface of the nitrocellulose filter of detection line right has nature controlling line, wherein：
Gold standard pad obtains in the steps below：The first step, appropriate gold chloride is dissolved in ultra-pure water and mass percent is made be
0.01% aqueous solution of chloraurate, gold chloride boiling liquid is obtained after aqueous solution of chloraurate is boiled, then boils in liquid and adds to gold chloride
Mass percent is 1% citric acid three sodium solution and stirring, and gold chloride boiling liquid after citric acid three sodium solution is added reddens
When, continue to obtain secondary gold chloride boiling liquid after gold chloride boiling liquid is boiled 5 minutes, secondary gold chloride boiling liquid is then cooled to 37
Coolant is obtained after DEG C, collaurum processing solution is obtained after sodium azide is dissolved in into coolant, collaurum processing solution is adopted
Colloidal gold solution is obtained after carrying out aseptic filtration with 0.22 μm of filter membrane, wherein：The addition of sodium azide according to every 100ml chlorine
The auric acid aqueous solution adds 20mg sodium azide；Second step, with 0.1mol/L solution of potassium carbonate by the pH value of colloidal gold solution
Regulation to basoid gold solution is obtained after 9.0, added into basoid gold solution be dissolved in it is mixed in borate buffer solution
Close antibody immunoglobulin and stir and obtain stirring liquid, stirring liquid adds quality after the stirring of 15 minutes into stirring liquid
Percentage be 10% the bovine albumin aqueous solution and stir 30 minutes after obtain a colloidal gold labeled monoclonal antibody solution, wherein：Per milli
Rise in basoid gold solution and add 18.5 μ g to 32.5 μ g mixed antibody immunoglobulin, mixed antibody immunoglobulin bag
Include rabbit-anti Echinococcus Granulosus Cysts protoscolex excretory-secretory antigen immunoglobulin and the excretion secretion of rabbit-anti Echinococcus granulosus adult is anti-
Former immunoglobulin, rabbit-anti Echinococcus Granulosus Cysts protoscolex excretory-secretory antigen immunoglobulin and rabbit-anti Echinococcus granulosus adult
The volume ratio of excretory-secretory antigen immunoglobulin is 1:1, quality of the bovine albumin in a colloidal gold labeled monoclonal antibody solution
Percentage composition is 1%；3rd step, a colloidal gold labeled monoclonal antibody solution is obtained after being centrifuged 20 minutes under rotating speed is 2000rpm
Supernatant, supernatant is then obtained into a clear liquid and primary sedimentation after 20000 × g is centrifuged 60 minutes at 4 DEG C, discards one
Secondary clear liquid, it is to contain weight percent to be added into primary sedimentation with the isometric re-suspension liquid I of aqueous solution of chloraurate, re-suspension liquid I
Than the borate buffer solution of the bovine albumin for 1%, after primary sedimentation is re-mixed, 20000 × g centrifuges 60 points at 4 DEG C
Secondary supernatant and secondary precipitation are obtained after clock, secondary supernatant is discarded, aqueous solution of chloraurate is then added into secondary precipitation
Colloidal gold labeled monoclonal antibody solution is obtained after the re-suspension liquid II and mixing of 1/5th of volume, wherein：Re-suspension liquid II is containing weight
Measure borate buffer solution of the percentage for 5% sucrose, 0.05% Tween-20 and 0.05% sodium azide；4th step, by glass
Glass fiber obtains pre-processing gold standard pad by degreasing casein immersion treatment and after drying, then, molten with colloidal gold labeled monoclonal antibody
Liquid carries out immersion coating to pretreatment gold standard pad, and soak time is 60 minutes, 150 μ l of pretreatment gold standard pad every square centimeter
Colloidal gold labeled monoclonal antibody solution be coated with, finally by by immersion be coated with after pretreatment gold standard pad be dried in vacuo after
Gold standard pad is obtained, vacuum drying vacuum is 200Pa；
Detection line obtains in the steps below：The first step, by rabbit-anti Echinococcus Granulosus Cysts protoscolex excretory-secretory antigen immunoglobulin
Mixed antibody is obtained after mixing in equal volume with the progress of rabbit-anti Echinococcus granulosus excretory-secretory antigen immunoglobulin to be immunized
Globulin；Second step, it is immunized with the carbonate buffer solution dilution mixed antibody that molar concentration is 0.01mol/L, pH value is 9.6
Mixed antibody immunoglobulin dilution is obtained after globulin, wherein：The carbon of mixed antibody immunoglobulin 1ml per 4mg
Phthalate buffer is diluted；3rd step, rule with mixed antibody immunoglobulin dilution on nitrocellulose filter coating
Obtain detection line；
Nature controlling line obtains in the steps below：The first step, with molar concentration it is 0.01mol/L, pH by goat anti-rabbit immunoglobulin
Goat anti-rabbit immunoglobulin dilution is obtained after being worth the carbonate buffer solution dilution for 9.6, in goat anti-rabbit immunoglobulin
In dilution, the concentration of goat anti-rabbit immunoglobulin is 2mg/ml；Second step, existed with goat anti-rabbit immunoglobulin dilution
Coating of being rule on nitrocellulose filter obtains nature controlling line.
Here is the further optimization and/or improvements to one of foregoing invention technical scheme：
Above-mentioned nitrocellulose filter can be the nitrocellulose filter after carbodiimide hydrochloride solution activation process；Or/
With sample pad is with bovine albumin, 1% sucrose, 0.1% Tween-20 and 0.02% nitrine for being 1% containing percentage by weight
Change sodium the pretreated sample pad of phosphate buffer, phosphate buffer be molar concentration be 0.01mol/L, pH value be
7.4 phosphate buffer；Or/and gold standard pad is the phosphate-buffered with the degreasing casein for being 1% containing percentage by weight
Liquid carries out pretreated gold standard pad, and phosphate buffer is that the phosphate that molar concentration is 0.05mol/L, pH value is 7.2 delays
Fliud flushing.
Above-mentioned will draw has the nitrocellulose filter of detection line and nature controlling line to soak 60 points of closing at 37 DEG C with confining liquid
Clock is simultaneously dried in vacuo, wherein：Confining liquid is the phosphate buffer that molar concentration is 0.02mol/L, pH value is 7.2, confining liquid
Contain bovine albumin, 1% PEG 20000 and 0.5% Tween-20 that percentage by weight is 2%.
Preferably, above-mentioned borate buffer solution can be that molar concentration is the boric acid salt buffer that 0.005mol/L, pH are 9.0
Liquid.
Preferably, above-mentioned glass fibre can be the glass fibres of Whatman glass 33；Or/and nitrocellulose filter is
The nitrocellulose filters of Millipore 135.
The purpose of another aspect of the present invention, it is to disclose a kind of dog excrement echinococcosis antigen quick detection kit
The application method of (colloidal gold method), it is achieved through the following technical solutions：
A kind of application method of dog excrement echinococcosis antigen quick detection kit (colloidal gold method), is carried out in the steps below：By 20 μ
L dog excrement suspension supernatant is added dropwise in sample pad, and detection line is not reached when dog excrement suspension supernatant spreads in sample pad
When, then to 100 μ l are added dropwise in sample pad to 150 μ l borate buffer solution, observe 15 minutes to 30 minutes, wherein：Borate
Buffer solution is the borate buffer solution that molar concentration is 0.005 mol/L, pH value is 9.0.
Here is two further optimization and/or improvements to foregoing invention technical scheme：
Above-mentioned dog excrement suspension supernatant can obtain as follows：The first step, dog excrement is added into the dog excrement after aseptic filtration
Preserve in liquid and obtain dog excrement suspension, wherein：Dog excrement preserve liquid for be 0.02% containing percentage by weight sodium azide, 0.3%
The phosphate buffer of Tween-20 and 1% bovine albumin, phosphate buffer be molar concentration be 0.15 mol/L, pH value be
7.2 phosphate buffer, the dog excrement per 2ml preserve the dog excrement that liquid adds 1g；Second step, dog excrement suspension by 15 minutes from
Dog excrement suspension supernatant is obtained after the heart.
The dog excrement echinococcosis antigen quick detection kit (colloidal gold method) of the present invention has higher sensitiveness, specificity
And stability, it can carry with, the test at the scene of carrying out i.e. immediately, it is not necessary to special test accessory, shorten
Testing time, testing efficiency is improved, the use in the farming and pastoral area that is particularly suitable for use in, in addition, the dog excrement echinococcosis antigen of the present invention is fast
Fast detection kit (colloidal gold method) is tested dog excrement, it is therefore prevented that the injury to dog so that detection work is more easy.
Brief description of the drawings
Accompanying drawing 1 is the overlooking the structure diagram of dog excrement echinococcosis antigen quick detection kit in the present invention.
Accompanying drawing 2 be accompanying drawing 1 in A-A to cross section structure diagram.
In figure, 1- sample pads, 2- gold standard pads, 3- nitrocellulose filters, 4- adsorptive pads, 5- bottom plates, 6- detection lines, 7- Quality Controls
Line.
Embodiment
The present invention is not limited by following embodiments, can technique according to the invention scheme and actual conditions it is specific to determine
Embodiment.
In invention, for the ease of description, the description of the relative position relation of each part is according to saying in embodiment
The Butut mode of bright book accompanying drawing 1 is described, such as：The position relationship of upper and lower, left and right etc. is according to Figure of description
Butut direction determines.
With reference to embodiment and accompanying drawing, the invention will be further described：
Embodiment 1
As shown in Fig. 1 to 2, the dog excrement echinococcosis antigen quick detection kit (colloidal gold method), including sample pad 1, gold standard pad
2nd, nitrocellulose filter 3, adsorptive pads 4 and bottom plate 5, sample pad 1 is fixed with the left part top of bottom plate 5, in the right part of sample pad 1
Gold standard pad 2 is fixed between bottom plate 5, adsorptive pads 4 are fixed with the right part top of bottom plate 5, gold standard pad 2 and adsorptive pads 4 it
Between be fixed with nitrocellulose filter 3, the height of nitrocellulose filter 3 is below the right part of gold standard pad 2 height and the left part of adsorptive pads 4
Highly, nitrocellulose filter 3 upper surface longitudinally draw have detection line 6, the right of detection line 6 nitrocellulose filter 3 it is upper
End face, which is longitudinally drawn, nature controlling line 7, wherein：
Gold standard pad 2 obtains in the steps below：
The first step, appropriate gold chloride is dissolved in ultra-pure water and the aqueous solution of chloraurate that mass percent is 0.01% is made, by chlorine
The auric acid aqueous solution obtains gold chloride boiling liquid after boiling, then boiled to gold chloride and the citric acid three that mass percent is 1% is added in liquid
Sodium solution simultaneously stirs, and when the gold chloride boiling liquid after adding citric acid three sodium solution reddens, continues gold chloride boiling liquid boiling 5 points
Secondary gold chloride boiling liquid is obtained after clock, coolant is obtained after secondary gold chloride boiling liquid then is cooled into 37 DEG C, into coolant
Collaurum processing solution is obtained after dissolving in sodium azide, after collaurum processing solution is carried out into aseptic filtration using 0.22 μm of filter membrane
Colloidal gold solution is obtained, wherein：The addition of sodium azide adds 20mg Azide according to every 100ml aqueous solution of chloraurate
Sodium；
Second step, the pH value of colloidal gold solution is adjusted to obtaining basoid gold after 9.0 with 0.1mol/L solution of potassium carbonate
Solution, the mixed antibody immunoglobulin being dissolved in borate buffer solution is added into basoid gold solution and stirring obtains
Liquid is stirred, stirring liquid adds the bovine albumin aqueous solution that mass percent is 10% into stirring liquid after the stirring of 15 minutes
And a colloidal gold labeled monoclonal antibody solution is obtained after stirring 30 minutes, wherein：18.5 μ are added in every milliliter of basoid gold solution
G to 32.5 μ g mixed antibody immunoglobulin, mixed antibody immunoglobulin are drained including rabbit-anti Echinococcus Granulosus Cysts protoscolex
Secretion antigen immunoglobulin and rabbit-anti Echinococcus granulosus excretory-secretory antigen immunoglobulin, rabbit-anti Echinococcus Granulosus Cysts
The body of protoscolex excretory-secretory antigen immunoglobulin and rabbit-anti Echinococcus granulosus excretory-secretory antigen immunoglobulin
Product is than being 1:1, weight/mass percentage composition of the bovine albumin in a colloidal gold labeled monoclonal antibody solution is 1%；
3rd step, a colloidal gold labeled monoclonal antibody solution is obtained into supernatant after being centrifuged 20 minutes under rotating speed is 2000rpm, so
Supernatant is obtained into a clear liquid and primary sedimentation after 20000 × g is centrifuged 60 minutes at 4 DEG C afterwards, discards a clear liquid, to
Added in primary sedimentation with the isometric re-suspension liquid I of aqueous solution of chloraurate, the ox that it is 1% containing percentage by weight that re-suspension liquid I, which is,
The borate buffer solution of albumin, after primary sedimentation is re-mixed, 20000 × g obtains two after centrifuging 60 minutes at 4 DEG C
Secondary supernatant and secondary precipitation, secondary supernatant is discarded, five points of aqueous solution of chloraurate volume are then added into secondary precipitation
One of re-suspension liquid II and mix after obtain colloidal gold labeled monoclonal antibody solution, wherein：Re-suspension liquid II is to be containing percentage by weight
The borate buffer solution of 5% sucrose, 0.05% Tween-20 and 0.05% sodium azide；
4th step, obtain pre-processing gold standard pad by glass fibre by degreasing casein immersion treatment and after drying, then, use glue
Body gold labelled antibody solution carries out immersion coating to pretreatment gold standard pad, and soak time is 60 minutes, pre- place every square centimeter
Reason gold standard pad is coated with 150 μ l colloidal gold labeled monoclonal antibody solution, finally by the pretreatment gold mark after immersion is coated with
Pad obtains gold standard pad 2 after being dried in vacuo, and vacuum drying vacuum is 200Pa；
Detection line 6 obtains in the steps below：
The first step, by rabbit-anti Echinococcus Granulosus Cysts protoscolex excretory-secretory antigen immunoglobulin and rabbit-anti Echinococcus granulosus adult
Excretory-secretory antigen immunoglobulin carries out obtaining mixed antibody immunoglobulin after mixing in equal volume；
Second step, ball is immunized with the carbonate buffer solution dilution mixed antibody that molar concentration is 0.01mol/L, pH value is 9.6
Mixed antibody immunoglobulin dilution is obtained after albumen, wherein：The carbonic acid of mixed antibody immunoglobulin 1ml per 4mg
Salt buffer is diluted；
3rd step, coating of being rule with mixed antibody immunoglobulin dilution on nitrocellulose filter 3 obtain detection line 6；
Nature controlling line 7 obtains in the steps below：
The first step, by goat anti-rabbit immunoglobulin with the carbonate buffer that molar concentration is 0.01 mol/L, pH value is 9.6
Goat anti-rabbit immunoglobulin dilution is obtained after liquid dilution, in goat anti-rabbit immunoglobulin dilution, goat antirabbit is exempted from
The concentration of epidemic disease globulin is 2mg/ml；
Second step, coating of being rule with goat anti-rabbit immunoglobulin dilution on nitrocellulose filter 3 obtain nature controlling line 7.
Rabbit-anti Echinococcus Granulosus Cysts protoscolex excretory-secretory antigen immunoglobulin and the excretion of rabbit-anti Echinococcus granulosus adult
Secretion antigen immunoglobulin obtains in the steps below：
The first step, Echinococcus Granulosus Cysts protoscolex excretory-secretory antigen (EgPES Ag) is prepared, from Echinococcus hydatid cyst intracapsular extraction cyst fluid, so
Afterwards, cut off the cyst wall of hydatid, rinse cyst wall with cyst fluid and the brood capsule and protoscolex that will be attached on cyst wall take out, by brood capsule and
In protoscolex injection sterile collection bottle and stand 1 hour at 4 DEG C and obtain primary sedimentation thing（Contain protoscolex）With a supernatant
Liquid, a supernatant is discarded, centrifugation obtains secondary precipitate in 15 minutes in the case where rotating speed is 2000 rpm by primary sedimentation thing
（Contain protoscolex）With secondary supernatant, secondary supernatant is discarded, is added by 5 times of secondary precipitate volume into secondary precipitate
Enter dual anti-physiological saline（0.9% sodium chloride solution containing 200 units of gentamicin/ml, 250 units of nystatin/ml）Washing two
Secondary sediment, protoscolex is obtained after washing 3 times repeatedly, then, with containing the dual anti-fluid nutrient mediums of RPMI 1640（Do not heal
Clearly）Suspension is made（Nutrient solution, containing the domesticated dog bile that percentage by weight is 1% to 5% in nutrient solution, domesticated dog bile is by removing
Bacterium is filtered）, every 20 ml to 30 ml nutrient solution adds 2000 protoscolexs, by the culture medium equipped with protoscolex 37 DEG C,
CO2Percent by volume be 5% incubator for tissue culture in cultivate, during culture, culture is respectively changed in 4 hours and 12 hours
Liquid once, changes nutrient solution once daily later, until stopping culture when nutrient solution is changed into yellow from red, by what is changed every time
Nutrient solution is combined to obtain the nutrient solution of the excretory-secretory antigen of protoscolex containing Echinococcus Granulosus Cysts, to protoscolex containing Echinococcus Granulosus Cysts
After the sodium azide and phenylmethylsulfonyl fluoride that percentage by weight is 0.1% are added in the nutrient solution of excretory-secretory antigen and is concentrated
To Echinococcus Granulosus Cysts protoscolex excretory-secretory antigen, wherein：Nutrient solution per 100ml adds 1mmol phenylmethylsulfonyl fluoride；
Second step, prepare Echinococcus granulosus excretory-secretory antigen（EgAES Ag）, Echinococcus granulosus adult is used double
After anti-physiological saline carrying out washing treatment 3 times, then by the Echinococcus granulosus adult nutrient solutions of RPMI 1640（Not increase serum）In
37 DEG C, CO2 percent by volume cultivated under conditions of being 5%, the addition of tapeworm adult is according to every 20ml to 30ml's
200 tapeworm adults are added in the nutrient solutions of RPMI 1640, during culture, nutrient solution is respectively changed in 4 hours and 12 hours
Once, nutrient solution being changed daily once later, stopping culture when nutrient solution is by red change Huang, the nutrient solution changed every time is concentrated
Get up to obtain the nutrient solution of the excretory-secretory antigen containing Echinococcus granulosus, it is anti-to the excretion secretion of adult containing Echinococcus granulosus
The sodium azide and phenylmethylsulfonyl fluoride that percentage by weight is 0.1% are added in former nutrient solution and obtains particulate spine ball after concentrating
Tapeworm excretory-secretory antigen, wherein：Nutrient solution per 100ml adds 1mmol phenylmethylsulfonyl fluoride；
3rd step, the preparation of rabbit-anti Echinococcus Granulosus Cysts protoscolex excretory-secretory antigen immunoglobulin and rabbit-anti Echinococcus granulosus
The preparation of excretory-secretory antigen immunoglobulin, determine the excretion point of Echinococcus Granulosus Cysts protoscolex respectively with Coomassie Brilliant Blue
After the protein content for secreting antigen and Echinococcus granulosus excretory-secretory antigen, respectively with the excretion point of Echinococcus Granulosus Cysts protoscolex
Secrete antigen and rabbit is immunized Echinococcus granulosus excretory-secretory antigen, drained and secreted with Echinococcus Granulosus Cysts protoscolex
One group of antigen immune rabbit is used as Echinococcus Granulosus Cysts protoscolex excretory-secretory antigen immune group, is arranged with Echinococcus granulosus adult
Let out secretion antigen immunizing rabbit one group is used as Echinococcus granulosus excretory-secretory antigen immune group, every group of immunizing rabbit 3
Only, rabbit plants male rabbit for the New Zealand of 2 kilograms of body weight（Birth 6 months to 1 year）.Every group is immunized by following procedure：
It is immune for the first time：Each group antigen 3mg is respectively taken, isometric Freund's complete adjuvant is added into every kind of antigen, is then distinguished
Antigen after being dissolved with Freund's complete adjuvant is respectively to respective corresponding immune group in 4 volas, muscle of back, the hind leg muscle of rabbit
Totally 10 point injections；
Second immune：After immune three weeks for the first time, 5ml freund 's incomplete adjuvant is added with 3mg antigens, is drenched in bilateral axillary regions
Fawning on surrounding subcutaneous and bilateral inguinal lymph node surrounding subcutaneous, totally 6 points carry out injecting immune；
Third time is immune：After second immune two weeks, add physiological saline with 1mg antigens, in bilateral oxter, groin and back skin
Under totally 6 points be subcutaneously injected it is immune；
Start within 7th day to try blood after third time is immune, in family's rabbit ear vein blood sampling separation serum, antibody is determined with agar gel diffusion test
Level, reach 1 in antibody titer；When more than 128, by immunizing rabbit bloodletting, serum is separated, is saltoutd legal system with saturated ammonium sulfate
Take the immunoglobulin in serum, every group by the immunoglobulin produced from serum sequentially celluloses of process DE- 52
（DEAE（DE-52）DEAE）After column chromatography purifies the content that immunoglobulin is determined with ultraviolet spectrometry,
Obtain every group of corresponding rabbit-anti Echinococcus Granulosus Cysts protoscolex excretory-secretory antigen immunoglobulin and rabbit-anti Echinococcus granulosus into
Worm excretory-secretory antigen immunoglobulin.
Embodiment 2：
As shown in accompanying drawing 1 to 2, the dog excrement echinococcosis antigen quick detection kit (colloidal gold method), including sample pad 1, Jin Biao
Pad 2, nitrocellulose filter 3, adsorptive pads 4 and bottom plate 5, sample pad 1 is fixed with the left part top of bottom plate 5, on the right side of sample pad 1
Gold standard pad 2 is fixed between portion and bottom plate 5, adsorptive pads 4 are fixed with the right part top of bottom plate 5, in gold standard pad 2 and adsorptive pads 4
Between be fixed with nitrocellulose filter 3, the height of nitrocellulose filter 3 is below the right part of gold standard pad 2 height and the left part of adsorptive pads 4
Height, longitudinally being drawn in the upper surface of nitrocellulose filter 3 has detection line 6, the nitrocellulose filter 3 in detection line 6 right
Upper surface, which is longitudinally drawn, nature controlling line 7, wherein：
Gold standard pad 2 obtains in the steps below：
The first step, appropriate gold chloride is dissolved in ultra-pure water and the aqueous solution of chloraurate that mass percent is 0.01% is made, by chlorine
The auric acid aqueous solution obtains gold chloride boiling liquid after boiling, then boiled to gold chloride and the citric acid three that mass percent is 1% is added in liquid
Sodium solution simultaneously stirs, and when the gold chloride boiling liquid after adding citric acid three sodium solution reddens, continues gold chloride boiling liquid boiling 5 points
Secondary gold chloride boiling liquid is obtained after clock, coolant is obtained after secondary gold chloride boiling liquid then is cooled into 37 DEG C, into coolant
Collaurum processing solution is obtained after dissolving in sodium azide, after collaurum processing solution is carried out into aseptic filtration using 0.22 μm of filter membrane
Colloidal gold solution is obtained, wherein：The addition of sodium azide adds 20mg Azide according to every 100ml aqueous solution of chloraurate
Sodium；
Second step, the pH value of colloidal gold solution is adjusted to obtaining basoid gold after 9.0 with 0.1mol/L solution of potassium carbonate
Solution, the mixed antibody immunoglobulin being dissolved in borate buffer solution is added into basoid gold solution and stirring obtains
Liquid is stirred, stirring liquid adds the bovine albumin aqueous solution that mass percent is 10% into stirring liquid after the stirring of 15 minutes
And a colloidal gold labeled monoclonal antibody solution is obtained after stirring 30 minutes, wherein：18.5 μ are added in every milliliter of basoid gold solution
G or 32.5 μ g mixed antibody immunoglobulin, mixed antibody immunoglobulin are drained including rabbit-anti Echinococcus Granulosus Cysts protoscolex
Secretion antigen immunoglobulin and rabbit-anti Echinococcus granulosus excretory-secretory antigen immunoglobulin, rabbit-anti Echinococcus Granulosus Cysts
The body of protoscolex excretory-secretory antigen immunoglobulin and rabbit-anti Echinococcus granulosus excretory-secretory antigen immunoglobulin
Product is than being 1:1, weight/mass percentage composition of the bovine albumin in a colloidal gold labeled monoclonal antibody solution is 1%；
3rd step, a colloidal gold labeled monoclonal antibody solution is obtained into supernatant after being centrifuged 20 minutes under rotating speed is 2000rpm, so
Supernatant is obtained into a clear liquid and primary sedimentation after 20000 × g is centrifuged 60 minutes at 4 DEG C afterwards, discards a clear liquid, to
Added in primary sedimentation with the isometric re-suspension liquid I of aqueous solution of chloraurate, the ox that it is 1% containing percentage by weight that re-suspension liquid I, which is,
The borate buffer solution of albumin, after primary sedimentation is re-mixed, 20000 × g obtains two after centrifuging 60 minutes at 4 DEG C
Secondary supernatant and secondary precipitation, secondary supernatant is discarded, five points of aqueous solution of chloraurate volume are then added into secondary precipitation
One of re-suspension liquid II and mix after obtain colloidal gold labeled monoclonal antibody solution, wherein：Re-suspension liquid II is to be containing percentage by weight
The borate buffer solution of 5% sucrose, 0.05% Tween-20 and 0.05% sodium azide；4th step, glass fibre is passed through
Degreasing casein immersion treatment simultaneously obtains after drying pre-processing gold standard pad, then, with colloidal gold labeled monoclonal antibody solution to pretreatment
Gold standard pad carries out immersion coating, and soak time is 60 minutes, 150 μ l of pretreatment gold standard pad every square centimeter collaurum mark
Note antibody-solutions are coated with, and gold standard pad is obtained after finally the pretreatment gold standard pad after immersion is coated with is dried in vacuo
2, vacuum drying vacuum is 200Pa；
Detection line 6 obtains in the steps below：
The first step, by rabbit-anti Echinococcus Granulosus Cysts protoscolex excretory-secretory antigen immunoglobulin and rabbit-anti Echinococcus granulosus adult
Excretory-secretory antigen immunoglobulin carries out obtaining mixed antibody immunoglobulin after mixing in equal volume；
Second step, ball is immunized with the carbonate buffer solution dilution mixed antibody that molar concentration is 0.01mol/L, pH value is 9.6
Mixed antibody immunoglobulin dilution is obtained after albumen, wherein：The carbonic acid of mixed antibody immunoglobulin 1ml per 4mg
Salt buffer is diluted；
3rd step, coating of being rule with mixed antibody immunoglobulin dilution on nitrocellulose filter 3 obtain detection line 6；
Nature controlling line 7 obtains in the steps below：
The first step, by goat anti-rabbit immunoglobulin with the carbonate buffer that molar concentration is 0.01mol/L, pH value is 9.6
Goat anti-rabbit immunoglobulin dilution is obtained after liquid dilution, in goat anti-rabbit immunoglobulin dilution, goat antirabbit is exempted from
The concentration of epidemic disease globulin is 2mg/ml；
Second step, coating of being rule with goat anti-rabbit immunoglobulin dilution on nitrocellulose filter 3 obtain nature controlling line 7.
Embodiment 3：
Be with the difference of above-described embodiment, nitrocellulose filter 3 be by carbodiimide hydrochloride solution activation at
Nitrocellulose filter after reason；Or/and sample pad 1 be be 1% containing percentage by weight bovine albumin, 1% sucrose,
The pretreated sample pad 1 of the phosphate buffer of 0.1% Tween-20 and 0.02% sodium azide, phosphate buffer are
The phosphate buffer that molar concentration is 0.01 mol/L, pH value is 7.4；Or/and gold standard pad 2 is with containing percentage by weight
Phosphate buffer for 1% degreasing casein carries out pretreated gold standard pad, and phosphate buffer is that molar concentration is
0.05 mol/L, the phosphate buffer that pH value is 7.2.
Embodiment 4：
It is with the difference of above-described embodiment, stroke nitrocellulose filter 3 for having detection line 6 and nature controlling line 7 is used into confining liquid
Immersion is closed 60 minutes and is dried in vacuo at 37 DEG C, wherein：Confining liquid be molar concentration be 0.02 mol/L, pH value 7.2
Phosphate buffer, confining liquid contains the bovine albumin that percentage by weight is 2%, 1% PEG 20000 and 0.5%
Tween-20.
Embodiment 5：
It is with the difference of above-described embodiment, borate buffer solution is that molar concentration is 0.005 mol/L, pH 9.0
Borate buffer solution.
Embodiment 6：
It is with the difference of above-described embodiment, glass fibre is the glass fibres of Whatman glass 33；Or/and nitric acid
Cellulose membrane 3 is the nitrocellulose filters of Millipore 135.Sample pad 1 is that the outstanding bio tech ltd in Shanghai provides
GL-1 type sample pads, adsorptive pads 4 are the Whatman 470 class adsorptive pads that the outstanding bio tech ltd in Shanghai provides.
Embodiment 7：
The application method of the dog excrement echinococcosis antigen quick detection kit (colloidal gold method), is carried out in the steps below：By 20 μ l
Dog excrement suspension supernatant be added dropwise in sample pad 1, do not reach detection line when dog excrement suspension supernatant spreads in sample pad 1
When 6, then to 100 μ l are added dropwise in sample pad 1 to 150 μ l borate buffer solution, observe 15 minutes to 30 minutes, wherein：Boric acid
Salt buffer is the borate buffer solution that molar concentration is 0.005 mol/L, pH value is 9.0.Dog excrement suspension supernatant is added dropwise
When in sample pad 1, dog excrement suspension supernatant（Sample）Under the sucking action of adsorptive pads 4, sample flows to the right rapidly, passes through
During gold standard pad 2, if antigen in sample be present（Echinococcus Granulosus Cysts protoscolex excretory-secretory antigen or Echinococcus granulosus adult row
Let out secretion antigen）, when antigen forms Antibody-antigen complex with corresponding antibody binding and advances to detection line 6, quilt
The seizure antibody being fixed in detection line 6 is intercepted and captured, and forms gold labeling antibody（Rabbit-anti Echinococcus Granulosus Cysts protoscolex excretory-secretory antigen is exempted from
Epidemic disease globulin and rabbit-anti Echinococcus granulosus excretory-secretory antigen immunoglobulin）- Antigen-capture antibody complex, sample
Product make detection line 6 that red be presented under chromatography effect due to the enrichment of gold labeling antibody, and gold labeling antibody advances to nature controlling line 7
When the goat anti-rabbit immunoglobulin antibody capture that is fixed on this line and nature controlling line 7 is shown red；The meaning of nature controlling line 7
Justice is to show that detection experiment is set up, so detection line 6 and nature controlling line 7 are all presented red expression experiment and set up, it is as a result the positive；And
When not having corresponding antigen in sample, detection line 6 does not develop the color, and the colour developing of nature controlling line 7 represents that experiment is set up, and is as a result feminine gender；Such as
Fruit nature controlling line 7 does not develop the color, then shows that strip fails.
Embodiment 8：
The application method of the dog excrement echinococcosis antigen quick detection kit (colloidal gold method), is carried out in the steps below：By 20 μ l
Dog excrement suspension supernatant be added dropwise in sample pad 1, do not reach detection line when dog excrement suspension supernatant spreads in sample pad 1
When 6, then to the borate buffer solution that 100 μ l or 150 μ l are added dropwise in sample pad 1, observe 15 minutes to 30 minutes, wherein：Boric acid
Salt buffer is the borate buffer solution that molar concentration is 0.005 mol/L, pH value is 9.0.
Embodiment 9：
It is with the difference of above-described embodiment, dog excrement suspension supernatant obtains as follows：
The first step, dog excrement is added in the preservation liquid of the dog excrement after aseptic filtration and obtains dog excrement suspension, wherein：Dog excrement preserves liquid
For be 0.02% containing percentage by weight sodium azide, 0.3% Tween-20 and 1% bovine albumin phosphate buffer,
Phosphate buffer is the phosphate buffer that molar concentration is 0.15 mol/L, pH value is 7.2, and the dog excrement per 2ml preserves liquid
Add 1g dog excrement；
Second step, dog excrement suspension obtain dog excrement suspension supernatant after the centrifugation of 15 minutes.
To the dog excrement echinococcosis antigen quick detection kit (colloidal gold method) that is obtained according to the above embodiment of the present invention
Sensitivity and Specificity is tested respectively.
By sensitivity testses, dog excrement echinococcosis antigen quick detection kit (colloidal gold method) detection in the present invention is thin
The least concentration most 25ng/ of grain echinococcus protoscolex excretory-secretory antigen or Echinococcus granulosus excretory-secretory antigen
Ml, meanwhile, to the experimental dog of artificial challenge's Echinococcus granulosus, the positive 80 parts of dog excrement samples of infection are turned out to be through arecaline expelling parasite
In, positive 74 parts are detected with the dog excrement echinococcosis antigen quick detection kit (colloidal gold method) in the present invention, positive rate reaches
To 92.5%, illustrate that the dog excrement echinococcosis antigen quick detection kit (colloidal gold method) in the present invention has higher sensitiveness.
All kinds of tapeworms in domesticated dog enteron aisle be present：Blister band tapeworm, more headband tapeworms, japanese double cord tapeworm, dog bend first ascarid
Worm, Echinococcus granulosus and Echinococcus multilocularis, in specific test, in 51 dogs of the natural infection blister with tapeworm, adopt
Positive 1, false positive rate are detected with the dog excrement echinococcosis antigen quick detection kit (colloidal gold method) in the present invention
1.96%；And in the more headband tapeworms of natural infection（3）, japanese double cord tapeworm（8）And Toxocara canis（13）Domesticated dog excrement
Just sample is in totally 24 parts, using dog excrement echinococcosis antigen quick detection kit (colloidal gold method) testing result in the present invention
All it is negative, thus the dog excrement echinococcosis antigen quick detection kit (colloidal gold method) in the explanation present invention has higher
Specificity.
In addition, to the testing result using ELISA method detection Echinococcus granulosus with using the dog excrement Echinococcus hydatid cyst in the present invention
The testing result of Echinococcus granulosus is contrasted in sick rapid antigen detection kit (colloidal gold method) detection dog excrement, detection pair
As for survival in Condition in North Xinjiang（Urumqi County southern suburbs farming and pastoral area, Fuhai County, Emin County, Yumin County and Hutubi County）405
Domesticated dog, arecaline catharsis 294 domesticated dogs of success, wherein 45 domesticated dogs detection Echinococcus granulosus, testing result shows, use
The testing result of dog excrement echinococcosis antigen quick detection kit (colloidal gold method) in the present invention and the detection knot with ELISA method
Fruit is the positive, and the two coincidence rate is 100%, illustrates the dog excrement echinococcosis antigen quick detection kit (collaurum in the present invention
Method) there is higher Detection accuracy and coefficient of stabilization.
In summary, the dog excrement echinococcosis antigen quick detection kit (colloidal gold method) in the present invention has higher quick
Perception, specificity and stability, can be carried with, the test at the scene of carrying out i.e. immediately, it is not necessary to special test auxiliary
Equipment, the testing time is shortened, improve testing efficiency, the use in the farming and pastoral area that is particularly suitable for use in, in addition, the dog excrement bag of the present invention
Parasitosis rapid antigen detection kit (colloidal gold method) is tested dog excrement, it is therefore prevented that the injury to dog so that detection work
It is more easy.
Above technical characteristic constitutes embodiments of the invention, and it has stronger adaptability and implementation result, can basis
The non-essential technical characteristic of increase and decrease is actually needed, to meet the needs of different situations.
Obviously, those skilled in the art can carry out the essence of various changes and modification without departing from the present invention to the present invention
God and scope.So, if these modifications and variations of the present invention belong to the scope of the claims in the present invention and its equivalent technologies
Within, then the present invention is also intended to comprising including these changes and modification.
Claims (7)
- A kind of 1. dog excrement echinococcosis antigen quick detection kit, it is characterised in that：It includes sample pad（1）, gold standard pad（2）、 Nitrocellulose filter（3）, adsorptive pads（4）And bottom plate（5）, in the bottom plate（5）Left part top be fixed with the sample pad （1）, in the sample pad（1）Right part and the bottom plate（5）Between be fixed with the gold standard pad（2）, in the bottom plate（5）'s Right part top is fixed with the adsorptive pads（4）, in the gold standard pad（2）With the adsorptive pads（4）Between be fixed with the nitric acid Cellulose membrane（3）, the nitrocellulose filter（3）Height be below the gold standard pad（2）Right part height and the adsorptive pads （4）The height of left part, in the nitrocellulose filter（3）Upper surface longitudinally draw have detection line（6）, in the detection line（6） The nitrocellulose filter of right（3）Upper surface longitudinally draw have nature controlling line（7）, wherein：The gold standard pad（2）Obtain in the steps below：The first step, appropriate gold chloride is dissolved in ultra-pure water and the aqueous solution of chloraurate that mass percent is 0.01% is made, by chlorine The auric acid aqueous solution obtains gold chloride boiling liquid after boiling, then boiled to gold chloride and the citric acid three that mass percent is 1% is added in liquid Sodium solution simultaneously stirs, and when the gold chloride boiling liquid after adding citric acid three sodium solution reddens, continues gold chloride boiling liquid boiling 5 points Secondary gold chloride boiling liquid is obtained after clock, coolant is obtained after secondary gold chloride boiling liquid then is cooled into 37 DEG C, into coolant Collaurum processing solution is obtained after dissolving in sodium azide, after collaurum processing solution is carried out into aseptic filtration using 0.22 μm of filter membrane Colloidal gold solution is obtained, wherein：The addition of sodium azide adds 20mg Azide according to every 100ml aqueous solution of chloraurate Sodium；Second step, the pH value of colloidal gold solution is adjusted to obtaining basoid gold after 9.0 with 0.1mol/L solution of potassium carbonate Solution, the mixed antibody immunoglobulin being dissolved in borate buffer solution is added into basoid gold solution and stirring obtains Liquid is stirred, stirring liquid adds the bovine albumin aqueous solution that mass percent is 10% into stirring liquid after the stirring of 15 minutes And a colloidal gold labeled monoclonal antibody solution is obtained after stirring 30 minutes, wherein：18.5 μ are added in every milliliter of basoid gold solution G to 32.5 μ g mixed antibody immunoglobulin, mixed antibody immunoglobulin are drained including rabbit-anti Echinococcus Granulosus Cysts protoscolex Secretion antigen immunoglobulin and rabbit-anti Echinococcus granulosus excretory-secretory antigen immunoglobulin, rabbit-anti Echinococcus Granulosus Cysts The body of protoscolex excretory-secretory antigen immunoglobulin and rabbit-anti Echinococcus granulosus excretory-secretory antigen immunoglobulin Product is than being 1:1, weight/mass percentage composition of the bovine albumin in a colloidal gold labeled monoclonal antibody solution is 1%；3rd step, a colloidal gold labeled monoclonal antibody solution is obtained into supernatant after being centrifuged 20 minutes under rotating speed is 2000rpm, so Supernatant is obtained into a clear liquid and primary sedimentation after 20000 × g is centrifuged 60 minutes at 4 DEG C afterwards, discards a clear liquid, to Added in primary sedimentation with the isometric re-suspension liquid I of aqueous solution of chloraurate, the ox that it is 1% containing percentage by weight that re-suspension liquid I, which is, The borate buffer solution of albumin, after primary sedimentation is re-mixed, 20000 × g obtains two after centrifuging 60 minutes at 4 DEG C Secondary supernatant and secondary precipitation, secondary supernatant is discarded, five points of aqueous solution of chloraurate volume are then added into secondary precipitation One of re-suspension liquid II and mix after obtain colloidal gold labeled monoclonal antibody solution, wherein：Re-suspension liquid II is to be containing percentage by weight The borate buffer solution of 5% sucrose, 0.05% Tween-20 and 0.05% sodium azide；4th step, glass fibre is passed through Degreasing casein immersion treatment simultaneously obtains after drying pre-processing gold standard pad, then, with colloidal gold labeled monoclonal antibody solution to pretreatment Gold standard pad carries out immersion coating, and soak time is 60 minutes, 150 μ l of pretreatment gold standard pad every square centimeter collaurum mark Note antibody-solutions are coated with, and golden mark is obtained after finally the pretreatment gold standard pad after immersion is coated with is dried in vacuo Pad, vacuum drying vacuum is 200Pa；The detection line（6）Obtain in the steps below：The first step, rabbit-anti Echinococcus Granulosus Cysts protoscolex excretory-secretory antigen is immunized Globulin and rabbit-anti Echinococcus granulosus excretory-secretory antigen immunoglobulin carry out obtaining mixing after mixing in equal volume resisting Body immunoglobulin；Second step, it is anti-with the carbonate buffer solution dilution mixing that molar concentration is 0.01mol/L, pH value is 9.6 Mixed antibody immunoglobulin dilution is obtained after body immunoglobulin, wherein：Mixed antibody immunoglobulin per 4mg is used 1ml carbonate buffer solution is diluted；3rd step, detection line is obtained with mixed antibody immunoglobulin dilution coating of being rule on nitrocellulose filter；The nature controlling line（7）Obtain in the steps below：The first step, by goat anti-rabbit immunoglobulin with the carbonate buffer solution that molar concentration is 0.01mol/L, pH value is 9.6 Goat anti-rabbit immunoglobulin dilution is obtained after dilution, in goat anti-rabbit immunoglobulin dilution, goat antirabbit is immunized The concentration of globulin is 2mg/ml；Second step, coating of being rule with goat anti-rabbit immunoglobulin dilution on nitrocellulose filter obtain nature controlling line.
- 2. dog excrement echinococcosis antigen quick detection kit according to claim 1, it is characterised in that the cellulose nitrate Plain film（3）It is by carbodiimide hydrochloride solution activation process；Or/and the sample pad（1）With containing weight percent Phosphate buffer than the bovine albumin for 1%, 1% sucrose, 0.1% Tween-20 and 0.02% sodium azide pre-processes, The molar concentration of phosphate buffer is 0.01mol/L, pH value 7.4；Or/and the gold standard pad（2）With containing weight percent Phosphate buffer than the degreasing casein for 1% is pre-processed, and the molar concentration of phosphate buffer is 0.05 mol/ L, pH value is 7.2.
- 3. dog excrement echinococcosis antigen quick detection kit according to claim 1 or 2, it is characterised in that have inspection by drawing Survey line（6）And nature controlling line（7）Nitrocellulose filter（3）Soaked with confining liquid at 37 DEG C and close 60 minutes and be dried in vacuo, Wherein：The confining liquid is the phosphate buffer that molar concentration is 0.02mol/L, pH value is 7.2, and the confining liquid contains weight Measure bovine albumin, 1% PEG 20000 and 0.5% Tween-20 that percentage is 2%.
- 4. dog excrement echinococcosis antigen quick detection kit according to claim 1 or 2, it is characterised in that the boric acid The molar concentration of salt buffer is 0.005mol/L, PH 9.0.
- 5. dog excrement echinococcosis antigen quick detection kit according to claim 1, it is characterised in that the glass fibre It is Whatman glass 33；Or/and the nitrocellulose filter（3）For Millipore 135.
- A kind of 6. application method of dog excrement echinococcosis antigen quick detection kit, it is characterised in that：It is carried out in the steps below： 20 μ l dog excrement suspension supernatant is added dropwise in sample pad（1）On, when the dog excrement suspension supernatant is in the sample pad（1）On Diffusion does not reach detection line（6）When, then to the sample pad（1）Upper dropwise addition 100 μ l to 150 μ l borate buffer solution, see Examine 15 minutes to 30 minutes, wherein：The molar concentration of borate buffer solution is 0.005mol/L, pH value 9.0.
- 7. the application method of dog excrement echinococcosis antigen quick detection kit according to claim 6, it is characterised in that：Institute Dog excrement suspension supernatant is stated to obtain as follows：The first step, dog excrement is added in the preservation liquid of the dog excrement after aseptic filtration and obtains dog excrement suspension, wherein：Dog excrement preserves liquid For be 0.02% containing percentage by weight sodium azide, 0.3% Tween-20 and 1% bovine albumin phosphate buffer, The molar concentration of phosphate buffer is 0.15mol/L, pH value 7.2；Dog excrement per 2ml preserves the dog excrement that liquid adds 1g；Second step, dog excrement suspension obtain dog excrement suspension supernatant after the centrifugation of 15 minutes.
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| CN108802364A (en) * | 2018-06-14 | 2018-11-13 | 河北特温特生物科技发展有限公司 | NC films and scribing line prepare the production technology of NC films |
| CN112755049A (en) * | 2021-02-05 | 2021-05-07 | 哈密市动物疫病预防控制中心 | Laxative formula for improving dog cestodiasis excrement detection sensitivity and preparation method thereof |
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| CN108802364A (en) * | 2018-06-14 | 2018-11-13 | 河北特温特生物科技发展有限公司 | NC films and scribing line prepare the production technology of NC films |
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