CN1116608C - Fast diagnostic test paper strip for livestock and poultry pestilence - Google Patents
Fast diagnostic test paper strip for livestock and poultry pestilence Download PDFInfo
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Abstract
The present invention relates to an appliance for diagnosing epidemic diseases of fowls, particularly to test paper for rapidly diagnosing epidemic diseases of fowls. The test paper contains a supporting layer and a reaction reagent carrier adsorbing layer, wherein the supporting layer is a sheet which does not absorb water; the reaction reagent carrier adsorbing layer is pasted on the supporting layer. A sample end is orderly provided with a fiber layer, a fiber layer for adsorbing gold mark antibodies Wi corresponding to some fowl epidemic disease to be tested and a cellulose membrane, and a handle end is a water absorbing material layer. A standard antigen solution Xi to test fowl epidemic diseases and an antibody solution Yi coated with some fowl epidemic disease respectively print a negative mark '-' or 'I' and a positive mark 'I' or '-' on the cellulose membrane. The test paper has the advantages of high specificity and sensitivity, simple operation, rapidness, low labor intensity, short testing time, vivid, visual and correct test result display, no false positive or false negative judgment, little investment, low cost and easy popularization and application.
Description
The present invention relates to livestock and poultry pestilence diagnosis utensil, particularly relate to a kind of fast diagnostic test paper strip for livestock and poultry pestilence.As everyone knows, along with the continuous development of science and technology and improving constantly of people's living standard, intensive management, the industrial aquaculture of animal husbandry are flourish, large quantities of livestock and poultry are concentrated and raise, in case outbreak of communicable diseases and spreading will cause huge harm and economic loss to culturing enterprise or company, the lighter causes immunity of livestock and production performance (lay eggs, produce meat and give milk) to descend and the price of deed reduces, the large quantities of livestock and poultry death of weight person cause the break of breeding enterprise and company.The economic loss that China every year causes because of livestock and poultry pestilence is in 10,000,000,000 yuan.The generation of livestock and poultry pestilence if can not in time make a definite diagnosis, is taked control and treatment measure, and consequence is hardly imaginable, will have a strong impact on animal husbandry development.
Current because relevant livestock and poultry pestilence cause of disease height variation, serotype is numerous, makes many eqpidemic diseases present new epidemic characteristic, and its symptom and pathological change tend to atypiaization gradually, bring big difficulty for the eqpidemic disease clinical diagnosis, be difficult to make a definite diagnosis according to clinical symptoms and pathological change.At present, the livestock and poultry pestilence diagnostic techniques that adopts mainly contains both at home and abroad: agar gel diffusion test (AGP), counter immunoelectrophoresis, serum virus neutralization test (VN), enzyme linked immunosorbent assay (ELISA), immunofluorescence antibody technique (IF), SPA test-Conglutination, polymerase chain reaction (PCR) and nucleic acid probe or the like.Though above-mentioned these methods and technology can the etiologic diagnosis livestock and poultry pestilences, but the test operation time is long, complicated, and need relevant particular instrument equipment and technical ability, the equipment that have even needs are expensive, as fluorescent microscope, microplate reader and PCR instrument etc., often be limited to and carry out in the laboratory, be difficult in basic unit and popularize and promote, can't in time accomplish rapidly to make a definite diagnosis, effectively control the eqpidemic disease development.In fact, China at present to the livestock and poultry pestilence clinical diagnosis still based on clinical symptoms and pathological change, the few adopt said method to diagnose or make a definite diagnosis, and because of needs suitable experience and technical ability, can't make a definite diagnosis sometimes.The antibody horizontal monitoring is then only carried out in some large-scale plants (factory), and only can monitor limited several eqpidemic disease, as newcastle disease, Bursal Disease etc.
The domestic livestock and poultry pestilence ELISA quick diagnosis boxes such as trichinosis, newcastle disease, Bursal Disease of having succeeded in developing are used to detect above-mentioned about eqpidemic disease cause of disease and its antiserum, and its sensitivity is higher tens of times than AGP respectively.This diagnosis box external packing is generally the rectangle carton, interior dress 4-8 bar reaction plate, the little plastic tube of the 4 kinds of reagent and the usefulness of taking a sample.Need during detection to add sample to be checked in each hole of reaction plate with little plastic tube, order is reacted with 1,2,3, No. 4 liquid then, each response procedures needs fully washing, chromogenic reaction needs to finish in 2-3 minute, and judge feminine gender or positive findings according to the depth degree of Show Color, whole operation needs 10 minutes.This diagnosis box has been done certain simplification to the ELISA detection rules of routine, have easy relatively, characteristics fast, but do not reach the easy, quick of " single stage method " as yet, and judge the positive and negative result with shade, not accurate enough reliable, false negative and false-positive misjudgement result appear sometimes.
The objective of the invention is: develop the serial test strips that is applicable to various livestock and poultry pestilence quick diagnosis, simple and efficient, accurately diagnose various livestock and poultry pestilences, cost is low, and is simple to operate, easily at clinical application.
The present invention is achieved in that a kind of fast diagnostic test paper strip for livestock and poultry pestilence, contain supporting layer, reaction reagent carrier absorption layer, supporting layer is the strip of foil that do not absorb water, reaction reagent carrier absorption layer sticks on the supporting layer, be followed successively by fibrage from the sample end, adsorb the corresponding golden labeling antibody of certain livestock and poultry pestilence to be measured (Wi) fibrage, the cellulose rete, handle end is an absorbent material layer, on the cellulose rete, print out negative trace "-" or " | " and the positive trace " | " or "-" that shows of showing respectively with certain livestock and poultry pestilence standard antigen solution to be measured (Xi) with corresponding certain livestock and poultry pestilence coated antibody solution (Yi).
The supporting layer strip of foil that do not absorb water can be used the hard plastic slip; or the cardboard slip that do not absorb water, fibrage useable glass cotton; absorbent material layer can be used thieving paper; the cellulose rete can be used nitrocellulose filter; gold labeling antibody fibrage can be with golden labeling antibody (Wi) glass wool; at glass wool, be coated with diaphragm on golden labeling antibody glass wool and the absorbent paper layer, negative show trace and positive show its combination of trace can be "+", " ‖ ", "=", "
", "
", "
", "
" trace, select for use these two kinds combinations of "+", " ‖ " trace better, print out mark line at the diaphragm deflection glass wool one side 0.5cm place of glass wool and golden labeling antibody (Wi) glass wool intersection correspondence.Corresponding golden labeling antibody of certain livestock and poultry pestilence or coated antibody are meant the corresponding monoclonal antibody of certain livestock and poultry pestilence.
Be adsorbed with certain livestock and poultry pestilence gold labeling antibody Wi glass wool to be measured, print with certain livestock and poultry pestilence standard antigen Xi and negatively to show trace and to show a kind of that trace can be in the following livestock and poultry pestilence: the golden labeling antibody glass wool of aftosa with the positive that certain corresponding livestock and poultry pestilence coated antibody Yi prints, aftosa standard antigen trace and corresponding aftosa coated antibody trace, rabies gold labeling antibody glass wool, rabies standard antigen trace and corresponding rabies coated antibody trace, pseudoabies gold labeling antibody glass wool, pseudoabies standard antigen trace and corresponding pseudoabies coated antibody trace, swine fever gold labeling antibody glass wool, swine fever standard antigen trace and corresponding swine fever coated antibody trace, pig reproduction and respiratory system syndrome gold labeling antibody glass wool, pig reproduction and respiratory system syndrome standard antigen trace and corresponding pig reproduction and respiratory system syndrome coated antibody trace, contagious equine abortion gold labeling antibody glass wool, contagious equine abortion standard antigen trace and corresponding contagious equine abortion coated antibody trace, bovine viral diarrhoea one mucosal disease gold labeling antibody glass wool, bovine viral diarrhoea-mucosal disease standard antigen trace and corresponding bovine viral diarrhoea-mucosal disease coated antibody trace, ovine blue tongue gold labeling antibody glass wool, ovine blue tongue standard antigen trace and corresponding ovine blue tongue coated antibody trace, Bursal Disease gold labeling antibody glass wool, Bursal Disease standard antigen trace and corresponding Bursal Disease coated antibody trace, Newcastle disease gold labeling antibody glass wool, Newcastle disease standard antigen trace and corresponding Newcastle disease coated antibody trace, chicken Marek's disease gold labeling antibody glass wool, chicken Marek's disease standard antigen trace and corresponding chicken Marek's disease coated antibody trace, chicken egg-decreasing syndrome gold labeling antibody glass wool, chicken egg-decreasing syndrome standard antigen trace and corresponding chicken egg-decreasing syndrome coated antibody trace, infectious bronchitis of chicken gold labeling antibody glass wool, infectious bronchitis of chicken standard antigen trace and corresponding infectious bronchitis of chicken coated antibody trace, infectious laryngotracheitis of chicken gold labeling antibody glass wool, infectious laryngotracheitis of chicken standard antigen trace and corresponding infectious laryngotracheitis of chicken coated antibody trace, duck virus hepatitis gold labeling antibody glass wool, duck virus hepatitis standard antigen trace and corresponding duck virus hepatitis coated antibody trace, gosling plague gold labeling antibody glass wool, gosling plague standard antigen trace and corresponding gosling plague coated antibody trace, bird flu gold labeling antibody glass wool, bird flu standard antigen trace and corresponding bird flu coated antibody trace, anthrax gold labeling antibody glass wool, anthrax standard antigen trace and corresponding anthrax coated antibody trace, Escherichia coli gold labeling antibody glass wool, Escherichia coli standard antigen trace and corresponding Escherichia coli coated antibody trace, salmonella gold labeling antibody glass wool, salmonella standard antigen trace and corresponding salmonella coated antibody trace, pasteurellosis gold labeling antibody glass wool, pasteurellosis standard antigen trace and corresponding pasteurellosis coated antibody trace, brucellosis gold labeling antibody glass wool, brucellosis standard antigen trace and corresponding brucellosis coated antibody trace.
Good effect of the present invention:
1, fast diagnostic test paper strip for livestock and poultry pestilence has following advantage
(1) specificity and susceptibility are good.Fast diagnose test paper bar is that the monoclonal antibody with the colloid gold label high-affinity is that the basis prepares, no covalent bond formation between gold grain and the antibody molecule in the gold labeling antibody, the two Van der Waals force by the charges of different polarity combines, colloid gold label is very little to the specificity and the affinity influence of monoclonal antibody, and has higher mark rate.Therefore, fast diagnose test paper bar has higher specificity and susceptibility.
(2) easy and simple to handle, quick.Use fast diagnose test paper bar to need not other any reagent,, in 2 minutes, can detect the result as long as test strips sample end was inserted sample to be checked about 5 seconds.
(3) show judged result image, directly perceived, accurate.Fast diagnose test paper bar is represented eqpidemic disease feminine gender and positive findings respectively with apparent "-", "+" or " | ", " ‖ ", show like this with the usual thinking of people and judge that custom is identical, vivid, true to nature, directly perceived, simple and clear, accurately, the misjudgement of false negative and false positive can not appear.
(4) cost is low, small investment.Use fast diagnose test paper bar, do not need to join in addition instrument and equipment, save great amount of investment, cost is low, operates very simply, need not train, and everybody can manipulate.
(5) apply easily.Be easy to on-the-spot the detection in the eqpidemic disease spot.
Fast diagnose test paper bar is widely used in rural area livestock and poultry cultivation self-employed worker, and large, medium and small type is cultured factory (or company), and the demand of each level such as veterinary station, epidemic prevention station and specialized laboratory has vast market prospect and than large economy, social benefit.
Fig. 1: fast diagnostic test paper strip for livestock and poultry pestilence structural representation (side-looking structure):
Fig. 2: one of fast diagnostic test paper strip for livestock and poultry pestilence plan structure synoptic diagram;
Fig. 3: two of fast diagnostic test paper strip for livestock and poultry pestilence plan structure synoptic diagram.
Embodiment:
Fast diagnostic test paper strip for livestock and poultry pestilence can be widely used in the clinical quick diagnosis of multiple livestock and poultry pestilence, as aftosa (Foot and mouth disease, FMD), rabies (Rabies), pseudoabies (Pseudorabies), swine fever (Swine fever, SF), pig reproduction and respiratory system syndrome (Porcine reproduuive andrespiratory syndrome, PRRS), contagious equine abortion (Equine infection anemia, EIA), bovine viral diarrhoea-mucosal disease (Bovine viral diarrhea-mucosal disease, BVD-MD), ovine blue tongue (Blue tongue BT), Bursal Disease (Infectious bursal disease, IBD), Newcastle disease (Newcastle disease ND), chicken Marek's disease (Marek ' s disease, MD), chicken egg-decreasing syndrome (Egg drop syndrome, EDS), avian infectious arm inflammation (Infections bronchitis, IB), infectious laryngotracheitis of chicken (Infectious laryngotracheitis, ILT), duck virus hepatitis (Duck virus hepatitis, DVH), gosling plague (Gosling plague, GP), bird flu (Avianinfluenza, AI), anthrax (Anthrax), colibacillosis (CoIibacillosis), salmonellosis (Salmonellosis), pasteurellosis (Pasteurellosis), brucellosis (Brucellosis) or the like.
Make certain fast diagnostic test paper strip for livestock and poultry pestilence, at first to make the standard antigen of certain livestock and poultry pestilence, be used for negative showing trace, and then the coated antibody of making corresponding certain livestock and poultry pestilence is used for positive trace and the corresponding livestock and poultry pestilence gold labeling antibody (being used for golden labeling antibody fiber) of showing.
(1) current domestic and international standard antigen (Xi) preparation technology to various livestock and poultry pestilences is divided into three kinds substantially:
(1.1) prepare certain livestock and poultry pestilence standard antigen (Xi) with cell culture and virus.
With the PRMI 1640 medium culture cell monolayers that contain 5% calf serum, with the above-mentioned cell monolayer of virus inoculation, inoculum concentration is 0.5-1.2ml, goes inoculation liquid behind 37 ℃ of absorption 60-90min, with Hanks damping fluid washing 1 time, the cell maintenance medium that is contained 0.5% calf serum is in 37 ℃ of 5%CO
2Incubator in cultivate 24-48h, collect virus-culturing fluid, behind sonicated or multigelation three times, the centrifugal 30min of 6000-8000g, get on the density gradient sucrose solution liquid level that viral supernatant adds 20-60%, with the centrifugal 3-5h of 50000-70000g, collect the viral band of purifying, its protein concentration is 10-30mg/ml, obtains certain livestock and poultry pestilence poison standard antigen, and the livestock and poultry pestilence that obtains viral standard antigen with this kind preparation method has Bursal Disease (IBD), aftosa (FMD), rabies, pseudoabies, swine fever (SF), pig reproduction and respiratory system syndrome (PRRS), contagious equine abortion (EIA), bovine viral diarrhoea-mucosal disease (BVD-MD), ovine blue tongue (BT), chicken Marek's disease (MD), infectious laryngotracheitis of chicken (ILT), duck virus hepatitis (DVH), gosling plague (GP), bird flu (AI) etc.
(1,2) obtains certain livestock and poultry pestilence standard antigen (Xi) with chicken (duck) embryo culture virus.
Choose fresh fertilization chicken (duck) egg, remove surface contaminants, use the thimerosal wiped clean, place 38-39 ℃ the interior hatching of incubator then, relative humidity remains on 60-70%, hatches to be used for virus inoculation after 9-10 days.Earlier certain virus is done 1: 10 times with sterile saline and be diluted to viral liquid, viral liquid is inoculated in the well-developed chicken of 9-10 age in days (duck) the embryo allantoic cavity, every embryonic breeding kind 0.1ml, after the inoculation inoculation hole on the eggshell is sealed with paraffin wax, chicken (duck) embryo of inoculating is placed continuation hatching 48-96h in 37 ℃ of incubators, according to egg and discard dead germ, at 4 ℃ of cooling 24h.With the air chamber position of eggshell with tincture of iodine sterilization after, the aseptic eggshell of opening the air chamber position, tear shell membrane, fine hair, chorioallantoic membrane and amnion off with pincet, extract chicken (duck) embryo allantoic liquid, behind ultrasonic Treatment or multigelation three times, the centrifugal 30min of 6000-8000g, get viral supernatant and be added on the 20-60% density gradient sucrose solution liquid level,, collect the virus band of purifying with the centrifugal 3-5h of 50000-70000g, its protein concentration is 10-20mg/ml, obtains certain viral standard antigen.The livestock and poultry pestilence for preparing viral standard antigen in this way has, chicken egg-decreasing syndrome (EDS), Newcastle disease (ND), infectious bronchitis of chicken (IB) etc.
(1.3) use microbe growth,, prepare certain bacillary livestock and poultry pestilence standard antigen (Xi) with LB medium culture bacterium and from wherein extracting bacterioprotein.
Select the single bacterium colony of certain bacterium to be cultivated, be inoculated in the LB fluid nutrient medium, 37 ℃ of 200r/min shakes are cultivated 16-20h, 4 ℃ of centrifugal 10-20min of 3000g, get centrifugation and be resuspended in the physiological saline,, make certain bacterium standard antigen with the ultrasonic disruption thalline, its protein concentration is 20-40mg/ml, the livestock and poultry pestilence for preparing bacillary standard antigen in this way has: colibacillosis, anthracnose, salmonellosis, brucellosis, pasteurellosis etc.
(2) various livestock and poultry pestilence bags are called for short the preparation method of coated antibody (Xi) with monoclonal antibody (Yi)
With 50 μ g-100 μ g/ certain livestock and poultry pestilence standard antigen (Xi) only, immunity BALB/C is mouse three times, each 15-30 days at interval, after booster immunization 3-4 days for the third time, with the eyeball of mouse bloodletting, draw neck to cause death, in 75% alcohol-pickled 5-10min, aseptic its spleen of getting, shred and through 100 order nylon net filters, the centrifugal 10min of 1000g collects splenocyte, with 1 * 10
8Splenocyte and the NSO plasmacytoma mixing with cells of 2-5 * 107,1000g is centrifugal, and 10min abandons supernatant, cell precipitation is in 37 ℃ of PEG4000 (pH8.5-9.0) effect 1min that slowly add the 40-50% of 0.7-1ml in water-soluble.Slowly add serum-free RPMI1640 nutrient culture media 15ml then, to stop the effect of PEG.37 ℃ of water-soluble 5-10min, 1000g is centrifugal, and 10min abandons supernatant, cell precipitation is resuspended in HAT selects in the nutrient culture media, and add 96 well culture plates (100 μ l-200 μ l/ hole), puts 37 ℃ of %CO
2Cultivate in the incubator after 7-10 days,,, choose strong positive cell clone (OD with the culture supernatant of enzyme linked immunosorbent assay (ELISA) detection hybridoma with 1: 500 dilution certain standard antigen (Yi) coated elisa plate (40 holes/piece)
492More than=0.8), carry out continuous three times limiting dilution assay cloning, the hybridoma chromosome number of being produced is 92-98, the monoclonal antibody of its secretion specifically with the standard antigen albumino reaction of corresponding certain livestock and poultry pestilence, and not with other irrelevant albumen generation cross reaction, affinity constant reaches 10
9-10, light chain subtype is κ or λ, the heavy chain hypotype is IgG
1, IgG
2a, IgG
2bOr IgG
3, forming Sheet clonal antibody at certain livestock and poultry pestilence standard antigen specific antigen determinant, the positive that is used for fast diagnose test paper bar shows trace, i.e. coated antibody trace, this kind preparation method is applicable to the preparation of any livestock and poultry pestilence coated antibody.
(3) preparation of certain livestock and poultry pestilence gold labelled antibody (Wi) and golden labeling antibody (Wi) glass wool.
Prepare aurosol with the sodium citrate reducing process, promptly add the 0.5-2% citric acid three sodium solution of 2-4ml in the 50-100ml0.01-0.05% aqueous solution of chloraurate of boiling, the acquisition diameter is the collaurum about 15nm, with 0.1mol/LK
2CO
3Transfer collaurum pH to 8.5-9.5, with 1: 1000-1: 3000 mark adds in the pH8.5-9.5 aurosol than the monoclonal antibody (Yi) with certain livestock and poultry pestilence to be marked, behind the mark 10min, add 20%PEG10000 to final concentration 0.05%, 4 ℃ of centrifugal 20min of 1500-3000g, remove the gold grain of last combination, 4 ℃ of centrifugal 1h of 15000g, abandon supernatant, after obtaining preliminary purification gold mark protein mixture, with propylene glucosan S-400 column chromatography, separation and purification gold mark protein obtains certain livestock and poultry pestilence colloid gold label antibody, with 1: 100-1: the glue gold labelled antibody of 500 dilutions is adsorbed in the processed glass cotton, drying makes sick golden labeling antibody (Wi) glass wool of certain livestock and poultry pestilence, and this preparation method is applicable to the preparation of any livestock and poultry pestilence gold labeling antibody cotton.
(4) fast diagnostic test paper strip for livestock and poultry pestilence is implemented structure
Referring to Fig. 1,2,3, among the figure, 1 for supporting layer with the strip of foil that do not absorb water, can adopt the plastic slice bar in the enforcement or adopt the hard paper sheet material that does not absorb water, reaction reagent carrier absorption layer is by 2,3,4,5, combine, from sample end 2, above handle end 5 sticked on supporting layer 1 successively, wherein 2 was sample end fibrage, can use glass fibre cotton to be called for short glass wool in the enforcement, 3 for being adsorbed with certain livestock and poultry pestilence gold labeling antibody (Wi) fibrage, can adopt the processed glass cellucotton, abbreviates (Wi) golden labeling antibody cotton as, 4 is the cellulose rete, can adopt nitrocellulose filter in the enforcement, 5 is the handle end absorbent material layer, adopts thieving paper, all can as filter paper or other thieving paper, test strips length overall 8cm, width 0.4cm, 6 for certain livestock and poultry pestilence standard antigen (Xi) but on cellulose membrane, make negative demonstration trace "-" or " | ", 7 for showing trace " | " or "-" with certain livestock and poultry pestilence coated antibody (Yi) positive on nitrocellulose filter, negative trace 6 and the positive trace 7 that shows of showing can be combined to "+" or is " ‖ ", or is "=", or be "
" or be "
" or be "
" or be "
".Select for use "+" best, or select for use " ‖ " also all well and good.
8 is diaphragm, and the diaphragm that covers glass wool and golden labeling antibody (Wi) glass wool is 8-1, and the diaphragm that covers on the water accepting layer filter paper is 8-2.On the diaphragm at deflection glass wool one side 0.5cm place on the white diaphragm of glass wool and golden labeling antibody (Wi) glass wool intersection correspondence position, be printed on a mark line 9; be printed on arrow and be printed on the max printed words on 9 lines the right; the handle end diaphragm can be used yellow or other color; 2; 3; the intersection fiber infiltration that crosses one another each other of 4,5 each layers.
(5) fast diagnostic test paper strip for livestock and poultry pestilence is implemented the diagnostic reaction principle
After test strips sample end inserts detected sample solution, solution to be checked spreads to cellulose membrane together by the golden labeling antibody (Wi) that siphon drives in certain livestock and poultry pestilence cause of disease and the golden labeling antibody cotton, and finally be penetrated in the filter paper layer, Wi in diffusion process (certain golden labeling antibody) combines with Xi (certain livestock and poultry pestilence detects antigen), form antigen-Jin labeling antibody compound, this compound can combine with trace coated antibody Yi on the cellulose rete again, in antibody, generate rufous " | " mark, part does not combine with standard antigen on tunica fibrosa with the golden labeling antibody of coated antibody combination, generate rufous mark "-" or " | ", two kinds of marks make up stack result mutually, form positive show "+" or " ‖ ", otherwise have only golden labeling antibody and standard antigen to combine, there are not antigen one gold medal labeling antibody compound and corresponding coated antibody to combine, then generate single negative show tags "-" or " | " if the test strips testing result, both there be not negative show tags "-" or " | ", do not have positive show tags "+" or " ‖ " yet, prove that test strips lost efficacy.
(6) fast diagnostic test paper strip for livestock and poultry pestilence embodiment detection method.
The preparation of test sample liquid, detection Bursal Disease (IBD) is got the disease chicken bursa and is organized as pathological material of disease, chicken egg-decreasing syndrome (EDS) gets disease ovum gallinaceum nest and fallopian tubal is a pathological material of disease, blood of the desirable livestock and poultry of other livestock and poultry pestilence or ight soil or tonsillotome or other pathological material of disease, viscera tissues such as the heart, liver,spleen,kidney and lung.Can coagulate pathological material of disease earlier and shred grinding, make 1: 10 sample solution to be checked.
Corresponding fast diagnostic test paper strip for livestock and poultry pestilence sample end is inserted in the sample solution, and several seconds approximately, insertion depth surpassed mark line 9, behind the taking-up diagnosis test paper, and horizontal positioned, about 2 minutes observationss.
The result judges: if having rufous "+" or " ‖ " mark to show on cellulose membrane, then be indicated as the positive, the proof livestock and poultry of examining suffer from certain eqpidemic disease: if having only the demonstration of rufous "-" or " | " mark on the nitrocellulose filter then be indicated as feminine gender, the proof livestock and poultry do not suffer from the disease of quarantining, if there is not mark to show, show that then test strips lost efficacy.
Embodiment one, Bursal Disease (IBD) fast diagnose test paper bar, referring to Fig. 1,2,3, at first prepare IBD standard antigen X1, prepare IBD coated antibody Y1, prepare IBO gold labeling antibody (W1) and golden labeling antibody (Wi) cotton by (3) method step among the embodiment by (2) method step among the embodiment by (1.1) among the embodiment or (1.2) method step.
Among the figure; 1 is the thin bar of plastic cement; as supporting layer; 2 is that glass wool (sample end) is a fibrage; 3 for being adsorbed with the glass wool of IBD gold labeling antibody (W1); promptly be adsorbed with the fibrage of golden labeling antibody W1, be called for short IBD W1 cotton, 4 is cellulose membrane; present embodiment is selected nitrocellulose filter for use; 5 is filter paper (handle end), i.e. absorbent material layer, 2; 3; 4,5 stick on the thin bar 1 of plastic cement from left to right successively, and 6 for being printed on IBD standard antigen (X1) trace "-" or " | " on the nitrocellulose filter 4; 7 is IBD coated antibody (Y1) trace " | " on nitrocellulose filter 4; two kinds of traces are formed "+" word, or " ‖ " style, and 8-1 is white diaphragm; cover on glass wool 2 and the IBD gold labeling antibody W1 cotton 3; the inclined to one side about 0.5cm of glass wool one side place is printed on the right seal arrow and the max printed words of mark line 9,9 on the corresponding diaphragm 8-1 of 2 and 3 intersections position, and yellow diaphragm 8-2 is arranged on the filter paper 5 (handle end).
Detect the disease chicken and whether suffer from IBD use IBD quick diagnosis paper slip, get the disease chicken bursa earlier, or spleen, kidney shreds grinding, makes 1: 10 sample solution to be checked, IBD diagnosis test paper sample end is inserted sample solution to be checked, insertion depth can not surpass mark line 9, takes out the test strips horizontal positioned after about 5 seconds, after 2 minutes, can observe testing result, if show the rufous mark of "+" or " ‖ " on nitrocellulose filter, expression is positive, show that chicken suffers from IBD, if show the rufous mark of "-" or " | " on the nitrocellulose filter 4, expression is negative, shows that chicken does not suffer from IBD, if show tags not on the plain film 4 shows that this test strips lost efficacy.
Embodiment two, chicken egg-decreasing syndrome (EDS) fast diagnose test paper bar.Referring to Fig. 1,2,3, preparation EDS gold labeling antibody W2 cotton (being the W2 cotton), EDS standard antigen X2, the method step of EDS coated antibody Y2 and embodiment one roughly the same, 1,2,4,5,, 8-1,8-2,9 labels do not repeat with embodiment one.3,6,7 is somewhat different with embodiment one.In the present embodiment, 3 is EDS gold labeling antibody (W2) cotton, and 6 is EDS standard antigen (X2) trace "-" or " | ", and 7 is EDS coated antibody (Y2) trace " | ", the combination of two traces or "+" or " ‖ ".
Pathological material of disease is got disease ovum gallinaceum nest, or cloaca, or ight soil, shreds to grind to produce 1: 10 sample solution.Detect and observations,, do not repeat with embodiment one.
Embodiment three, Newcastle disease (ND) fast diagnose test paper bar, referring to Fig. 1,2,3, method for making, structure, detection, observations and embodiment one are similar, do not repeat, some is not both: 2 are ND gold labeling antibody cotton (being the W3 cotton), and 6 is ND standard antigen (X3) trace, and 7 is ND coated antibody (Y3) trace.Pathological material of disease is got and a kind ofly in the heart, liver,spleen,kidney, lung of disease chicken is shredded grinding, produces sample solution.
Embodiment four, chicken Marek's disease (MD) fast diagnose test paper bar, and roughly the same embodiment one, do not repeat.Somewhat differently be, 2 is MD gold labeling antibody (W4) cotton, and 6 is MD standard antigen (X4) trace, and 7 is MD coated antibody (Y4) trace.Samples solution is got disease chicken organ organ or nerve is produced.
Embodiment five, infectious bronchitis of chicken (IB) fast diagnose test paper bar, and roughly the same embodiment one, do not repeat.Somewhat differently be: 2 are IB gold labeling antibody (W5) cotton, and 6 is IB standard antigen (X5) trace, and 7 is IB coated antibody (Y6) trace.Pathological material of disease solution is got the chicken lung, tracheae, and exudate is produced.
Embodiment six, infectious laryngotracheitis of chicken (ILT) fast diagnose test paper bar, and roughly the same embodiment one, do not repeat.Somewhat differently be, 2 is ILT gold labeling antibody (W6) cotton, and 6 is ILT standard antigen (X6) trace, and 7 is ILT coated antibody (Y6) trace.Samples solution is got the chicken larynx, tunica mucosa tracheae, and secretion is produced.
Embodiment seven, duck virus hepatitis (DVH) fast diagnose test paper bar, and roughly the same embodiment one, do not repeat.Somewhat differently be, 2 is DVH gold labeling antibody (W7) cotton, and 6 is DVH standard antigen (X7) trace, and 7 is DVH coated antibody (Y7) trace.Samples solution is got disease duck liver and is produced.
Embodiment eight, bird flu (AI) fast diagnose test paper bar, and roughly the same embodiment one, do not repeat.Somewhat differently be, 2 is AI gold labeling antibody (W8) cotton, and 6 is AI standard antigen (X8) trace, and 7 is AI coated antibody (Y8) trace.Samples solution is got disease fowl internal organs and is produced.
Embodiment nine, gosling plague (GP) fast diagnose test paper bar, and roughly the same embodiment one, do not repeat.Somewhat differently be, 2 is GP gold labeling antibody (W9) cotton, and 6 is GP standard antigen (X9) trace, and 7 is GP coated antibody (Y9) trace.Samples solution is got disease goose spleen, pancreas, and liver is produced.
Embodiment ten, the anthrax fast diagnose test paper bar, and roughly the same embodiment one, do not repeat.Somewhat differently be, anthrax standard antigen X10 adopts among the embodiment (1,3) with the method for cultivation of bacteria preparation, and 2 be golden labeling antibody (W10) cotton of anthrax, and 6 is anthrax standard antigen (X10) trace, and 7 is anthrax coated antibody (Y10) trace.Samples solution is got ill domestic animal blood, and transudate is produced.
Embodiment 11, the colibacillosis fast diagnose test paper bar, and roughly the same embodiment ten, do not repeat.2 is colibacillosis gold labeling antibody (W11) cotton, and 6 is colibacillosis standard antigen (X11) trace, and 7 is colibacillosis coated antibody (Y11) trace.Samples solution is got ill domestic animal fowl blood or viscera tissue is produced.
Embodiment 12, the salmonellosis fast diagnose test paper bar, and roughly the same embodiment ten, do not repeat.Somewhat differently be, 2 is salmonellosis gold labeling antibody (W12) cotton, and 6 be salmonellosis standard antigen (X12) trace, and 7 be that salmonellosis bag quilt resists (Y12) trace.Samples solution is got ill domestic animal fowl blood, internal organs, and ight soil is produced.
Embodiment 13, the pasteurellosis fast diagnose test paper bar, and roughly the same embodiment ten, do not repeat.Somewhat differently be, 2 is pasteurellosis gold labeling antibody (W13) cotton, and 6 is pasteurellosis standard antigen (X13) trace, and 7 is pasteurellosis coated antibody (Y13) trace.Samples solution is got the heart of ill domestic animal fowl, liver, spleen, body cavity exudate.
Embodiment 14, the brucellosis fast diagnose test paper bar, and roughly the same embodiment ten, do not repeat.Somewhat differently be, 2 is brucellosis gold labeling antibody (W14) cotton, and 6 is brucellosis standard antigen (X14) trace, and 7 is brucellosis coated antibody (Y14) trace.Samples solution is got fetus, the afterbirth (miscarriage) of ill domestic animal fowl and is produced.
Embodiment 15, aftosa (FMD) fast diagnose test paper bar, and roughly the same embodiment one, do not repeat.Somewhat differently be, 2 is FMD gold labeling antibody (W15) cotton, and 6 is FMD standard antigen (X15) trace, and 7 is FMD coated antibody (Y15) trace.Samples solution is got blister skin or the blister liquid of ill domestic animal fowl and is produced.
Embodiment 16, the rabies fast diagnose test paper bar, and roughly the same embodiment one, somewhat differently is, and 2 is rabies gold labeling antibody (W16) cottons, and 6 is rabies standard antigen (X16) trace, and 7 is rabies coated antibody (Y16) trace.Samples solution is got disease dog brain tissue, and salivary gland is produced.
Embodiment 17, the pseudoabies fast diagnose test paper bar, and roughly the same embodiment one, do not repeat.Somewhat differently be, 2 is pseudoabies gold labeling antibody (W17) cotton, and 6 is pseudoabies standard antigen (X17) trace, and 7 is pseudoabies coated antibody (Y17) trace.Samples solution is got disease dog brain tissue, tonsillotome, and fetus (miscarriage) is produced.
Embodiment 18, swine fever (SF) fast diagnose test paper bar, and roughly the same embodiment one, do not repeat.Yes have a few difference, 2 is SF gold labeling antibody (W18) cotton, and 6 is SF standard antigen (X18) trace, and 7 is SF coated antibody (Y18) trace.Samples solution is got tonsillotome, spleen, and kidney is produced.
Embodiment 20, contagious equine abortion (EIA) fast diagnose test paper bar, and roughly the same embodiment one, do not repeat.Somewhat differently be, 2 is EIA gold labeling antibody (W20) cotton, and 6 is EIA standard antigen (X20) trace, and 7 is EIA coated antibody (Y20) trace.Samples solution is got disease horse blood or spleen, liver, and the heart is produced.
Embodiment 21, bovine viral diarrhoea-mucosal disease (BVD-MD) fast diagnose test paper bar, and roughly the same embodiment one, do not repeat.Somewhat differently be, 2 is BVD-MD gold labeling antibody (W21) cotton, and 6 is the standard antigen trace of BVD-MD, and 7 is BVD-MD coated antibody (Y21) trace.Samples solution is got the disease bovine blood, spleen, and lymph node is produced.
Embodiment 22, ovine blue tongue (BT) fast diagnose test paper bar, and roughly the same embodiment one, do not repeat.Somewhat differently be, 2 is BT gold labeling antibody (W22) cotton, and 6 is BT standard antigen (X22) trace, and 7 is BT coated antibody (Y22) trace.Samples solution is got the disease sheep blood or the heart, spleen, and kidney, lymph node is produced.
Claims (3)
1, a kind of fast diagnostic test paper strip for livestock and poultry pestilence, contain supporting layer, reaction reagent carrier absorption layer, feature of the present invention is: supporting layer is the strip of foil that do not absorb water, reaction reagent carrier absorption layer sticks on the supporting layer, be followed successively by fibrage from the sample end, adsorb the corresponding golden labeling antibody fibrage of certain livestock and poultry pestilence to be measured, the cellulose rete, handle end is an absorbent material layer, on the cellulose rete, print out negative trace "-" or " | " and the positive trace " | " or "-" that shows of showing respectively with certain livestock and poultry pestilence standard antigen solution to be measured with corresponding certain livestock and poultry pestilence coated antibody solution.
2, diagnosis test paper according to claim 1 is characterized in that: the supporting layer strip of foil that do not absorb water can be used the hard plastic slip, or the cardboard slip that do not absorb water; fibrage useable glass cotton; absorbent material layer can be used thieving paper, and the cellulose rete can be used nitrocellulose filter, and golden labeling antibody fibrage can be with golden labeling antibody glass wool; at glass wool; be coated with diaphragm on gold labeling antibody glass wool and the absorbent paper layer, the negative trace and the positive of showing shows that its combination of trace can be "+", " ‖ "; "=", "
", "
", "
", "
" trace, print out mark line at the diaphragm deflection glass wool one side 0.5cm place of glass wool and golden labeling antibody glass wool intersection correspondence, corresponding golden labeling antibody of certain livestock and poultry pestilence or coated antibody are meant the corresponding monoclonal antibody of certain livestock and poultry pestilence.
3, diagnosis test paper according to claim 1 and 2, it is characterized in that: be adsorbed with certain livestock and poultry pestilence gold labeling antibody glass wool to be measured, print with certain livestock and poultry pestilence standard antigen and negatively to show trace and to show a kind of that trace can be in the following livestock and poultry pestilence: the golden labeling antibody glass wool of aftosa with the positive that certain corresponding livestock and poultry pestilence coated antibody is printed, aftosa standard antigen trace and corresponding aftosa coated antibody trace, rabies gold labeling antibody glass wool, rabies standard antigen trace and corresponding rabies coated antibody trace, pseudoabies gold labeling antibody glass wool, pseudoabies standard antigen trace and corresponding pseudoabies coated antibody trace, swine fever gold labeling antibody glass wool, swine fever standard antigen trace and corresponding swine fever coated antibody trace, pig reproduction and respiratory system syndrome gold labeling antibody glass wool, pig reproduction and respiratory system syndrome standard antigen trace and corresponding pig reproduction and respiratory system syndrome coated antibody trace, contagious equine abortion gold labeling antibody glass wool, contagious equine abortion standard antigen trace and corresponding contagious equine abortion coated antibody trace, bovine viral diarrhoea one mucosal disease gold labeling antibody glass wool, bovine viral diarrhoea one mucosal disease standard antigen trace and corresponding bovine viral diarrhoea one mucosal disease coated antibody trace, ovine blue tongue gold labeling antibody glass wool, ovine blue tongue standard antigen trace and corresponding ovine blue tongue coated antibody trace, Bursal Disease gold labeling antibody glass wool, Bursal Disease standard antigen trace and corresponding Bursal Disease coated antibody trace, Newcastle disease gold labeling antibody glass wool, Newcastle disease standard antigen trace and corresponding Newcastle disease coated antibody trace, chicken Marek's disease gold labeling antibody glass wool, chicken Marek's disease standard antigen trace and corresponding chicken Marek's disease coated antibody trace, chicken egg-decreasing syndrome gold labeling antibody glass wool, chicken egg-decreasing syndrome standard antigen trace and corresponding chicken egg-decreasing syndrome coated antibody trace, infectious bronchitis of chicken gold labeling antibody glass wool, infectious bronchitis of chicken standard antigen trace and corresponding infectious bronchitis of chicken coated antibody trace, infectious laryngotracheitis of chicken gold labeling antibody glass wool, infectious laryngotracheitis of chicken standard antigen trace and corresponding infectious laryngotracheitis of chicken coated antibody trace, duck virus hepatitis gold labeling antibody glass wool, duck virus hepatitis standard antigen trace and corresponding duck virus hepatitis coated antibody trace, gosling plague gold labeling antibody glass wool, gosling plague standard antigen trace and corresponding gosling plague coated antibody trace, bird flu gold labeling antibody glass wool, bird flu standard antigen trace and corresponding bird flu coated antibody trace, anthrax gold labeling antibody glass wool, anthrax standard antigen trace and corresponding anthrax coated antibody trace, Escherichia coli gold labeling antibody glass wool, Escherichia coli standard antigen trace and corresponding Escherichia coli coated antibody trace, salmonella gold labeling antibody glass wool, salmonella standard antigen trace and corresponding salmonella coated antibody trace, pasteurellosis gold labeling antibody glass wool, pasteurellosis standard antigen trace and corresponding pasteurellosis coated antibody trace, brucellosis gold labeling antibody glass wool, brucellosis standard antigen trace and corresponding brucellosis coated antibody trace.
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| Application Number | Priority Date | Filing Date | Title |
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| CN99101537A CN1116608C (en) | 1999-01-21 | 1999-01-21 | Fast diagnostic test paper strip for livestock and poultry pestilence |
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| Application Number | Priority Date | Filing Date | Title |
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| CN99101537A CN1116608C (en) | 1999-01-21 | 1999-01-21 | Fast diagnostic test paper strip for livestock and poultry pestilence |
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| CN1261670A CN1261670A (en) | 2000-08-02 |
| CN1116608C true CN1116608C (en) | 2003-07-30 |
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| CN99101537A Expired - Fee Related CN1116608C (en) | 1999-01-21 | 1999-01-21 | Fast diagnostic test paper strip for livestock and poultry pestilence |
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| FR2967497B1 (en) * | 2010-11-17 | 2014-12-12 | Biomerieux Sa | DEVICE AND METHOD FOR IMMUNOASSAY |
| CN102778557B (en) * | 2011-04-28 | 2016-01-06 | 美艾利尔（上海）诊断产品有限公司 | Biological detection reagent coating liquid |
| CN102495216A (en) * | 2011-12-16 | 2012-06-13 | 吉林大学 | Colloidal gold test paper for rapidly detecting antibody of porcine reproductive and respiratory syndrome virus |
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