CN1403813A - Fast trichinelliasis diagnosing test paper strip - Google Patents
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- CN1403813A CN1403813A CN 02101927 CN02101927A CN1403813A CN 1403813 A CN1403813 A CN 1403813A CN 02101927 CN02101927 CN 02101927 CN 02101927 A CN02101927 A CN 02101927A CN 1403813 A CN1403813 A CN 1403813A
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Abstract
The fast trichinelliasis diagnosing test paper strip includes support layer and reaction reagent carrier absorbing layer. The support layer in unhydroscopic and the reaction reagent carrier adsorbing layer stuck to the support layer has fiber layer, trichiotic gold mark antibody W1 or W2 or trichinotic gold mark antigen cellulose layer, cellulose film layer, and hydroscopic material layer successively from the sample end to the handle end. The test print is printed with trichinotic monocloned antibody W1 or W2, solution or antigen, and the reference print is printed with anti-mouse IgG antibody solution or trichinotic ES antigen monocloned solution. The test paper strip has strong specificity, high sensitivity and intuitive diagnosis result and may be used in livestock farms, meat works, customs, etc.
Description
(1) technical field: the present invention relates to a kind of trichinosis diagnostic reagent displaying appliance, particularly relate to a kind of fast trichinelliasis diagnosing test paper strip.
(2) background technology: well-known, trichinosis is a kind of parasitic disease of infecting both domestic animals and human.Its infection host is numerous, and popular scope is very extensive, and routes of infection complexity has more than 150 kind of animal capable to infect approximately and revolves parasitosis, and human body health is caused very big threat.Trichinosis is to animal husbandry (particularly pig industry), the meat product processing industry, and foreign export already causes enormous economic loss.Trichina contain the worm packing to external world environment have very strong resistivity, in at--12 ℃ environment, can survive 57 days, to the meat that contains trichina fire-cure, pickle, quick-fried and the larva that is difficult to kill the trichina in the packing such as tan by the sun, the approach of human infection trichina, eaten the trichina muscle larvae in the meat product that maintains vigour often, as boiled dumpling, quick-fried sliced meat or contaminated cold dish etc.After the people was infected, its clinical manifestation was a low-grade fever, and in poor health, fatiguability has diarrhoea sometimes, vomiting, stomachache phenomenon; Severe patient can occur chewing, swallow with parathria, and hoarseness is breathed and eyeball pain, and the most serious may involve heart and nervous centralis, causes heart failure, stupor even death.So it is knowing altogether of people that trichinosis causes harm to human health.But a lot of animals such as pig etc. have very big tolerance to trichina, and the sick pig that infects trichina shows clinical symptoms hardly.This brings certain difficulty again for human prevention trichinosis.Certain trichinosis infection rate of economizing pig was 0.4% in 1979, and to nineteen eighty-two, infection rate rises to 0.85%,～nineteen eighty-two in 1980 wherein, the trichinosis infection rate of somewhere pig was 13.8%, and the trichina serology positive rate of pig in 1986 rises to 18.7%, according to incomplete statistics in 1996 years, only certain province trichinosis patient has reached people more than 10,000, can find out that trichinosis is serious day by day to the healthy harm of people.For a long time,, get every flesh compressing tablet microscopy method after veterinary sanitary inspection department mainly relies on tradition to butcher, or collect the method DIAGNOSIS OF TRICHINOSIS WITH of polypide with pepsin digestion muscle for guaranteeing meat food safety.But said method, at the bottom of the recall rate, the omission of part trichinosis pig flows to market, and the harm health of people influences food security.Above-mentioned two kinds of methods of inspection can not be carried out before domestic animal is slaughtered simultaneously, and the scene quarantine of work production line is butchered by incompatible modernized meat-packing plant, and also incompatibility market quarantine also can't provide the live hog export quarantine, bring big difficulty for the quarantine of live pig.Domestic and international for this reason scientific worker creates the method for other several DIAGNOSIS OF TRICHINOSIS WITH again.
(1) intracutaneous test, this method are to use allergic reaction to come DIAGNOSIS OF TRICHINOSIS WITH.This diagnostic method required time is shorter, and to the diagnosis of human body about 15 minutes, diagnosis needed 30～45 minutes approximately to the pig body.But the outstanding defective of this method is to have serious false positive reaction and cross reaction, has only 60% inspection rate, and there be limited evidence currently of adopts.
(2) complement fixation reaction (CF), owing to be not so that all antibody all can this method of complement-fixing can only detect a part of trichina antibody, its susceptibility and specificity are relatively poor, and, the complement fixation reaction complicated operation, time-consuming taking a lot of work there are differences bigger between the different operating person, influence the result and judge, cause to be difficult to apply.
(3) agglutination test, the trichina soluble antigen is adsorbed in some carrier surface, carrier granular and the antibody in the serum with adsorption antigen carries out indirect agglutination test then, to detect trichinosis, the trichinosis agglutination test detection method that grew up in recent years, mainly contain the cotton-shaped agglutination test of bentonite (BFT), latex agglutination test (LA), indirect hemagglutination test (IHA) etc.The advantage of these methods is to have improved trichinous recall rate greatlyyer, but complicated operation requires operating personnel's quality higher, and can only carry out in the laboratory, can't arrive on-the-spot the detection.
(4) precipitation test mainly contains microprecipitation test, counter immunoelectrophoresis, agar gel diffusion test (AGP) etc.The sensitivity assessment of these methods differs, and agents useful for same and method are owed standardization.
(5) immunofluorescence antibody technique (IF), IF not only can be used for trichinous detection, also can be used for the location of trichina antigen.Susceptibility and the specificity of using antigen-antibody reaction combine with the microscope morphological observation, have sensitivity, special, stable and result judges advantages such as accurate and visual, is the trichinous a kind of better method that detects at present.But exist film-making work loaded down with trivial details, workload is big, and microscopic examination has subjectivity, and the interference of non-specific fluorescence simultaneously also usually influences result's judgement, needs better microscopy apparatus.
(6) enzyme linked immunosorbent assay (ELISA) is applied to trichinous detection with elisa technique, has improved recall rate greatly, and very responsive; False positive rate descends greatly, increases substantially accuracy, and trichinous immunology detection is made a breakthrough.The fast diagnostic kit of the current pigs trichina disease that we succeed in developing is used widely, but complicated operation, and need subsidiary 4 kinds of reagent just can finish experimental implementation, and technical level of operators is had relatively high expectations, can't realize on-the-spot the detection.
(7) DNA detection technology is a kind of new and high technology that detects trichinzation from molecular level, has the susceptibility and the specificity of height.New approach has been opened up in classification and evaluation to trichina, makes trichinous research more deep.But because trichina and itself in the singularity at host endoparasitism position.Make to be difficult to the trichina DNA that from infection animal blood and tissue or excreta, increases, thereby be restricted with of the detection of technique of gene detection such as PCR to trichina.
Comprehensively above-mentioned, since nineteen forties so far, many scholars have explored trichinous a lot of detection method, constantly make progress, improve constantly the accuracy rate of detection, but it is strong and the false positive phenomenon occurs to fail to overcome specificity all the time, all there is complicated operation, loaded down with trivial details in said method, what have also needs specialized instrument and equipment, needs the professional to operate.The production-lines operation of the modernized meat packing plant of incompatibility causes the omission of trichinosis pig, and harm people's edible safety threatens human health.
(3) summary of the invention: the objective of the invention is: utilize trichina antigen, antibody technique, set up special, responsive, quick, the easy trichinosis diagnostic method of trichina, develop two kinds and (II) the trichinous fast diagnose test paper bar of humans and animals (I).
Technical scheme of the present invention is: a kind of fast trichinelliasis diagnosing test paper strip (I): contain supporting layer, reaction reagent carrier absorption layer, the lamella of supporting layer for not absorbing water, reaction reagent carrier absorption layer is fixed on the supporting layer, adsorbed layer is followed successively by fibrage from the sample end, trichina gold labeling antibody W
1Or W
2Fibrage, cellulose rete, handle end are absorbent material layer, use the monoclonal antibody W of trichina respectively
1Or W
2Solution and anti-mouse IgG antibody solution are printed diagnosis trace " | " or " one " and contrast trace " " or " | " on the cellulose rete.
Supporting layer can be the hard plastic slip, or is the cardboard bar that do not absorb water, and fibrage can be glass wool, and the cellulose rete can be used nitrocellulose filter, and absorbent material layer can be used thieving paper, trichina gold labeling antibody W
1Or W
2Fibrage, for the monoclonal antibody of the glue gold anti-trichina secretion antigen of mark (ES) is adsorbed on the glass wool, diagnosis trace and the combination of contrast trace can be "+", " ‖ ", "=",
In a kind of, select for use "+" or " ‖ " or "=" combination trace better, at fibrage, gold mark W
1Or W
2Glass wool is coated with the plastic cement diaphragm on the absorbent material layer, at fibrage and gold mark W
1Or W
2The about 0.5cm of corresponding diaphragm deflection fibrage one side of fibrage intersection prints the sample mark line in the place.
A kind of fast trichinelliasis diagnosing test paper strip (II), contain supporting layer, reaction reagent carrier absorption layer, the lamella of supporting layer for not absorbing water, reaction reagent carrier absorption layer is fixed on the supporting layer, adsorbed layer is followed successively by fibrage from the sample end, trichina gold mark antigen M fibrage, the cellulose rete, handle end is an absorbent material layer, on the cellulose rete, print detection trace " | " or " one " with anti-people or anti-certain animal IgG antibody Xi solution, on the cellulose rete, print contrast trace " " or " | " with the monoclonal anti liquid solution of anti-trichina ES antigen.
The supporting layer that does not absorb water can be used the duroplasts film strip, or the cardboard bar that does not absorb water, fibrage useable glass cotton, gold mark antigen M fibrage is the glass wool of ADSORPTION OF GOLD mark ES antigen, gold mark antigen M is the ES antigen of glue gold mark trichina, and the combination that detects trace and contrast trace can be "+", or " ‖ ", or "=", or
Or
, or
, or
In a kind of; select for use "+" or " ‖ " or "=" combination trace better; at fibrage, cover on gold mark M fibrage and the absorbent material layer and be fixed with the plastic cement diaphragm, print the sample mark line at fibrage and the about 0.5cm of gold mark M corresponding inclined to one side fibrage one side of diaphragm of fibrage intersection place.
The antibody Xi of anti-people or anti-certain animal IgG prints and detects trace " | " or " one " and can be a kind of in the following antibody; Anti-human IgG antibody X
1, anti-pig IgG antibody X
2, anti-cat IgG antibody X
3, anti-dog IgG antibody X
4... anti-certain animal IgG antibody Xn, trichina ES antigen comprise polypide secretion ES antigen and express ES antigen.
The present invention has positive beneficial effect.
(1) fast trichinelliasis diagnosing test paper strip (I) and (II), high specificity, susceptibility height.It is that basic reagent preparation forms that two kinds of diagnosis test papers all use with glue gold mark high-affinity monoclonal antibody and reorganization ES antigen, no covalent bond formation between gold grain and antibody or the ES antigen molecule in gold labeling antibody and the antigen, the two combines by the Van der Waals force between the charges of different polarity, glue gold mark is to the specificity and the affinity and very little to the antigenicity influence of ES antigen of monoclonal antibody, and has very high mark rate, make two kinds of fast diagnose test paper bars have high specificity, the characteristic that susceptibility is high.
(2) easy and simple to handle, quick, shorten Diagnostic Time significantly.As long as fast diagnose test paper bar was inserted in the testing sample about 10 seconds, in 2 minutes, can judge diagnostic result.
(3) show the diagnostic result image, directly perceived, accurate, clear.Two kinds of diagnosis test papers are with demonstration rufous and " ‖ " or "-" and "+" feminine gender or the positive mark as diagnostic result, and the result judges very image, and is directly perceived, and accurate, clear, easily note false positive and erroneous judgement can not occur.
(4) need not instrument and equipment, need not other any reagent, need not professional testing staff, small investment, cost is low, will be welcomed by customers.
(5) realize the execute-in-place diagnosis, settle at one go, will be subjected to feed lot deeply, meat packing plant, departments such as customs welcome, and are fit to China's national situation.
(6) apply easily,, need not special training, can say so that everybody can operate, apply easily because diagnostic operation is simple.
(4) description of drawings:
Fig. 1 fast trichinelliasis diagnosing test paper strip (I) and (II) structural representation
Fig. 2 fast trichinelliasis diagnosing test paper strip (I) and (II) one of plan structure synoptic diagram
Fig. 3 fast trichinelliasis diagnosing test paper strip (I) and (II) the plan structure synoptic diagram two
(5) embodiment:
Fast trichinelliasis diagnosing test paper strip (I) and (II) can be widely used in the trichinosis quick diagnosis of humans and animals (pig, cat, dog etc.).For finishing enforcement of the present invention, at first must finish preparation trichina secretion antigen ES, prepare the monoclonal antibody of anti-ES antigen, prepare anti-people, animal IgG antibody such as anti-mouse, anti-pig, anti-cat, anti-dog.Be used to prepare gold mark W1 and M fibrage.
(1) preparation of trichina ES antigen
PCDM8 is a carrier with the expressivity plasmid, has made up pig trichina muscle larvae expressivity cDNA gene library.Behind this gene library rotaring redyeing COS 7 cell, identify the positive expression cell with the SABC method, positive cell is drawn with micro pipette and is obtained, extract material DNA transformed into escherichia coli and quick clone in the preparation cell, using this method, to have obtained two encoding genes of holographic ES antigen (molecular weight is 45kda, 49kda) exactly be TSESI and TSESII.With individual layer COS in 0.25% pancreatin (containing 0.02%EDTA) the vitellophag culture flask
7Cell, with vitellophag 1000r/min, centrifugal 10min abandons supernatant, and precipitation adds DMEM-10 nutrient culture media (containing 10% hyclone) suspension cell again, carries out cell count.With cell suspending liquid rare be 10
5Individual/ml, inoculate 24 porocyte culture plates, every hole inoculation 1ml puts 37 ℃ of 5%CO
2Cultivate 24h. in the training case, cultivating back cell coverage rate is 60%.With DMEM-SF (serum-free DMEM) washing COS
7Cell once, every hole adds 1ml.Express material DNA and liposome with the EMEM dilution, every hole transfectional cell is got Eppendorf 1 arm by following procedure operation, adds DMEM-SF 100 μ l, and library DNA 3 μ l (0.5 μ g/ μ l) mix.Other gets Eppendorf 1 arm and adds successively, EMEM-SF 100 μ l, and liposome 10 μ l mix, and room temperature after placing 40min mixes two Eppendorf pipes, and room temperature is placed 15min, adds 800 μ l DMEM-SF, mixes the back gently and adds COS
7In the cell hole.Put 37 ℃ of 5% CO
2Cultivate 20h. in the incubator, sucking-off DMEM-SF nutrient culture media, with the DMEM-10 washing once, add the every hole 1ml of DMEM-10, after continuing to cultivate 48～60h, collect cell culture supernatant and concentrated,, be used to prepare anti-ES antigen monoclonal antibody and gold mark antigen (M) with nickel chelating resin purification of Recombinant ES antigen.
(2) the anti-ES antigen monoclonal antibody of preparation
With 50mg-100mg/ ES antigen immune BALB/c only is mouse three times, each 15～30 days at interval, for the third time behind the booster immunization 3～4 days with the bloodletting of immune mouse eyeball, draw neck to cause death, in 75% alcohol-pickled 5-10min, aseptic its spleen of getting shreds and through 100 order nylon net filters, the centrifugal 10min of 1000r/min collects splenocyte; With 1 * 10
8Splenocyte and 2-5 * 10
7NS0 plasmacytoma mixing with cells, the centrifugal 10min of 1000r/min, abandon supernatant, cell precipitation slowly adds 40%-59%PEG4000 (pH8.5-9.0) the effect 1min of 0.7-1ml in 37 ℃ of water-baths, slowly add serum-free 1640 nutrient culture media 15ml then, to stop the effect of PEG, 37 ℃ of water-bath 5-10min, 1000r/min is centrifugal, and 10min abandons supernatant, cell precipitation is resuspended in HAT selects in the nutrient culture media, and add 96 well culture plates (100ul-200ul/ hole) and put 37 ℃ of 5%CO
2Cultivate in the incubator.Cultivate after 7-10 days, with the culture supernatant of the antigen coated ELISA Plate of the ES of 5mg-10mg/ml (40 holes/piece) with enzyme linked immunosorbent assay (ELISA) detection hybridoma, picking strong positive cell clone (CD
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 ES antigen-reactive, and not with other irrelevant albumen generation cross reaction, affinity constant reaches 109-10, and light chain subtype is K or goes into that the heavy chain hypotype is IgG
1, IgG
2a, IgG
2bOr IgG
3, at the monoclonal antibody of ES antigenic determinant, be used to prepare golden labeling antibody cotton.
(3) prepare golden labeling antibody (W1 or W2), gold mark antigen M and golden labeling antibody W1 or W2 glass wool or gold mark M glass wool.
Prepare aurosol with the sodium citrate reducing process, promptly in the 50-100ml0.01%-0.05% aqueous solution of chloraurate of boiling, add the 0.5%-2% citric acid three sodium solution of 2-4ml, obtain the glue gold about diameter 15nm.With 0.1mol/L K
2CO
3Impregnation gold pH to 8.5-9.5, with 1: 1000-1: 3000 mark ratio is with anti-ES monoclonal antibody W to be marked
1Or W
2Or ES antigen adds in the pH8.5-9.5 aurosol, behind the mark 10min, add 20%PEG10000 to 0.05%, 4 ℃ of centrifugal 20min of 1500-3000g of final concentration, remove unconjugated gold grain, 4 ℃ of centrifugal 1h of 15000g, abandon supernatant, obtain preliminary purification gold mark protein mixture after, with propylene glucosan S-400 column chromatography, separation and purification gold mark albumen obtains glue gold labelled antibody or antigen.With 1: 100-1: the glue gold labelled antibody or the antigen of 500 dilutions are adsorbed in the processed glass cotton, and 4 ℃ of low-temperature vacuum dryings are prepared into the cotton or gold mark antigen cotton of golden labeling antibody.
(4) animal IgG antibody such as anti-people of preparation and anti-mouse, anti-pig, anti-cat, anti-dog extract human serum IgG albumen with saturated ammonium sulfate, get 1 part of human blood and reset and add 2 parts of PBS (7.2) mixing, add equal-volume saturated ammonium sulfate mixing, put 4 ℃ of refrigerator 2h, 4 ℃ of centrifugal 15min of 12000r/min abandon supernatant; PBS (7.2) dissolution precipitation with twice serum volume, add saturated ammonium sulfate to final concentration 33%, put 4 ℃ of refrigerator 2h, 4 ℃ of centrifugal 15min of 12000r/min, abandon supernatant, with a small amount of PBS (7.2) dissolution precipitation, put 4 ℃ of refrigerators dialyzed overnight in PBS (7.2), change liquid 2-3 time, 4 ℃ of centrifugal 15min of 12000r/min, collect supernatant, measure its protein concentration with ultraviolet spectrophotometer, through subcutaneous and intramuscular injection immune health sheep or rabbit 3-4 time, last is after immune 10 days with the human serum IgG albumen of 50mg-100mg/kg body weight, venous blood collection, measure its serum antibody titer more than 1: 2000 with ELISA, its hyper-immune serum is collected in heart blood sampling or arteria carotis bloodletting, extract sheep or rabbit anti-human igg's antibody (method is identical with the extraction human serum IgG, does not repeat) with saturated ammonium sulfate.This method is applicable to that the preparation of animal IgG antibody such as anti-mouse, anti-pig, anti-cat, anti-dog does not repeat.
(5) reaction principle of fast trichinelliasis diagnosing test paper strip (I) and reaction principle fast trichinelliasis diagnosing test paper strip (I) (II) is, after fast trichinelliasis diagnosing test paper strip (I) sample end inserted detected sample solution, solution to be checked drove trichina ES antigen and the golden golden labeling antibody W that marks in the glass wool by siphon
1Or W
2Together to nitrocellulose filter diffusion, and finally infiltrate (water accepting layer) in the filter paper, in diffusion process ES antigen can with the golden labeling antibody W of anti-ES antigen
1Or W
2Combine, form ES antigen-Jin labeling antibody compound, this compound again with the cellulose rete on anti-ES monoclonal antibody detect trace and combine, generate rufous " " or " | " mark, part not with detect the golden labeling antibody W that trace combines
1Or W
2Combine with the anti-mouse IgG antibody contrast trace on the fiber rete, generate rufous " | " or "-" mark, two kinds of marks make up stack result mutually, form positive show "+" or " ‖ ", otherwise, golden labeling antibody (W
1Or W
2) owing to there is not form ES antigen-Jin labeling antibody compound, can not detect trace with the anti-ES monoclonal antibody on the cellulose rete combines, can only combine with the anti-mouse IgG antibody contrast trace on the cellulose rete, generate single rufous negative marker " | " or "-", if the test strips testing result had not both had positive show tags "+" or " ‖ " on the cellulose membrane, there are not negative show tags " | " or "-" yet, test strips lost efficacy when then showing, and can not be used further to detect.
The reaction principle of fast trichinelliasis diagnosing test paper strip (II) is, after fast trichinelliasis diagnosing test paper strip II sample end inserts sample solution to be checked, solution to be checked drives trichina antibody by siphon and gold mark antigen M spreads to nitrocellulose filter together, and finally infiltrate in the water accepting layer filter paper, trichina antibody can combine with ES gold mark antigen M in diffusion process, form ES gold mark antigen-antibody complex, this compound again can with anti-people or the anti-pig on the cellulose rete, anti-cat, antibody such as anti-dog (Xi) detect trace and combine, generate rufous "-" or " | " mark, part does not combine with anti-ES antigen monoclonal antibody contrast trace on the cellulose rete with the gold mark antigen M that detects the trace combination, generate rufous " | " or "-" mark, two kinds of marks make up stack result mutually, form positive show "+" or " ‖ ", otherwise, gold mark antigen M is not owing to form gold mark ES antigen-antibody complex, can not with anti-people or the anti-pig on the cellulose rete, or anti-cat, or antibody (Xi) such as anti-dog detects trace and combines, can only combine with the anti-ES monoclonal antibody contrast trace on the cellulose rete, generate single rufous negative marker " | " or "-", if test strips testing result, both there be not positive show tags "+" or " ‖ " on the cellulose membrane, there are not negative show tags " | " or "-" yet, show that then test strips lost efficacy, can not continue to use.
(6) preparation of preparation fast trichinelliasis diagnosing test paper strip (I) and testing sample (II) and detection method test sample liquid, fast trichinelliasis diagnosing test paper strip is used to detect blood, meat and meat products trichinzation, gather people or pig, cat, animal anticoagulations such as dog, or with pig, cat, the meat of animals such as dog and the sample of meat products shred, grind, make 1: 2～1: 10 sample suspension to be checked with physiological saline, trichinosis antibody test test strips is used to detect serum antibody, gather people or pig, cat, animal venous blood such as dog prepare serum, with physiological saline sample is done 1: 2,1: 4,1: 8..... doubling dilution, preparation test sample solution.
Fast trichinelliasis diagnosing test paper strip (I) or sample end (II) are inserted sample solution to be checked, and insertion depth does not surpass mark line 9, takes out test strip after about 10 seconds, about 2 minutes of horizontal positioned, observations.
The result judges: if having rufous positive mark "+" or " ‖ " to show that the expression testing result is positive, promptly detect trichina antigen or trichina antibody in institute's sample product on cellulose membrane, represent that detected human or animal suffers from trichinosis.If have rufous negative marker "-" or " | " to show on the cellulose membrane, the expression testing result is negative, does not promptly detect trichina antigen or trichina antibody in institute's sample product, represents that detected human or animal does not suffer from trichinosis.If there is not the rufous mark to show on the cellulose membrane, show that then test strips lost efficacy, stop using, scrap.
Embodiment one: people's fast trichinelliasis diagnosing test paper strip (I), and referring to Fig. 1 and 2 or Fig. 1 and 3.At first prepare trichina ES antigen, prepare trichina gold labeling antibody W by embodiment (2) step by embodiment (1) step
1Or W
2, and corresponding gold mark W
1Or W
2Cellucotton, i.e. gold mark W
1Or W
2Glass wool.Prepare anti-mouse IgG antibody solution by (4) step among the embodiment.
Among the figure, 1 is supporting layer, adopts plastic slice to make, and 2 for glass wool (sample end) is a fibrage, and 3 for being adsorbed with trichina gold labeling antibody W
1Or W
2Glass wool, i.e. gold mark W
1Or W
2Cotton, 4 is the cellulose rete, present embodiment is selected nitrocellulose filter for use, 5 is absorbent material layer, select for use on the plastic slice that the above-mentioned label 2,3,4,5 of filter paper (being handle end) cements on the supporting layer 1 successively, constitute reaction reagent carrier absorption layer, use trichina ES antigen monoclonal antibody solution on cellulose nitrate rete 4, to print diagnosis trace 6 " | " or "-", use anti-mouse IgG antibody solution to print contrast trace 7 "-" or " | " at cellulose nitrate rete 4.Be "+" or " ‖ " after two kinds of trace stacks, at glass wool 2, trichina gold mark W
1Or W
2Be coated with diaphragm 8-1 on cotton 3, the top covering of filter paper water accepting layer 5 is fixed with diaphragm 8-2, and present embodiment adopts light yellow overlay.At glass wool 2 and trichina gold mark W
1Or W
2The right of cotton 3 the about 0.5cm of inclined to one side glass wool 2 one sides of intersection place marking line 9,9 is printed with the arrow points mark line, is printed on the max printed words under the arrow.The about 8cm of test strips total length, wide about 0.3cm.
The preparation testing sample solution carries out venous blood collection 1～2ml to people to be checked, adds an amount of anti-coagulants, makes 1: 5 to 1: 10 testing sample suspension with physiological saline.
Diagnosis detecting method is: with the sample end of people's fast trichinelliasis diagnosing test paper strip (I) is microglass fiber layer 2, insert in the sample solution to be checked, insertion depth does not surpass mark line 9, after about 10 seconds, take out test strips, horizontal positioned, can see the quick diagnosis result about 2 minutes, if on cellulose nitrate rete 4, show rufous "+" or " ‖ " mark, the expression test sample is positive, promptly have trichina ES antigen in the people's that adopts the blood sample, the detected person suffers from trichinosis; If show the rufous mark of "-" or " | " on the cellulose nitrate rete 4, expression institute sample product are negative, and illustrate there is not trichina ES antigen in the detected blood that promptly the detected person does not suffer from trichinosis.If do not show mark on the cellulose nitrate rete 4, represent that promptly this test strips lost efficacy.
Embodiment two: people's fast trichinelliasis diagnosing test paper strip (II).The fast diagnose test paper bar of present embodiment (II), its structure and embodiment one fast diagnose test paper bar (I) are basic identical, and identical structure is repeated description no longer.Different is: 3 are trichina gold mark antigen M cotton, and 6 is detection trace " | " or "-" that anti-human IgG antibody's solution is printed, and 7 is contrast trace "-" or " | " of the monoclonal anti liquid solution printing of the anti-ES antigen of trichina.
Testing sample solution preparation: treat the people and carry out venous blood collection 1～2ml and prepare serum, do 1: 2 with physiological saline, 1: 4,1: 8 ... doubling dilution, make test sample solution.
The step of its detection method, the display result mode, and determination methods is not heavily chatted with embodiment one.
Embodiment three, pigs trichina disease fast diagnose test paper bar (I), and referring to Fig. 1 and 2, or Fig. 1 and 3, the test strips of present embodiment (I) structure is identical with embodiment one test strips (I) not to be repeated.
The testing sample of pig is adopted pig blood, presses the identical disposal methods of human blood, not repeating, is sample to be checked if adopt pork or pork product, earlier pork or pork product is shredded or grind to make 1: 5～1: 10 sample solution, its detection method and result observe with embodiment one, do not repeat.
Embodiment four: pigs trichina disease fast diagnose test paper bar (II), the basic identical embodiment two of the structure of present embodiment test strips (II), have be: the diagnosis trace 6 at cellulose nitrate rete 4 uses anti-pig IgG antibody (X2) solution to print, the preparation of the testing sample solution of pig does not repeat with embodiment three.
Embodiment five: its structure of cat fast trichinelliasis diagnosing test paper strip (I) and detection method, display result are identical with embodiment one, do not repeat.The testing sample preparation of cat does not repeat with the testing sample of pig among the embodiment three.
Embodiment six: cat fast trichinelliasis diagnosing test paper strip (II), with its structure detection method, display result does not repeat with embodiment two.Somewhat differently be: the diagnosis trace 6 on cellulose nitrate rete 4 uses anti-cat IgG antibody (X3) solution to print.The preparation of testing sample does not similarly repeat with embodiment two and three.
Embodiment seven: dog fast trichinelliasis diagnosing test paper strip (I), and its structure detection method, display result with embodiment one, does not repeat.The preparation of its testing sample solution does not repeat with embodiment three.
Embodiment eight: its structure of dog fast trichinelliasis diagnosing test paper strip (II), and detection method, display result does not repeat with embodiment two.Have be: the diagnosis trace on the cellulose nitrate rete 4 adopts anti-dog IgG antibody (X4) to print.The preparation of its testing sample solution is similar with embodiment two and embodiment three, does not repeat.
Other animal fast trichinelliasis diagnosing test paper strip (I) and (II) available similar approach designing for manufacturing go out, here no longer repeat.
Claims (5)
1. a fast trichinelliasis diagnosing test paper strip (I): contain supporting layer, reaction reagent carrier absorption layer, the lamella of supporting layer for not absorbing water, reaction reagent carrier absorption layer is fixed on the supporting layer, it is characterized in that: adsorbed layer is followed successively by fibrage from the sample end, trichina gold labeling antibody W
1Or W
2Fibrage, cellulose rete, handle end are absorbent material layer, use the monoclonal antibody of trichina respectively
Or W
2Solution and anti-mouse IgG antibody solution are printed diagnosis trace " | " or " one " and contrast trace " " or " | " on the cellulose rete.
2. test strips according to claim 1 (I) is characterized in that: supporting layer can be the hard plastic slip, or is the cardboard bar that do not absorb water, fibrage can be glass wool, the cellulose rete can be used nitrocellulose filter, and absorbent material layer can be used thieving paper, trichina gold labeling antibody W
1Or W
2Fibrage, for the monoclonal antibody of the glue gold anti-trichina secretion antigen of mark (ES) is adsorbed on the glass wool, diagnosis trace and the combination of contrast trace can be "+", " ‖ ", "=",
In a kind of, select for use "+" or " ‖ " or "=" combination trace better, at fibrage, gold mark W
1Or W
2Glass wool is coated with the plastic cement diaphragm on the absorbent material layer, at fibrage and gold mark W
1Or W
2The about 0.5cm of corresponding diaphragm deflection fibrage one side of fibrage intersection prints the sample mark line in the place.
3. a fast trichinelliasis diagnosing test paper strip (II): contain supporting layer, reaction reagent carrier absorption layer, the lamella of supporting layer for not absorbing water, reaction reagent carrier absorption layer is fixed on the supporting layer, it is characterized in that: adsorbed layer is followed successively by fibrage from the sample end, trichina gold mark antigen M fibrage, the cellulose rete, handle end is an absorbent material layer, on the cellulose rete, print detection trace " | " or " one " with anti-people or anti-certain animal IgG antibody Xi solution, on the cellulose rete, print contrast trace " " or " | " with the monoclonal anti liquid solution of anti-trichina ES antigen.
4. test strips according to claim 3 (II), it is characterized in that: the supporting layer that does not absorb water can be used the duroplasts film strip, or the cardboard bar that does not absorb water, fibrage useable glass cotton, gold mark M fibrage is the glass wool of ADSORPTION OF GOLD mark ES antigen, gold mark antigen M is the ES antigen of glue gold mark trichina, the combination that detects trace and contrast trace can be "+", or " ‖ ", or "=", or
Or
Or
Or
In a kind of; select for use "+" or " ‖ " or "=" combination trace better; at fibrage; cover on gold mark antigen M fibrage and the absorbent material layer and be fixed with the plastic cement diaphragm, print the sample mark line at fibrage and the about 0.5cm of gold mark antigen M corresponding inclined to one side fibrage one side of diaphragm of fibrage intersection place.
5. according to claim 3 or 4 described test strips (II), it is characterized in that: the antibody Xi of anti-people or anti-certain animal IgG prints and detects trace " | " or " one " and can be a kind of in the following antibody; Anti-human IgG antibody X
1, anti-pig IgG antibody X
2, anti-cat IgG antibody X
3, anti-dog IgG antibody X
4... anti-certain animal IgG antibody Xn, trichina ES antigen comprise polypide secretion ES antigen and express ES antigen.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN 02101927 CN1403813A (en) | 2001-12-15 | 2002-01-14 | Fast trichinelliasis diagnosing test paper strip |
Applications Claiming Priority (3)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN01138337 | 2001-12-15 | ||
| CN01138337.2 | 2001-12-15 | ||
| CN 02101927 CN1403813A (en) | 2001-12-15 | 2002-01-14 | Fast trichinelliasis diagnosing test paper strip |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| CN1403813A true CN1403813A (en) | 2003-03-19 |
Family
ID=25740605
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN 02101927 Pending CN1403813A (en) | 2001-12-15 | 2002-01-14 | Fast trichinelliasis diagnosing test paper strip |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN1403813A (en) |
Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN103314297A (en) * | 2010-09-30 | 2013-09-18 | 积水医疗株式会社 | Test strip for immunochromatography, and process for production thereof |
| CN101339193B (en) * | 2008-08-28 | 2015-06-24 | 河南省农业科学院 | Eperythrozoonosis rapid diagnosis test paper |
| CN110221066A (en) * | 2019-07-05 | 2019-09-10 | 吉林大学 | A kind of trichinosis fluorescence immune chromatography test strip and the preparation method and application thereof |
| CN112526125A (en) * | 2020-11-30 | 2021-03-19 | 吉林大学 | Fluorescence immunochromatography test strip capable of detecting early infection of trichina suis and preparation method thereof |
-
2002
- 2002-01-14 CN CN 02101927 patent/CN1403813A/en active Pending
Cited By (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101339193B (en) * | 2008-08-28 | 2015-06-24 | 河南省农业科学院 | Eperythrozoonosis rapid diagnosis test paper |
| CN103314297A (en) * | 2010-09-30 | 2013-09-18 | 积水医疗株式会社 | Test strip for immunochromatography, and process for production thereof |
| CN110221066A (en) * | 2019-07-05 | 2019-09-10 | 吉林大学 | A kind of trichinosis fluorescence immune chromatography test strip and the preparation method and application thereof |
| CN112526125A (en) * | 2020-11-30 | 2021-03-19 | 吉林大学 | Fluorescence immunochromatography test strip capable of detecting early infection of trichina suis and preparation method thereof |
| WO2022110733A1 (en) * | 2020-11-30 | 2022-06-02 | 吉林大学 | Fluorescent immunochromatographic test strip capable of detecting early infection of swine trichinella spiralis and preparation method therefor |
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