CN101038289A - Test paper for rapid diagnosing livestocks schistosomiasis japonica - Google Patents
Test paper for rapid diagnosing livestocks schistosomiasis japonica Download PDFInfo
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- CN101038289A CN101038289A CN 200610024699 CN200610024699A CN101038289A CN 101038289 A CN101038289 A CN 101038289A CN 200610024699 CN200610024699 CN 200610024699 CN 200610024699 A CN200610024699 A CN 200610024699A CN 101038289 A CN101038289 A CN 101038289A
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Abstract
The present invention discloses a medical test paper strip for a fast diagnosis of schistosomiasis japonicum in domestic animals. Said medical test paper strip in accordance with the present invention adopts SEA antibody and SEA coated nitrocellulose membrane as a quality control line and a detection line and is manufactured using a double antigen sandwich method, wherein the SEA antigen is labelled by colloidal gold. The present invention has a remarkable specificity and a high sensitivity, and is operated conveniently, simply and quickly. The present invention has a low manufacturing cost and then is suitable for a mass production.
Description
Technical field
The present invention relates to biological technical field, be specifically related to a kind of quick diagnosis domestic animal Japanese schistosomiasis immuno-chromatographic test paper strip and preparation method thereof with to anti schistosoma detection of antibodies in domestic animal (animal) body.
Background technology
Snail fever is a kind of parasitic disease of serious harm people, animal health, and its susceptible animal comprises domestic animal and wild animals such as people, ox, sheep, relates to more than 40 kind of mammal.The animal infection blood fluke is not only caused heavy losses to animal husbandry production, what is more important, and ill domestic animal still is the topmost infection sources of human body snail fever, and therefore carrying out that looking into of domestic animal schistosome disease control is the key of control and elimination schistosoma disease.At present the diagnostic method of domestic animal Japanese schistosomiasis mainly is immunological detection methods such as aetology detection such as excrement inspection and IHA, ELISA, Dot-ELISA, and these methods or waste time and energy or need instrument and equipment are not suitable for extensively promoting the use of at the scene.Aspect quick diagnosis human body snail fever, existing relevant patent and bibliographical information, but these technology serve as that the basis is set up with anti-people's antibody of colloid mark or SPA mostly can not be used and the domestic animal quick diagnosis of bos sinicus particularly.Study for a long period of time and facts have proved, on the basis of using existing immunology diagnosis technology, develop a kind of novelty, quick, easy, the amynologic diagnostic method that can diagnose multiple animal snail fever that is suitable for basic unit, to be applicable to on-the-spot extensive examination domestic animal Japanese schistosomiasis.
Summary of the invention
Technical matters to be solved by this invention is to overcome above-mentioned weak point, designs a kind of easy and simple to handle, quick diagnosis domestic animal Japanese schistosomiasis test strips.
The invention provides a kind of quick diagnosis domestic animal Japanese schistosomiasis test strips.
The object of the present invention is achieved like this: be prepared into gold mark pad with colloid gold label Schistosoma japonicum soluble egg antigen (SEA) as probe, respectively SEA antigen and anti-SEA antibody sandwich NC film are developed test strips as detection line and control line, by the anti-SEA antibody in the method test sample of immunochromatography.Specific as follows:
Material therefor of the present invention is as follows: Schistosoma japonicum (Chinese mainland strain): by this laboratory by the support the family history circulation of oncomelania and new zealand white rabbit.Chlorauride, trisodium citrate, sal tartari, accurate pH test paper are Shanghai traditional Chinese medicines group product; BSA, Amresco company packing product; 206 adjuvants are the French Derby gram Montanide ISA of company product; The high-speed low temperature desk centrifuge, Sigma company produces; The low-temperature freeze-dry machine, FTC company product; ZX1000 Membrane jetter, XYZ3200 specking machine, the Biodot product; Cellulose nitrate (NC) film (model: AE98FAST), German Schleicher﹠amp; Schuell company product; All-glass paper, thieving paper, double faced adhesive tape plastic bottom board genome company product.
Schistosoma japonicum soluble egg antigen (SEA) is prepared as: utilize 2000～2500 schistosoma japonicum cercariaes of cover glass method new zealand white rabbit abdominal infection, infect the back and got the rabbit liver in 40-50 days, collect the purifying worm's ovum, multigelation for several times under liquid nitrogen/water bath condition, add an amount of phosphate buffer (PBS),, get supernatant through grinding, Ultrasonic Pulverization, low-temperature centrifugation, desalt the back by CBB-SDS method mensuration concentration through deionized water dialysis, and it is standby to be sub-packed in-20 ℃ of preservations.
The preparation of anti-SEA antibody: with SEA antigenic solution and 206 adjuvants than mixing, with each 1mg dosage leg muscle injecting immune New Zealand white rabbit, after the immunity 3 times, get blood, detect antibody titer, when titre reaches 1: 32 when above with the agar double diffusion test, cut open rabbit extremely, collect and separation of serum.Use the ammonium sulfate salting-out process antibody purification, PBS dialyses repeatedly to there not being SO42-, and the CBB-SDS method is measured packing after the concentration, and-20 ℃ of preservations are standby.
Being prepared as of colloidal gold probe: adopt the sodium citrate reducing process to prepare the collaurum of 15-40nm, collaurum pH is regulated to being worth 7.0～8.0 with the sal tartari of 0.1-0.5M in the cooling back; And get 1mL with Tris[three (methylol) aminomethane]-SEA that the HCl damping fluid is diluted to 60-100ug/mL dropwise adds under magnetic agitation and mixes up in the 10mL colloidal gold solution of pH value, add stabilizing agent after stirring 5-20min, continue to stir 5-20min, 10000-15000rpm, 4 ℃ of centrifugal 5-30min abandon supernatant, with the resuspended precipitation of stabilizing agent, the same method repeats to wash after 3 times resuspended to 2mL, adds 10% NaN3 (final concentration is 0.2%) back in 4 ℃ of preservations.
The top condition of gold mark pad preparation is: with homogenate agent pre-service glass fibre, drying; The SEA-colloidal gold probe is added in the XYZ3200 specking machine is sprayed on the glass fibre with 5-20uL/cm2, low-temperature freeze-dry seals in aluminium foil bag, and 4 ℃～20 ℃ preservations are standby.
The optimum preparating condition of cellulose nitrate reaction film is: with the Tris-HCl damping fluid with SEA, anti-SEA antibody dilution to desired concn, be added on respectively in the ZX1000 Membrane jetter with 1uL/cm spray film bag quilt, 20-40 ℃ of dry 5min, use Tris-HCl damping fluid rinsing 3 times behind the confining liquid sealing 30min, the about 30min of 37 oven dry, the sealing back is standby in 4 ℃～20 ℃ preservations.
Being assembled into of test strip: thieving paper, bag are marked pad, another water adsorption glass fiber etc. by being fixed on the double faced adhesive tape plastic bottom board from top to bottom successively shown in native Fig. 1-3 by good NC film, gold, be cut into the test strips of 25 * 50mm with shearing knife or cutting cutter, ready-made test strips is packed into drying agent is sealed in 4 ℃ of-20 ℃ of preservations in the aluminium foil bag.
Domestic animal Japanese schistosomiasis diagnosis test paper of the present invention, its trace routine is: get the 20uL test serum and be added in (sample end) on the water adsorption glass fiber, insert immediately in the plastics enzyme mark cuvette that damping fluid (the 0.01M Tris-HCl of pH7.4) is housed and (note gold mark pad not being inserted in the damping fluid) observations behind about 10～15min.
Its criterion that detects sample of the present invention is: nature controlling line and detection line all occur red positive, only occur at nature controlling line red negative, two equal redfrees of line be test strips inefficacy (Fig. 4).
Effect of the present invention is: the special instruments and equipment that fast, do not need easy and simple to handle, do not need professional training to operate, can better satisfy the needs in vast basic unit epidemic-stricken area, as the screening of eqpidemic disease monitoring, chemotherapy target drove etc., the value that has vast market prospect and apply on a large scale.
The present invention has following advantage:
(1) high specificity, the susceptibility height, security is good, and the scope of application is extensive.Test strips of the present invention is that label is prepared from the collaurum, does not have the participation of objectionable impuritiess such as biphenyl two ammoniums and radioelement, and its security is good; SEA is a soluble protein antigen, has good antigenicity, with colloid gold label SEA antigen, utilize the principle of double antigens sandwich, can detect multiple animal (comprising the people) anti-schistosome antibody, therefore have very high specificity and susceptibility, its range of application is very extensive.
(2) easy and simple to handle quick.When using test strips of the present invention to diagnose the domestic animal Japanese schistosomiasis, need not to join other reagent in addition, sample need not special processing, can declare diagnostic result by trace routine in 10～15min.
(3) result judges image, accurate, reliable.Test strips of the present invention has inaction to detect judged result with colour developing, and promptly the promptly positive of redness appears in detection line, and colourless is promptly negative, and the result judges image, and background is clear, and accurately and reliably, unmanned is operate miss.
(4) cost is low, small investment.Test strips of the present invention can be produced in batches, and single single part of detection is with low cost, small investment, instant effect.
Description of drawings
Fig. 1 is a Japanese schistosomiasis diagnosis test paper side schematic view, and 1 is the PVC base plate among the figure, and 2 are sample suction pad, and 3 is gold mark pad, and 4 is detection line (T line), and 5 is control line (C line), and 6 is the NC film, and 7 is absorption pad.The absorption of sample pad is an all-glass paper, and absorption pad is a thieving paper.
Fig. 2 is the test strips functional areas, comprises the sample end, results display area and tab end; 1 is the PVC base plate among the figure, and 2 is gold mark pad, and 3 is the absorption of sample pad, and 4 is absorption pad, and 5 is control line (C line), and 6 is detection line (T line), and 7 is the NC film.
Fig. 3 is that the test strips testing result is judged, 1 positive result among the figure, and 2 negative results, 3 are the test strips inefficacy;
Fig. 4 is the testing result of test strips sheep, buffalo, small white mouse, New Zealand white rabbit serum, 1,2 for detecting the sheep positive and negative serum result among the figure, 3,4 for detecting the buffalo positive and negative serum result, 5,6 for detecting the small white mouse positive and negative serum result, and 7,8 for detecting the New Zealand white rabbit positive and negative serum result.
Specific embodiment one
According to technical scheme of the present invention, be prepared as follows:
(1) preparation of Schistosoma japonicum soluble egg antigen (SEA)
With coverslip method in 2000～2500 schistosoma japonicum cercariaes of new zealand white rabbit abdominal infection, killed rabbit in 42 days, get liver and remove bile duct, blood vessel and connective tissue, 1%NaCl solution with 4 ℃ of precoolings is cleaned blood stains, shred the PBS that adds 600mL, after homogenizer homogenate is pulverized, cross 40 orders respectively, 80 orders, 120 orders, 160 orders, 200 order bolters, extracting screen underflow is used 260 order nylon mesh collection ovum again, after sediment diluted with PBS, 3500rpm5min was centrifugal repeatedly, supernatant discarded and upper strata Liver paste, be golden yellow until precipitation, microscopically observe to background clean till, centrifugal 30 seconds again, inhale and remove upper water solution with 12000rpm, lower floor's worm's ovum is the multigelation several under liquid nitrogen/tap water condition, add an amount of PBS, grind, sonicated is 6 times under the condition of ice bath, each 30see, interval 2min, the centrifugal 1hr of 4,40 000g gets supernatant, with deionized water dialysis 2 days, change water every day 3 times.The supernatant of dialysis after desalting is pure SEA antigen, measures its concentration by the CBB-SDS method, and it is standby to be sub-packed in-20 ℃ of preservations.
(2) preparation of anti-SEA antibody
Mixed by weight 1: 1 with 206 adjuvants with SEA antigen, with each 1mg antigen dose, leg muscle injecting immune New Zealand white rabbit, three exempt from back ear vein blood sampling, and two-way agar diffusion method is measured antibody titer, when reaching 1: 32, antibody titer cuts open rabbit blood sampling extremely when above, collect and separation of serum, use the ammonium sulfate salting-out process purifying, in 4 PBS, dialyse till do not have NH4+ or do not have SO42-, packing after the CBB-SDS method mensuration concentration ,-20 ℃ of preservations are standby.
(3) preparation of damping fluid, confining liquid, stabilizing agent, homogenate agent
0.01M phosphate buffer (KH2PO4 0.24g, Na2HPO412H2O 17.9g, NaCl 8g, KCl 0.2g is settled to 1000ml, transfers pH to 7.4); 0.01MTris-HCl damping fluid (1.414gTris is dissolved in the 1000mL deionized water, transfers pH to 7.4 with HCl); Confining liquid (the 3g skimmed milk power is dissolved among the Tris-HCl of 100mL0.01M); Stabilizing agent (seralbumin is dissolved among the 100mLTris-HCl among the 1gBSA); Homogenate agent (3g sucrose is dissolved among the 100mLTris-HCl).
(4) preparation of colloidal gold probe
Adopt the sodium citrate reducing process to prepare the collaurum of 40nm.
The trisodium citrate of getting 500uL1% adds in 0.005% gold chloride boil, treats that solution boils to claret to continue to boil the collaurum that 5min prepares 40nm, and collaurum pH to 7.0～7.5 are regulated with the sal tartari of 0.2M in the cooling back; Under magnetic agitation, in the 10mL colloidal gold solution that mixes up the pH value, dropwise add 1mL is diluted to 80ug/mL with the 0.01MTris-HCl of pH7.4 SEA, add stabilizing agent 1mL after stirring 10min, continue to stir 10min, 12000rpm, 4 ℃ of centrifugal 20min abandon supernatant, the resuspended precipitation of stabilizing agent, the same method is centrifugal, washes repeatedly that to add stabilizing agent after 3 times resuspended to 2mL, adds 10% NaN3 (final concentration is 0.2%) back in 4 ℃ of preservations.
(5) gold mark pad preparation
Glass fibre is soaked a moment, 37 ℃ of dry 30min with the homogenate agent; The SEA-colloidal gold probe is added in the XYZ3200 specking machine is sprayed on the glass fibre with 10uL/cm2, low-temperature freeze-dry seals in the aluminium foil bag of packing into, and 4 ℃～20 ℃ preservations are standby.
(6) the bag quilt of NC film
Selecting the aperture for use is 5um, and climbing speed is the NC film of 120～160sec/4cm.0.01M Tris-HCl solution with pH7.4 is diluted to 1.5mg/mL, anti-SEA antibody dilution to 3.2mg/mL with SEA, be added on respectively in the ZX1000 Membrane jetter with 1uL/cm spray film, 37 ℃ of dry 5min, with the confining liquid sealing 30min that contains 3% skimmed milk power, and waft with the 0.01MTris-HCl eluent and to wash 3 times, 37 ℃ of oven dry 30min seal in the aluminium foil bag that contains drying agent of packing into after the sealing, and 4～20 ℃ of preservations are standby.
(7) assembling of test strips
From top to bottom successively be fixed on double faced adhesive tape plastic bottom board on by good NC film, gold mark pad, another water adsorption glass fiber thieving paper, bag, be cut into the test strips of 25 * 50mm with shearing knife or cutting cutter, ready-made test strips is packed into drying agent is sealed in 4～20 ℃ of preservations in the aluminium foil bag.
(8) test strips detecting operation program
Get the 20uL test serum and be added in test strips sample end, immediately the sample end is inserted in the plastics enzyme mark cuvette that damping fluid (0.01MTris-HCl damping fluid) is housed and (note gold mark pad not being inserted in the damping fluid), observations behind about 10～15min.
(9) criterion as a result
In paper slip being inserted plastics enzyme mark cuvette, observe behind about 10～15min, if the positive of redness all occur at nature controlling line and detection line, what the redness that detection line occurs was darker is strong positive, what redness was more shallow is the weak positive, at nature controlling line the negative of red and detection line redfree appears only, article two, the equal redfree of line is the test strips inefficacy, needs detection again.
Specific embodiment two:
(1) test strips with the present invention's preparation detects for each 5 parts the positive and the negative serum of sheep, buffalo, New Zealand white rabbit, small white mouse respectively, positive serum all red stripes all occurs at C line and T line as a result, negative serum only the red zone (see figure 4) occurs at the C line, shows to utilize the test strips of dual-antigen sandwich method preparation can detect multiple animal Japanese schistosomiasis.
(2) susceptibility of test strips detection and specificity experiment
Detect the positive sheep serum of 107 parts of artificial challenge infections, 80 parts of strong healthy sheep serums respectively with test strips of the present invention, its susceptibility and specificity are respectively 91.6% and 87.5% as a result.
(3) cross reaction test
Detect 24 parts of fascioliasis sheep blood serums and 18 parts of trypanosomiasis cow's serums with test strips of the present invention, the cross reacting rate of result and Fasciola hepatica is 12.5%, and trypanosome no cross reaction.
(4) detect the on-the-spot buffalo serum that infects with test strips
Herd buffalo positive serum and 15 parts of negative serums with the epidemic-stricken area that 20 parts of excrement inspections of test strips detection are made a definite diagnosis, the coincidence rate of its positive serum is 100% as a result, and detecting has two examples positive in 15 parts of negative serums.
(5) replica test
Each 5 parts of 5 sheep positive and negative of test strips duplicate detection serum of making of different batches, the positive, negative match-rate are 100% as a result.
3.6 detect effect and the relation of being with the worm amount
Detecting band worm amount respectively with test strips of the present invention is that each portion of buffalo serum of 2,12,21,32,47,51,63,97,209,765/detects, band worm amount is that 2,12,21,32/buffalo serum is shown as feminine gender as a result, band worm amount is that 47 and 51 serum is shown as the weak positive, and band worm amount is the strong positive that is shown as more than 63.
Claims (3)
1, a kind of diagnosis domestic animal Japanese schistosomiasis test strips, it is characterized in that this test strips be by anti schistosoma soluble egg antigen antibody and Schistosoma japonicum soluble egg antigen bag by nitrocellulose membrane as quality control line and detection line, with colloid gold label Schistosoma japonicum soluble egg antigen, utilize dual-antigen sandwich method to make.
2, a kind of preparation method who diagnoses domestic animal Japanese schistosomiasis test strips according to claim 1, this method comprises the following steps:
(1) preparation of 40nm colloidal gold solution:
Adopt the sodium citrate reducing process to prepare the collaurum of 40nm.
(2) buffer solution system of colloid gold label Schistosoma japonicum soluble egg antigen and labeling process:
After treating the collaurum cooling, regulate pH to being worth 7.0-～8.0 with the sal tartari of 0.1-0.5M; And get Schistosoma japonicum soluble egg antigen that 1mL is diluted to 60-100ug/mL with the Tris-HCl damping fluid and under magnetic agitation, dropwise add and mix up in the 10mL colloidal gold solution of pH value, add stabilizing agent after stirring 5-20min, continue to stir 5-20min, 12000rpm, 4 ℃ of centrifugal 5-30min, abandon supernatant, with the resuspended precipitation of stabilizing agent, the same method repeats to wash after 3 times resuspended to 2mL, adds 10% NaN3, final concentration is 0.2%, and the back is in 4 ℃ of preservations;
(3) preparation of gold mark pad:
Glass fibre is soaked a moment, 20-40 ℃ of dry 30min with the homogenate agent; Schistosoma japonicum or dissolubility egg antigen-colloidal gold probe are sprayed on the glass fibre low-temperature freeze-dry with 5-20uL/cm2;
(4) the bag quilt of NC film:
But with the 0.01-0.1M Tris-HCl solution dilution Schistosoma japonicum of pH7.0-7.5 or property egg antigen, anti schistosoma soluble egg antigen antibody, respectively with 1uL/cm spray film, 20-40 ℃ of drying, with the confining liquid sealing 30min that contains the 1-5% skimmed milk power, with the rinsing of 0.01-0.1M Tris-HCl eluent, 20-40 ℃ of oven dry 30min seals in the aluminium foil bag that contains drying agent of packing into after the sealing, and 4～20 ℃ of preservations are standby;
(5) assembling of test strips
Thieving paper, bag are filled up, are fixed on successively from top to bottom on the double faced adhesive tape plastic bottom board with the water adsorption glass fiber by good nitrocellulose membrane, gold mark, be cut into test strips with shearing knife or cutting cutter.
3, a kind of detection method of diagnosing domestic animal Japanese schistosomiasis test strips to be used to diagnose the domestic animal Japanese schistosomiasis according to claim 1, it is characterized in that this method is, test serum is added in test strips sample end, immediately the sample end is inserted in the plastics enzyme mark cuvette that damping fluid is housed, gold mark pad is not inserted in the damping fluid, observations behind 10～15min: the positive of redness all appears in nature controlling line and detection line, only occur at nature controlling line red negative, two equal redfrees of line be the test strips inefficacy.
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| CN101930003A (en) * | 2010-08-19 | 2010-12-29 | 武汉中博生物股份有限公司 | Anti-rabies virus IgG antibody colloidal gold immunochromatographic assay reagent plate and preparation method |
| CN102944675A (en) * | 2012-11-07 | 2013-02-27 | 浙江省农业科学院 | Preparation of test strip for detection of antibody of bovine schistosoma japonicum katsurada and application method thereof |
| CN103076447A (en) * | 2012-12-27 | 2013-05-01 | 上海市疾病预防控制中心 | Schistosoma egg crude antigen purification method, related purified antigen and schistosoma antibody detection colloidal gold immunoassay kit |
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| CN1342902A (en) * | 2000-09-11 | 2002-04-03 | 同济医科大学 | Test paper for quickly diagnosing Schistosomiasis japonica |
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| CN102944675A (en) * | 2012-11-07 | 2013-02-27 | 浙江省农业科学院 | Preparation of test strip for detection of antibody of bovine schistosoma japonicum katsurada and application method thereof |
| CN103076447A (en) * | 2012-12-27 | 2013-05-01 | 上海市疾病预防控制中心 | Schistosoma egg crude antigen purification method, related purified antigen and schistosoma antibody detection colloidal gold immunoassay kit |
| CN103076447B (en) * | 2012-12-27 | 2015-04-08 | 上海市疾病预防控制中心 | Schistosoma egg crude antigen purification method, related purified antigen and schistosoma antibody detection colloidal gold immunoassay kit |
| CN104730239A (en) * | 2015-03-04 | 2015-06-24 | 中国农业科学院兰州兽医研究所 | Ovinetheileriasis immuno colloidal gold detection test paper strip and preparation method thereof |
| CN107153114A (en) * | 2017-06-28 | 2017-09-12 | 滨州医学院 | A kind of snail fever quick diagnosis reagent kit and preparation method thereof |
| WO2019029083A1 (en) * | 2017-08-10 | 2019-02-14 | 深圳先进技术研究院 | Test strip for detecting chemerin and derived polypeptide thereof, and preparation method therefor |
| CN109406798A (en) * | 2018-10-29 | 2019-03-01 | 美康生物科技股份有限公司 | Creatinine enzyme process detection kit |
| CN110531062A (en) * | 2019-08-26 | 2019-12-03 | 中国农业科学院上海兽医研究所(中国动物卫生与流行病学中心上海分中心) | The preparation of the isolation and purification method and worm's ovum soluble antigen and its colloidal gold immunochromatographimethod card of Schistosoma mansoni worm's ovum |
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