CN102944675A

CN102944675A - Preparation of test strip for detection of antibody of bovine schistosoma japonicum katsurada and application method thereof

Info

Publication number: CN102944675A
Application number: CN2012104429924A
Authority: CN
Inventors: 卢福庄; 张雪娟; 顾昀; 付媛; 季敬余; 冯尚连; 俞国乔; 杨玉焕; 周煜; 阳爱国; 董国栋; 郭莉; 毛光琼; 石团员
Original assignee: Zhejiang Academy of Agricultural Sciences
Current assignee: Zhejiang Academy of Agricultural Sciences
Priority date: 2012-11-07
Filing date: 2012-11-07
Publication date: 2013-02-27

Abstract

The invention discloses preparation of a test strip for detection of antibody of bovine schistosoma japonicum katsurada and an application method thereof, which belong to the technical field of diagnosis of animal parasitic diseases. The test strip consists of a sample gasket, an immune probe dry plate, a chromatographic film, water absorbing paper, a PVC (Polyvinyl Chloride) base plate, and an arrow label coating and a handle label coating which are combined. The immune probe dry plate is prepared by forming a covalent bond conjugate by carboxyl of polystyrene latex beads and amido of rabbit anti-bovine IgG antibody protein and absorbing on a polyester film. The chromatographic film is prepared by using a schistosome soluble antigen anchored on a nitrocellulose membrane as a detection line and goat anti-rabbit IgG antibody as a quality control line. The schistosome antibody in bovine serum can be quickly detected. The method is simple and convenient to operate, and the results are intuitional and accurate. The method can be widely applied to diagnosis, general survey and quarantine of bovine schistosome.

Description

Preparation and the application process thereof of ox Japanese schistosomiasis antibody test test strips

[technical field]

The present invention relates to the diagnostic techniques field of animal parasite disease, be specifically related to preparation and the application process thereof of ox Japanese schistosomiasis antibody test test strips.

[background technology]

Snail fever is the parasitic disease of infecting both domestic animals and human.Current China still has more than 100 county of 7 provinces (autonomous region) not reach the propagation control criterion of snail fever.Ox is the important infection sources of snail fever.Because be subjected to the flow impact of the social factor such as frequent of the natural cause such as the Changjiang river Seasonal flood and people and animals, China's schistosomiasis epidemic has occurred again repeatedly in recent years, ill people and animals increase.China all classifies snail fever as the important object of animal epidemic monitoring and quarantine always.Therefore, to the transit quarantine of the snail fever of allocation and transportation domestic animal, be a long-term and difficult task to the generaI investigation of the snail fever of existing epidemic-stricken area domestic animal and to the monitoring that the livestock of Endemic Areas of Schistosomiasis Japonica once occured in history.Therefore, the method for research domestic animal snail fever fast detecting is significant to anti-system and elimination schistosoma disease.

The method of current diagnosis domestic animal schistosome disease mainly contains excrement and incubates schistosoma miracidium method, ELISA method, IHA method and Dot Immunogold Filtration Assay.Excrement is incubated the miracidium method only could hatch schistosoma miracidium by excrement more than 5 weeks behind the zoogenetic infection blood fluke, and the worm's ovum quantity in the ight soil has fluctuation, the ill domestic animal of low-grade infection is especially easily undetected, and detect a sample with the excrement method of incubating and need 5～6 hours consuming time, along with labour's wage sharp rises, this method seldom is used in the generaI investigation of domestic animal snail fever.ELISA method complex operation, time length, enzyme reagent are difficult to preserve, influence factor is many, and detecting needs some material of use in specialized instrument and equipment, the test easily to cause environmental pollution, healthy harmful to the people.IHA less stable, susceptibility are not as the ELISA method, and the experiment required time is also longer.ELISA method and IHA method all can not satisfy the requirement of on-the-spot quarantine.Dot Immunogold Filtration Assay has fast, the easy and simple to handle characteristics such as specific apparatus that do not need of detection speed, is suitable for scene quarantine, diagnosis and the generaI investigation of snail fever.But its diagnostic reagent is liquid, and less stable is preserved and required high (needing 4 ℃ of Refrigerator stores), storage life short (only 6 months), and reagent and reaction plate volume are large, and mailing and air transportation are limited, have increased the time that preservation, trucking costs and transport point need.The gold-marking immunity chromatography can overcome these shortcomings of Dot Immunogold Filtration Assay.Domestic have several people from units that snail fever gold-marking immunity chromatography was carried out research, but come owing to a variety of causes does not all have to promote in batch production and the practice.

Analyze theoretically, the combination of collaurum and antigen or antibody all is that the electrostatic attraction by positive and negative charge combines.The firm degree of this combination easily is subjected to the impact of pH and other ionic strengths, so its optimal pH scope is often very little, detection repeatable relatively poor.The combination of collaurum and antigen or antibody also may be by the impact of competitive binding in the blood or between other albumen that is referred to as stabilizing agent that adds in the reagent and the collaurum, reagent was comparatively fast lost efficacy or false positive occurs.Can say that this is the limitation that golden mark method itself has.

In theory, make the firm degree of combination between the molecule be better than intensity by the electrostatic attraction combination by covalent bond.Milk-globule immunochromatography technique a kind of immune labeled new technology take covalent bonds as characteristics that newly-developed gets up behind immune colloidal gold technique just.The anti-yellowing buffalo IgG of the rabbit antibody that detection reagent of the present invention is the latex beads mark, between the anti-yellowing buffalo IgG of latex beads and the rabbit antibody with covalent bonds, so this stable composite is good, and large 10～20 times than collaurum of the diameters of latex beads, signal intensity is large, can improve the sensitivity of reagent and the recall rate of positive sample.

[summary of the invention]

The present invention seeks to, have for ox Japanese schistosomiasis ELISA and IHA diagnosis that operation steps is many, speed slow, can not satisfy on-the-spot quarantine demand, detecting reagent with Dot Immunogold Filtration Assay is liquid, storage life is shorter, can not air transport or the defective such as mailing, for diagnosis, quarantine and the generaI investigation of ox Japanese schistosomiasis provides a kind of sensitivity, special, safety, good stability, signal intensity large, and be solid state, do not have the test strip polluted, and be achievable quick in 5～15 minutes, easy detection method.

Principle of the present invention is: after employing organic chemistry method makes the polystyrene latex microballoon of redness or other color carboxylated, make the carboxyl of the amido of the anti-yellowing buffalo IgG of rabbit antibody protein and latex beads form covalent bond, make the bond of the anti-yellowing buffalo IgG of rabbit antibody of this dyeing latex beads mark as detection reagent (immunological probe); Again the anti-yellowing buffalo IgG of this aqueous latex beads-rabbit antibody conjugates is made it to be adsorbed on the polyester film with spray-on process, drying becomes the immunological probe dry plate of solid shape; In addition with nitrocellulose filter as chromatographic film, and draw schistosome ovum soluble antigen and two straight lines of goat anti-rabbit igg antibody with a stroke film gold spraying instrument at the diverse location of this film, respectively as detection line (T line) and nature controlling line (C line); Then according to the order of sample pad, immunological probe dry plate, chromatographic film and absorbent filter, be assemblied on the PVC substrate with partly overlapping form end to end, after sticking again arrow label glued membrane and handle label glued membrane, with cutting cutter by after setting width and cutting ox Japanese schistosomiasis antibody test test strips.

During detection, dilute serum to be checked is dropped on the sample pad of test strips and (maybe exposed sample pad is inserted 10s in the dilute serum to be checked), serum solution is by the capillarity immunological probe dry plate of flowing through, the anti-yellowing buffalo IgG of the latex beads-rabbit antibody conjugates that is adsorbed on the polyester film is dissolved into free shape, if have ox anti-schistosome antibody (being positive ox blood sample) in the blood sample, then the antibody in this blood sample will combine with the anti-yellowing buffalo IgG of the latex beads-rabbit antibody conjugates of the free shape of part, behind three redness of the anti-yellowing buffalo IgG of formation ox anti-schistosome antibody-rabbit antibody-latex beads or the compound of other color, and continue to divide a word with a hyphen at the end of a line forward along nitrocellulose filter, when the in advance anchoring of dividing a word with a hyphen at the end of a line of the compound of this redness or other color the detection line (T line) of schistosome antigen when upper, this schistosome antigen will combine with the ox anti-schistosome antibody in the compound, and forms larger tetrad compound and make the T line manifest the band of redness or other color; And the remaining anti-yellowing buffalo IgG of the free shape latex beads-rabbit antibody conjugates that does not combine with ox anti-schistosome antibody will continue to divide a word with a hyphen at the end of a line forward, when be anchored at goat anti-rabbit igg antibody nature controlling line (C line) on the nitrocellulose filter when meeting, just form three red complex of the anti-yellowing buffalo IgG of goat anti-rabbit igg antibody-rabbit antibody-latex beads, make the C line present red stripes; If there is not ox anti-schistosome antibody (being negative ox blood sample) in the blood sample, then just can not form three compounds of the anti-yellowing buffalo IgG of ox anti-schistosome antibody-rabbit antibody-latex beads, and the anti-yellowing buffalo IgG of simple latex beads-rabbit antibody conjugates is to combine with the schistosome antigen on the T line, so detection line (T line) can not present the band of redness or other color, and only has nature controlling line (C line) still to manifest the band of redness or other color; If red stripes does not all appear in detection line and nature controlling line, then illustrate this test strips lost efficacy or detect the operation wrong.

The object of the invention is achieved by the following technical programs:

1, a kind of test strips that detects ox Japanese schistosomiasis antibody, this test strips is combined by parts sample pad, immunological probe dry plate, chromatographic film, thieving paper, PVC substrate and arrow label glued membrane and handle label glued membrane; Wherein, the immunological probe dry plate is the carboxyl with red or other color carboxylic polystyrene latex beads, forms behind the covalent bonds thing with the amido of the anti-yellowing buffalo IgG of rabbit antibody protein and makes after detection reagent solution as immunological probe is adsorbed in the polyester film; Chromatographic film is take nitrocellulose filter as the film base, schistosome ovum soluble antigen and goat anti-rabbit igg antibody are drawn at this film respectively detects the T line and Quality Control C line forms with drawing the film metal spraying machine; First with overlapping bonding being assemblied on the PVC substrate of above-mentioned front four parts head portion, again with the arrow label glue-film stickup on the immunological probe dry plate, in addition with handle label glue-film stickup after on the thieving paper, with cutting cutter by after setting width and cutting ox Japanese schistosomiasis antibody test test strips.

2, a kind of method for preparing ox Japanese schistosomiasis antibody test test strips, the method is carried out according to the following steps:

(1) preparation of immunological probe dry plate: comprise

1) preparation of the anti-yellowing buffalo IgG of rabbit:

1. the preparation of yellow water ox IgG: gather healthy ox, buffalo blood, getting respectively each 5ml of serum behind the separation of serum mixes, add equivalent 0.85%NaCl, extract yellow water ox IgG with 30% ammonium sulfate precipitation method, the yellow water ox IgG of precipitation dissolves with the 0.85%NaCl of 2 times of amounts of former serum volume, repeat to extract 2 times with 35% ammonium sulfate precipitation method again, at last with the yellow water ox IgG of 5ml 0.85%NaCl dissolving centrifugation, remove NH in the solution with dialysis ₄ ⁺And SO ₄ ^2-After, put into the concentrated method of PEG with distilled water diluting or with bag filter again, regulate protein concentration to 10mg/ml, for subsequent use in-20 ℃ of sealed type storages;

2. yellow water ox IgG immunize rabbit: with yellow water ox IgG and Freund's complete adjuvant by volume the 3:7 ratio make oil emu, press 6mg/ rabbit consumption of yellow water ox IgG, to the healthy male rabbit of body weight 2.5kg, carry out first immunisation at the subcutaneous and rabbit bilateral metapedes Plantar lift hemostasis of rabbit neck part; Except substituting the Freund's complete adjuvant with incomplete Freunds adjuvant, by above-mentioned same proportioning, method, every carrying out booster immunization two weeks one time, carry out altogether three times more afterwards; After the 4th 10 days, get a small amount of blood system of ear vein from serum, measure immune serum with agar diffusion method and tire, when serum titer 〉=1:64, can a large amount of Bian blood of heart, behind the separation of serum, for subsequent use;

3. the preparation of the anti-yellowing buffalo IgG of rabbit antibody: get the anti-yellowing buffalo IgG of rabbit serum 10ml, extract the anti-yellowing buffalo IgG of rabbit antibody with 35% ammonium sulfate precipitation method, with 2 times to the anti-yellowing buffalo IgG of rabbit of the 0.85%NaCl of serum amount dissolution precipitation antibody, after so repeatedly extracting 3 times, the centrifugal sediment of the anti-yellowing buffalo IgG of the rabbit antibody of the 3rd sulphoxylic acid ammonium precipitation with 5ml 0.85%NaCl dissolving, is removed NH in the solution with dialysis ₄ ⁺And SO ₄ ^2-After the protein determination instrument is measured the anti-yellowing buffalo IgG of rabbit antibody protein content, put into the method that PEG concentrates with the borate buffer dilution of pH7.200.1M or with bag filter, protein concentration is adjusted to 4mg/ml after, for subsequent use in-20 ℃ of sealed type storages;

2) washing of latex beads: with the polystyrene latex microballoon liquid of diameter 200nm redness or other color, at 10000rpm, centrifugal 10min under 4 ℃ of conditions, remove supernatant, sediment is reduced to original volume with high purity water, after disperseing the latex beads of precipitation to become unit for uniform suspension with ultrasound wave, use again above-mentioned identical method repeated centrifugation, resuspension 1～2 time;

3) activation of latex beads carboxyl: with pH6.50.1mol MES, 10% suspension with the look latex beads, the EDCHCl of 20mg/mL and the NHS of 20mg/mL, by volume 2hr is mixed, reacted to the ratio of 9:2:1:0.75; Then centrifugal 25min under 4 ℃ of conditions of 11000rpm removes supernatant, and sediment is resuspended with the borate buffer of used pH7.200.1M with 5 times of amounts of look latex volume, impacts with ultrasound wave and is reduced into uniform suspending liquid; With vertical mixed instrument mixing 15min, at 11000rpm, centrifugal 25min removes supernatant under 4 ℃ of conditions, and sediment is resuspended with the borate buffer of used pH7.200.1M with 2.5 times of amounts of look latex volume, impact with ultrasound wave and to be reduced into uniform suspending liquid, be the latex beads of activated carboxylic;

4) the anti-yellowing buffalo IgG of latex beads mark rabbit antibody: add equal-volume in the centrifuge tube that activated carboxylic latex beads suspending liquid is housed and be diluted to protein concentration as the anti-yellowing buffalo IgG of the rabbit of 1～4mg/mL antibody take the borate buffer of pH7.200.1M, on vertical mixed instrument more than the hybrid reaction 2hr; Add the 20%BSA oscillating reactions 1hr of cumulative volume 1/10th amounts, reacted potpourri is removed supernatant with 4 ℃ of centrifugal 25min of 11000rpm again, and sediment is with casein containing protein 0.2%～0.5%, sucrose 5%～10%, NaN ₃The confining liquid original volume that 0.02%～0.05% pH7.20.05M Tris-HCl damping fluid consists of is resuspended, ultrasound wave impacts and is reduced into uniform suspending liquid, at vertical mixed instrument mixing 15min, again with 4 ℃ of centrifugal 25min of 11000rpm, remove supernatant, sediment is resuspended with the same confining liquid that is equivalent to the anti-yellowing buffalo IgG of latex beads suspending liquid and rabbit antibody cumulative volume 75%, ultrasound wave impacts and is reduced into uniform suspending liquid, and the immunological probe that this liquid is redness or other color detects reagent solution;

5) preparation of immunological probe dry plate: above-mentioned immunological probe is detected reagent solution be sprayed on and pass through casein containing protein 0.2%～0.5%, sucrose 5%～10%, NaN with drawing the flow velocity of film metal spraying machine with 2～5 μ l/cm ₃On the polyester film of the pretreated long 300mm of polyester film treating fluid that the pH7.20.05M Tris-HCl damping fluid of 0.02%～0.05%, Tween-200.2%～0.5% consists of * wide 8.5mm, through 37 ℃, the 1hr oven dry is the immunological probe dry plate;

(2) preparation of chromatographic film:

1) preparation of schistosome ovum soluble antigen: by the healthy new zealand rabbit of body weight 2.5～3kg, inoculate 2000 of blood fluke cercarias for every, the experimental infection rabbit was butchered in inoculation and gets liver in rear 45 days, separate and the purifying japonice ovum, grind fragmentation after freeze drying, be made into 3%～5% suspending liquid with 0.85%NaCl solution, 2～8 ℃ were soaked 3, multigelation is 10 times again, the broken 30min of ice-bath ultrasonic ripple, the centrifugal 1hr of 10000r/min gets supernatant, the dress bag filter, with pH7.00.01M PB dialysis 16 hours, changed dislysate 1 time every 4 hours, after dialysis finishes, with the centrifugal 60min of 10000rp, supernatant is the schistosome ovum soluble antigen; After detecting its protein content, be diluted to 1～7mg/mL concentration with pH8.2～8.50.05M TBS, namely can be used for detecting on the chromatographic film drafting of T line;

2) preparation of goat anti-rabbit igg:

1. the preparation of rabbit igg: the blood that gathers healthy new zealand rabbit with the heart blood collection method, separation of serum, get rabbit anteserum 10ml, add again 10ml 0.85%NaCl mixing, extract rabbit igg with 35% ammonium sulfate precipitation method, repeated precipitation is extracted 3 times, after sediment dissolves with 5ml 0.85%NaCl, remove ammonium sulfate with dialysis, and measure the protein content of rabbit igg at the protein determination instrument;

2. the preparation of goat anti-rabbit igg antibody: with Freunds adjuvant the rabbit igg solution preparation is become 1mg/ml, make emulsion with ultrasound wave; Dosage hypodermic injection immune sheep according to every kg body weight 2mg, 2 weeks of every interval, be prepared into again the emulsion of same concentrations with incomplete Freunds adjuvant and rabbit igg, with same dose and method immune sheep, 10d jugular vein blood sampling separation of serum after the 3rd immunity, with goat anti-rabbit igg antibody in the agar immunodiffusion determination serum tire 〉=when 1:32 is above, can gathers in a large number sheep blood and produce serum;

Separate goat anti-rabbit igg antibody and extract thick goat anti-rabbit igg antibody once with 30% ammonium sulfate precipitation method first, extract 2 times with 45% ammonium sulfate precipitation method, centrifugal sediment usefulness 5ml 0.85%NaCl dissolves, and removes NH in the solution with dialysis ₄ ⁺And SO ₄ ^2-, the centrifugal 30min of 3000rpm behind the detection supernatant protein content, is diluted to supernatant with pH8.2～8.50.05M TBS the concentration of 0.1～2mg/mL, namely can be used for the drafting of Quality Control C line on the chromatographic film;

3) making of detection line and nature controlling line: nitrocellulose filter that will long 25mm * wide 300mm is placed on the operating platform of drawing the film metal spraying machine, in the reagent bottle of this machine 1# pump, add the schistosome ovum soluble antigen, add goat anti-rabbit igg in the reagent bottle of 2# pump, flow velocity with 1～3 μ l/cm, mark detection T line and Quality Control C line at nitrocellulose filter, two lines are put indoor drying and are chromatographic film at a distance of 5～6mm;

(3) preparation of sample pad: with 23mm * 300mm glass fibre membrane by casein containing protein 0.01%～0.05%, sucrose 5%～10%, NaN ₃0.02%～0.05% and the pH7.20.04MTris-HCl damping fluid of the Tween-200.2%～0.5% sample pad treating fluid that consists of in soak 1hr, take out 37 ℃ of oven dry;

(4) preparation of thieving paper: CH37 thieving paper is cut into the paper slip of long 300mm * wide 28mm, put 37 ℃ the oven dry;

(5) preparation of PVC substrate: its structure of PVC plate is three layers, and ground floor is protection sheet, and the second layer is adhesive sticker, and the 3rd layer is the PVC substrate; Be carved with three indentations at protection sheet, lay respectively at 20mm, 27mm and the 53mm place on the long limit of distance one side; With the PVC plate by for subsequent use after long 300mm * wide 80mm specification cutting;

(6) preparation of label glued membrane: form with the printing of adhesive sticker paster; Wherein,

1) arrow label glued membrane: its specification is long 300mm * wide 18mm, being printed on straight line apart from 1mm place, the long limit of a side, above this straight line, is printed on " ← MAX " printed words every 1.3mm, and arrow is vertical with straight line; The depth capacity that Indicator Paper bar sample pad can immerse solution to be measured must not surpass this straight line;

2) handle label glued membrane: its specification is long 303mm * wide 30mm, wrongly written or mispronounced character of the green end, be 15 ° of angles with long limit and be printed on 4 row " SjAb " printed words, SjAb is the abbreviation of Schistosoma japonicum Antibody, represent this test strips for detection of antibody of Schistosoma japonicum, this position is the handle part of test strips when taking;

(7) assembling of each parts of test strips, slitting, packing and storage:

1) assembling of each parts of test strips: test strips is take the PVC of long 300mm * wide 80mm as substrate, successively with the sample pad of 300mm * 23mm, the immunological probe dry plate of 300mm * 8.5mm, the chromatographic film of 300mm * 25mm and the thieving paper of 300mm * 28mm, by the overlapping connection that 1～2mm is respectively arranged end to end, stick on the PVC substrate with the adhesive sticker face; Paste upward arrow label glued membrane at afterbody and the immunological probe dry plate of adsorptive pads again; Paste upper handle label glued membrane in sample pad in addition; Each parts flattens, firm pasting the test strips kilocalorie;

2) slitting: the test strips kilocalorie is placed on the cutting cutter, cuts continuously by the 3mm width, the test strips of wide 3mm * long 80mm;

3) packaging and storage: after test strips sampling observation is qualified, pack in the aluminium foil bag by packing specification requirements, put into 1 parcel drying agent after, seal, product; 2～8 ℃ of preservations, the term of validity is 12 months.

3, use the method for ELISA test strip ox Japanese schistosomiasis antibody, the method is carried out according to the following steps:

(1) preparation of serum dilution: with Na ₂HPO ₄, KH ₂PO ₄, NaCl and distilled water volume 0.199g: 0.082g: 1g by weight: the 200ml ratio is mixed, after 37～40 ℃ of heating water baths dissolvings, the cooling serum dilution;

(2) dilution of cow's serum to be checked: with cow's serum to be checked and serum dilution 1:1～2 ratio mixed dilutings by volume;

(3) detect operation: the serum 0.05ml to be checked that draws dilution drops on the sample pad of the test strips that keeps flat, or after the sample pad end of test strips inserted in the dilute serum to be checked the vertical element 15s that prints to the arrow label glued membrane, lies on the operating table surface;

(4) result judges: observations behind 5～15min, detect the homochromy band that T line and Quality Control C line all present redness or other color, and then be judged to the positive; Only Quality Control C line presents the band of 1 redness or other color, then is judged to feminine gender; Detect T line and Quality Control C line and all do not present band, illustrate that then this test strips had lost efficacy or test operation is wrong.

The invention has the beneficial effects as follows:

(1) need not complex instrument and special knowledge technical ability with ELISA test strip bos sinicus of the present invention, easy and simple to handle, quick, 5～15min just can obtain a result.

(2) test strips volume of the present invention is little, lightweight, all solid state, can conveniently post or air consignment.

(3) the test strips term of validity can reach more than 1 year, detected the long shelf-life of reagent than immune-gold labeled percolation.

(4) detection specificity of test strips of the present invention and IHA are suitable with the ELISA method, and good reproducibility is suitable for batch production production.

(5) the present invention's Huang, buffalo IgG immunize rabbit are with the anti-yellowing buffalo IgG of preparation rabbit antibody, compare with the method for simple use ox IgG immunize rabbit, 1. the anti-yellowing buffalo IgG of the rabbit antibody titer high 4 times (1:64vs 1:16) that produces, the anti-yellowing buffalo IgG of rabbit antibody titer is high can to reduce consumption relatively, reduces cost; 2. use the anti-yellowing buffalo IgG of rabbit antibody yellow, the preparation of buffalo IgG immunize rabbit can improve the susceptibility that the positive buffalo Serum Antibody of blood fluke detects.2 times of dilution water cow's serums of 49 days of ELISA test strip experimental infection blood fluke cercaria with the preparation of the anti-yellowing ox IgG of rabbit antibody, present positive reaction, detect 3 times of dilute serums and only present weak positive reaction (+), but with the same 3 times of dilute serums of ELISA test strip of the anti-yellowing buffalo IgG of rabbit antibody preparation, but present strong positive reaction (+++).Have more advantage so prepare the immunological probe reagent solution with the anti-yellowing buffalo IgG of rabbit of the present invention antibody than using the anti-yellowing ox IgG of common rabbit antibody.

[description of drawings]

The label structural representation of test strips before the glued membrane of Fig. 1

The label structural representation of test strips behind the glued membrane of Fig. 2

Wherein, 1 sample pad, 2 immunological probe dry plates, 3 chromatographic film, 4 thieving papers, 5PVC substrate, 6 detect T line, 7 Quality Control C lines, 8 arrow label glued membranes, 9 handle label glued membranes

The present invention with Fig. 2 as Figure of abstract.

[embodiment]

By following examples and test example and the present invention is described in further detail by reference to the accompanying drawings, but content of the present invention is not limited to this.

Embodiment 1:(detects the preparation 1 of ox Japanese schistosomiasis antibody test strips)

The preparation of 1 test strips

1.1 immunological probe dry plate preparation

1.1.1 the preparation of the anti-yellowing buffalo IgG of rabbit

⑴ the extraction of yellow water ox IgG

1. sterile working gathers healthy ox and buffalo blood, respectively separation of serum.

2. getting each 5ml of ox and buffalo serum mixes, add again 10ml 0.85%NaCl mixing, dropwise add saturated ammonium sulfate (SAS) 8.6ml and make it to reach 30% saturation degree, the limit edged stirs, and regulates pH value to 7.0 with strong aqua, puts 4 ℃ and spends the night, take out equilibrium at room temperature 1hr, with the centrifugal 30min of 3000r/min, abandon supernatant, sediment adds 20ml 0.85%NaCl dissolving.Dropwise add SAS 10.8ml again and make it to reach 35% saturation degree, the limit edged stirs, and regulates pH value to 7.0 with strong aqua, takes out after putting 4 ℃ of 3hr, and equilibrium at room temperature 1hr with the centrifugal 30min of 3000r/min, abandons supernatant, and sediment adds 20ml 0.85%NaCl and dissolves.Repeat again to extract once with 35% saturation degree ammonium sulfate.After the centrifuged deposit thing dissolves with 5ml 0.85%NaCl, sealing in the bag filter dialysis of the diameter 2cm that packs into.

3. dialyse under 4 ℃ of conditions, beginning was used first distill water dialysis 8 hours, changed liquid 4 times, used 0.01mol pH7.0 phosphate buffer (PB) dialysis 6 hours again, changed liquid 2 times, to remove NH ₄ ⁺And SO ₄ ²Yellow water ox IgG after the dialysis can reach the purity requirement of immunize rabbit.

4. recording yellow water ox IgG protein concentration with ultraviolet spectrophotometry is 5.3mg/ml, the concentrated 10mg/ml that is adjusted into of PEG.

⑵ yellow water ox IgG immunize rabbit

1. experimental animal selects 2 of the healthy male rabbits of body weight 2.5kg.

2. the emulsification head of the yellow water ox IgG oil emu of exempting from is mixed in the 3:7 ratio with Freund's complete adjuvant by yellow water ox IgG, and ultrasound wave impacts and makes it emulsification, until yellow water ox IgG oil emu is dripped in the cold water surface, can keep complete the long period and does not disperse, and can use.The oil emu that 2-4 exempts from is made in the 3:7 ratio by yellow water ox IgG and incomplete Freunds adjuvant.

3. respectively inject yellow water ox Ig emulsion 4ml (Tot Prot of yellow water ox IgG is 12mg) under immune first immunisation, the rabbit bilateral metapedes Plantar lift subcutaneous in the rabbit incidence, after two weeks, use weekly yellow water ox IgG booster immunization 1 time, the Tot Prot of immune yellow water ox IgG is 12mg.As follows repeat 3 times, last immunity is after 10 days, and the ear vein blood that takes a morsel is isolated serum, measures tiring of immune serum with agar diffusion method, treats that serum titer reaches 1:64 or heart blood sampling when above, separation of serum.

⑶ the extraction of the anti-yellowing buffalo IgG of rabbit antibody

1. get the anti-yellowing buffalo IgG of rabbit serum 10ml, the 0.85%NaCl mixing that adds equivalent, dropwise add SAS 10.8ml and make it to reach 35% saturation degree, the limit edged stirs, and regulates pH value to 7.0 with strong aqua, puts 4 ℃ of rear taking-ups of spending the night, equilibrium at room temperature 1 hour, with the centrifugal 30min of 3000rpm, abandon supernatant, sediment adds 20ml 0.85%NaCl dissolving.Take out after putting 4 ℃ of 3hr, equilibrium at room temperature 1hr, dropwise add SAS10.8ml and make it to reach 35% saturation degree, the limit edged stirs, and regulates pH value to 7.0 with strong aqua, takes out after putting 4 ℃ of 3hr, equilibrium at room temperature 1 hour, repeat again to extract once with 35% saturation degree ammonium sulfate, after the centrifuged deposit thing dissolves with 5ml 0.85%NaCl, in the bag filter of the diameter 2cm that packs into.

⑷ dialysis is purified to dialyse and is carried out under 4 ℃, and beginning was used first distill water dialysis 8 hours, changed liquid 4 times, uses 0.01mol pH7.0 phosphate buffer (PB) dialysis 6 hours again, changes liquid 2 times, to remove NH ₄ ⁺And SO ₄ ^2-

⑸ determining the protein quantity is 5.0mg/ml with the protein concentration that ultraviolet spectrophotometry records the anti-yellowing buffalo IgG of rabbit, is diluted to the pH7.20.1M borate buffer to contain albumen 4mg/ml, and-20 ℃ of storages are for subsequent use.

1.1.2 reagent preparation

⑴ 1N NaOH takes by weighing NaOH 2.000g, and distilled water is settled to 50ml.

⑵ pH6.50.1mol MES takes by weighing MES 0.4881g, and distilled water is settled to 25ml, drips 1N NaOH and makes pH value of solution reach 6.5.

⑶ 20mg/ml NHS takes by weighing NHS 0.050g, adding distil water 2.5ml dissolving.

⑷ 20mg/ml EDCHCl takes by weighing EDCHCl 0.050g, adding distil water 2.5ml dissolving

⑸ 0.1M BAS takes by weighing boric acid 0.6183g, is settled to 100ml with distilled water.

⑹ 0.025M borax soln takes by weighing borax 0.4767g, is settled to 50ml with distilled water.

⑺ pH7.20.1M borate buffer is got 0.1mol BAS 95ml and is added 0.025mol borax soln 5ml mixing.

⑻ 0.2M Tris takes by weighing Tris 12.114g, is settled to 500ml with distilled water.

⑼ 0.1N HCl draws 12N concentrated hydrochloric acid 4.167ml, puts into volumetric flask, is settled to 500ml with distilled water

⑽ 0.05M Tris-HCl damping fluid

1. pH7.20.05M Tris-HCl damping fluid is got 0.2M Tris 25ml and is added 0.1N HCl 45ml and mix, and adding distil water is to 100ml again.

2. pH8.20.05M Tris-HCl damping fluid is got 0.2M Tris 25ml and is added 0.1N HCl 22.5ml and mix, and adding distil water is to 100ml again.

⑾ pH7.2 confining liquid takes by weighing casein 0.500g, sucrose 5.00g, NaN ₃0.020g all put into 100ml pH7.20.05M Tris-HCl damping fluid, 37～40 ℃ of heating water baths are to fully dissolving.Casein containing protein 0.5%, sucrose 5%, NaN ₃The confining liquid that 0.02% pH7.20.05M Tris-HCl damping fluid consists of.

⑿ the anti-yellowing buffalo IgG of the 2.0mg/ml rabbit antibody absorption anti-yellowing buffalo IgG of rabbit (5mg/ml) 8ml joins in the 12ml pH7.20.05mol borate buffer and mixes.

⒀ 20%BSA solution takes by weighing import BSA 2.000g, uses the 10ml dissolved in distilled water.

1.1.3 the preparation of the anti-yellowing buffalo IgG of latex beads mark rabbit bond

⑴ the washing red latex microballoon of latex beads, diameter 300nm, at 10000rpm, centrifugal 10min under 4 ℃ of conditions, remove supernatant, precipitation is reduced to original volume with an amount of high purity water, disperses the latex beads of precipitation with ultrasound wave, make it to become uniform suspending liquid, use again above-mentioned identical parameter repeated washing (being centrifugal and resuspension) 1～2 time.

⑵ get 24 of 1.5ml plastic centrifuge tubes, adds 0.9ml pH6.50.1molMES in every centrifuge tube, and 0.2ml red latex microballoon mixes.Add again 0.1ml EDCHCl (20mg/ml) and 0.075mlNHS (20mg/ml) mixes.

⑶ mix 2hr in vertical mixed instrument vibration.

⑷ 10000rpm, 4 ℃, centrifugal 10min removes supernatant, and the precipitation of every centrifuge tube is resuspended with 1ml pH7.20.1M borate buffer, and ultrasound wave (2s * 5 time) is homogeneous.Mix 15min in vertical mixed instrument vibration.

⑸ repeat the operation of ⑷, and the precipitation that is every centrifuge tube is resuspended with the 0.5ml borate buffer.

⑹ add the anti-yellowing buffalo IgG0.5ml of rabbit of protein content 1mg/ml in every centrifuge tube, mixes 2hr in vertical mixed instrument vibration.

⑺ add 20%BSA 0.1ml in every centrifuge tube, mix 1hr in vertical mixed instrument vibration.

⑻ 11000rpm, 4 ℃, centrifugal 25min removes supernatant, and the precipitation of every centrifuge tube is resuspended with the 1ml confining liquid, and ultrasound wave (2s * 5 time) is homogeneous.Mix 15min in vertical mixed instrument vibration.

⑼ repeat the operation of ⑺, and the precipitation that is every centrifuge tube is resuspended with 0.75ml pH7.2 confining liquid.Ultrasound wave (2s * 5 time) is homogeneous.The opaque red suspension that obtains at last is the anti-yellowing buffalo IgG of the rabbit bond of latex beads mark, and namely immunological probe detects reagent solution.

1.1.4 the preparation of immunological probe dry plate

1.1.4.1 the preparation 0.2M Tris 125ml of polyester film treating fluid, 0.1N HCl 225ml, casein 2.5g, sucrose 25g, NaN ₃0.1g mix, 37 ℃ of water-baths dissolvings add Tween-20 1.0ml again and stir, adding distil water mixes to 500ml, casein containing protein 0.5%, sucrose 5%, NaN ₃0.02%, the polyester film treating fluid that the pH7.20.05M Tris-HCl damping fluid of Tween-20 0.2% consists of.

1.1.4.2 the pre-service of polyester film is cut into polyester film little of 300mm * 17mm, puts into the polyester film treating fluid and soaks 4hr, takes out to lie in the enamel basin, 37 ℃ of oven dry in the sealing polybag of packing into, are put 4～8 ℃ of refrigerator and cooled Tibetan and are saved backup.

1.1.4.3 the immunological probe dry plate is made of drawing the film metal spraying machine flow velocity of the anti-yellowing buffalo IgG of latex beads mark rabbit bond with 5 μ l/cm is sprayed on the polyester film of having processed, every polyester film (300mm * 17mm) take longitudinal centre line as axle, spray symmetrically 2 the anti-yellowing buffalo IgG of latex beads mark rabbit bond bands, 37 ℃ of oven dry, be cut into little of 300mm * 8.5mm, then become the immunological probe dry plate.The immunological probe dry plate is sealed in the polybag, puts 4～8 ℃ of refrigerator and cooled and hide preservation, for subsequent use.

1.2 the preparation of chromatographic film

1.2.1 the preparation of schistosome antigen

1.2.1.1 the preparation of japonice ovum

1.2.1.1.1 blood fluke cercaria inoculation rabbit will be kept at 4 ℃ of Schistosoma japonicum Snails in the refrigerator, with 200 of front taking-ups, put 25 ℃ of room temperature 1～2hr after, being placed on escapes in the 100ml beaker that dechlorination water is housed puts cercaria.The rabbit belly is shaved hair, makes skin exposed.With oese carefully at liquid level picking liquid, place on the microslide, after microscopically is to the cercaria counting, will have the microslide of cercaria to be affixed on rabbit (the healthy new zealand rabbit of body weight 2.5～3kg) skin of abdomen exposure place 5～10min, finish infection, every rabbit infects approximately 2000 of cercarias.1.2.1.1.2 will infecting the rabbit of 45d, the separation of japonice ovum takes out liver, remove gall-bladder, bile duct, blood vessel and connective tissue, 1%NaCl with 4 ℃ of precoolings cleans blood stains, liver is cut into fragment, add 1%NaCl, pour in the bruiser in the lump and smash fast (8000rpm) process of smashing to pieces to pieces and divide 4 times, carry out at the interval, smash 2min at every turn, stop 2min.The liver of smashing to pieces adds the 1%NaCl dilution, filters with 60,80,100 and 120 purpose standard sieves.Filtrate is used 260 order nylon net filters again.With worm's ovum crude extract 1%NaCl wash-out on the nylon wire.

Eluent is poured in the sterilization centrifuge tube of 50ml, with the centrifugal 1min of 3500rpm, discarded, the middle level, the golden yellow worm's ovum of lower floor is repeatedly with the 1%NaCl washing, and is centrifugal, until can't see till the dun hepatic tissue.The worm's ovum that obtains is used the overlapping filtration of nylon-tissue bag (140,260 order) again, remove residual hepatic tissue cell, thing on the 260 hole nylon-tissue bag is removed white brown floccus repeatedly through centrifugation, namely gets pure schistosome ovum.Pure worm's ovum is sub-packed in the 1.5ml Eppendorf pipe, and the centrifugal 30s of 12000r/min is with the moisture in the further removal worm's ovum.

1.2.1.1.2 the drying of japonice ovum and preservation are used the freeze drier freeze-drying with in the worm's ovum horizontalization blood.-20 ℃ of preservations are put in sealing.

1.2.1.2 the extraction of schistosome antigen

⑴ get japonice ovum 1g and grind 30min with glass homogenizer first, add again a small amount of 0.85%NaCl and grind 30min, then be made into 3% suspension, put 4 ℃ of refrigerators and soak 3d,-20 ℃ of multigelations 10 times, ice-bath ultrasonic cracking 30min (400W), the centrifugal 1hr of 13000rpm, get supernatant the blood fluke soluble antigen 1 of not dialysing.-20 ℃ of preservations perhaps directly are for further processing.

⑵ resuspended with 5ml 0.85%NaCl with ⑴ step centrifugal sediment, and ice-bath ultrasonic cracking 10min (400W) acts on 15min on the vertical mixed instrument, and the centrifugal 1hr of 10000rpm gets supernatant and is the blood fluke soluble antigen 2 of not dialysing.

⑶ get japonice ovum 1g and grind 30min with glass homogenizer first, add again a small amount of 0.85%NaCl and grind 30min, then be made into 3% suspension, put 4 ℃ of refrigerators and soak 3d,-20 ℃ of multigelations 10 times, ice-bath ultrasonic cracking 30min (400W), the centrifugal 1hr of 10000r/min, get supernatant the blood fluke soluble antigen 3 of not dialysing.

1.2.1.3 the dialysis of schistosome antigen

Get schistosome antigen an amount of, the bag filter of packing into pH7.00.01M PB dialysis 16hr, changes dislysate 1 time every 4hr, and after dialysis finished, with the centrifugal 60min of 10000r/min, supernatant was the schistosome antigen of dialysing.After surveying protein content, or quantitative separating by volume, put-20 ℃ of preservations, or press the protein content quantitative separating, freeze-drying ,-20 ℃ save backup, or direct preparation production antigen.

1.2.1.4 antigen protein assay (measuring principle of protein determination instrument is identical, has realized sequencing and traceization mensuration)

Get at random 3 bottles of the schistosome antigens of having dialysed, as dilution and blank, measure absorption value (A in ultraviolet spectrophotometer with pH7.00.01molPB ₂₈₀And A ₂₆₀).By formula: protein content (mg/ml)=(1.45 * A ₂₈₀-0.74 * A ₂₆₀) * extension rate calculates the protein content of every 1ml antigen, averages.

1.2.1.5 produce the preparation with schistosome antigen

1.2.1.5.1 the preparation of antigenic dilution

⑴ 0.2mol Tris solution preparation takes by weighing Tris 2.4228g, and distilled water is settled to 100ml, is 0.2M Tris solution.

⑵ 0.1N HCl solution preparation is drawn and is analyzed pure concentrated hydrochloric acid 4.167ml, and distilled water is settled to 500ml.

⑶ pH8.20.05M TBS damping fluid preparation 0.2M Tris solution 25ml and 0.1N HCl 22.5ml mix, add NaCl 0.85g, and dissolving adds to 100ml with distilled water again.

1.2.1.5.2 produce the preparation with schistosome antigen

According to the content of antigen protein after the dialysis treatment, the antigen diluent that will dialyse with pH8.20.05M TBS damping fluid becomes to contain the production antigen of albumen 1mg/ml.

1.2.2 the preparation of goat anti-rabbit igg

1.2.2.1 the preparation of rabbit igg

⑴ the preparation of rabbit anteserum gathers the blood of healthy New Zealand rabbit, separation of serum by the sterile working requirement with the heart blood collection method.

⑵ rabbit anteserum 10ml is got in the extraction of rabbit igg, add again 10ml 0.85%NaCl mixing, dropwise add SAS 10.8ml and make it to reach 35% saturation degree, the limit edged stirs, and regulates pH value to 7.0 with strong aqua, puts 4 ℃ of rear taking-ups of spending the night, equilibrium at room temperature 1 hour, with the centrifugal 30min of 3000rpm, abandon supernatant, sediment adds 20ml 0.85%NaCl dissolving.Precipitate 2 times with method with 35% saturation degree ammonium sulfate again.Just 4 ℃ of time of repose change 3hr into.After the 3rd time the centrifuged deposit thing dissolves with 5ml 0.85%NaCl, in the bag filter dialysis of the diameter 2cm that packs into.

⑶ dialysis is purified to dialyse and is carried out under 4 ℃ of conditions, and beginning was used first distill water dialysis 8 hours, changed liquid 4 times, uses pH7.00.01M phosphate buffer (PB) dialysis 6 hours again, changes liquid 2 times, to remove NH ₄ ⁺And SO ₄ ^2-

⑷ it is 7.7mg/ml that determination of protein concentration records the rabbit igg protein concentration with the protein determination instrument, is adjusted into 10mg/ml after concentrating with PEG.-20 ℃ save backup.

1.2.2.2 rabbit igg immune sheep

⑴ animal used as test selects 2 of healthy male boer goats, and body weight 20kg/ only.

⑵ the emulsification head of rabbit igg exempt from the rabbit igg oil emu by rabbit igg and Freund's complete adjuvant in the emulsification of 3:7 ratio, the ice-bath ultrasonic ripple impacts to the rabbit igg oil emu being dripped in the cold water surface, the long period keeps complete and does not disperse, and can use.The IgG oil emu that 2-4 exempts from is prepared in the 3:7 ratio by rabbit igg and incomplete Freunds adjuvant.

⑶ immunity first immunisation is subcutaneous at the sheepshead neck, respectively inject rabbit igg oil emu 6.4ml (protein content of rabbit igg is 19.2mg) under the sheep bilateral metapedes Plantar lift, after two weeks, per 2 weeks are used rabbit igg booster immunization 1 time, and the Tot Prot of immune rabbit igg is 19.2mg.So repeat 3 times, the last immunity is after 8 days, and the ear vein blood that takes a morsel is isolated serum, measures tiring of immune serum with agar diffusion method, can Bian blood when treating that serum titer reaches 1:32, and separation of serum.Be the serum that contains goat anti-rabbit igg.

1.2.2.3 the extraction of goat anti-rabbit igg

⑴ goat anti-rabbit igg is got goat anti-rabbit igg serum 10ml, add again 10ml 0.85%NaCl mixing, dropwise add SAS 8.6ml and make it to reach 30% saturation degree, the limit edged stirs, and regulates pH value to 7.0 with strong aqua, puts 4 ℃ of rear taking-ups of spending the night, equilibrium at room temperature 1 hour, with the centrifugal 30min of 3000rpm, abandon supernatant, sediment adds 20ml 0.85%NaCl dissolving.Dropwise add SAS16.4ml again and make it to reach 45% saturation degree, the limit edged stirs, and regulates pH value to 7.0 with strong aqua, takes out after putting 4 ℃ of 3hr, and equilibrium at room temperature 1hr with the centrifugal 30min of 3000rpm, abandons supernatant, and sediment adds 20ml 0.85%NaCl and dissolves.Extract once with 45% saturation degree ammonium sulfate again, after the centrifuged deposit thing dissolves with 5ml 0.85%NaCl, in the bag filter dialysis of the diameter 2cm that packs into.

⑵ dialysis is purified to dialyse and is carried out under 4 ℃ of conditions, and distill water dialysis 8hr is used first in beginning, changes water 4 times, uses pH7.00.01M phosphate buffer (PB) dialysis 6hr again, changes liquid 2 times.Dislysate 3000rpm is centrifugal, and 30min gets supernatant.Be goat anti-rabbit igg antibody solution.

⑶ determination of protein concentration is 10mg/ml with the protein concentration that ultraviolet spectrophotometry records goat anti-rabbit igg antibody, and-20 ℃ of storages are for subsequent use.

1.2.2.4 produce the preparation with goat anti-rabbit igg

⑴ the preparation goat anti-rabbit igg dilution of goat anti-rabbit igg dilution is identical with antigenic dilution, and compound method is seen 1.2.1.5.1.

⑵ produce the preparation with goat anti-rabbit igg

0.5mg/ml the goat anti-rabbit igg 0.5ml of the preparation protein content 10mg/ml of goat anti-rabbit igg mixes with pH8.20.05M TBS damping fluid 9.5ml.

1.2.3 the cutting of nitrocellulose filter and paster

The nitrocellulose filter of selecting is the YN120BLF film of her energy film industry company limited; first film is cut into the segment of 300mm * 25mm; get PVC base plate (300mm * 80mm); protection sheet in the middle of throwing off; expose non-drying glue layer, have the one side of plastic foil backing to stick on the PVC base plate nitrocellulose filter.

1.2.4 mark T line and C line at nitrocellulose filter

Use stroke film metal spraying machine with the flow velocity of 3 μ l/cm the schistosome antigen after the dilution and goat anti-rabbit igg antibody, in the middle of the nitrocellulose filter that sticks on the PVC base plate, draw and detect T line and Quality Control C line, wherein schistosome antigen is drawn in close immunological probe dry plate one side of pasting in the future, goat anti-rabbit igg antibody is drawn in close thieving paper one side of pasting, and two lines are at a distance of 5～6mm.

1.2.5 baking film and preservation

The nitrocellulose filter that pulls T line and C line is put indoor drying together with base plate, save backup in mid-4～8 ℃ of refrigerators of the sealed plastic bag of packing into.

1.3 the making of sample pad

1.3.1 the preparation of sample pad treating fluid

1.3.1.1pH7.2 sample pad treating fluid 0.2M Tris 100ml, 0.1N HCl 180ml, casein 0.250g, sucrose 25.000g, NaN ₃0.100g mix, 37 ℃ of water-baths dissolvings add the 1.0ml Tween-20 again and stir, adding distil water mixes to 500ml, casein containing protein 0.5%, sucrose 5%, NaN ₃0.02%, the sample pad treating fluid that the pH7.20.04M Tris-HCl damping fluid of Tween-20 0.2% consists of.

1.3.2 the making of sample pad is cut into little of 23mm * 300mm with glass fibre element film, soaks 1hr with the sample pad treating fluid, takes out, in 37 ℃ of baking ovens, dry, sample pad.

1.4 the making of thieving paper

With thieving paper (model: CH37) be cut into little of 28mm * 300mm, in 37 ℃ of baking ovens, dry.

1.5 the making of label glued membrane

1.5.1 the making of arrow label glued membrane

The arrow label glued membrane is the adhesive sticker adhesive paper of a kind of 300mm * 18mm, apart from 1mm place, a long limit straight line being arranged, is close to this straight line, is printed on " ← MAX " printed words every 1.3mm, and arrow is vertical with straight line.Its effect is the depth capacity that the indication test strips is inserted sample liquid to be checked on the one hand; That protection immunological probe dry plate is not contaminated on the other hand.

1.5.2 the making of handle label glued membrane

Green-ticket protection paster is printed by the adhesive sticker adhesive paper of 300mm * 30mm, is 15 ° of angles with minor face, is printed on " SjAb " printed words of 4 row's wrongly written or mispronounced characters of the green end, and expression is for detection of schistosome antibody, and protection thieving paper is avoided polluting when hand is taken.

1.6 the assembling of test strips kilocalorie

1.6.1 get the PVC base plate that 2.2 steps have pasted chromatographic film, take a block protection paper of width maximum on the PVC edges of boards off, expose non-drying glue layer, thieving paper is pasted up, make it and the overlapping approximately 1～2mm of chromatographic film.

1.6.2 get handle label glued membrane, throw off protection sheet, expose non-drying glue layer, it is sticked on the thieving paper.

1.6.3 take on the PVC plate one little protection sheet on the chromatographic film limit off, expose non-drying glue layer, (300mm * 8.5mm) stick on the adhesive sticker of PVC plate makes the overlapping approximately 1mm of immunological probe dry plate and chromatographic film with the immunological probe dry plate.

1.6.4 take the protection sheet on immunological probe dry plate limit on the PVC plate off, expose non-drying glue layer, pretreated sample pad is pasted up, make it and the overlapping approximately 1～2mm of polyester film.

1.6.5 get the arrow label glue-film stickup at the immunological probe dry plate with it on the adjacent sample segment pad; make the blank side of diaphragm be close to approximately 1mm of chromatographic film imbricate; arrow side is close to sample pad, and the immunological probe dry plate is covered by the label glued membrane fully, and each parts flattens compacting and namely forms kilocalorie.

1.7 slitting, packing and storage

1.7.1 slitting

The test strips kilocalorie is cut into the test strips of 80mm * 3mm with cutting cutter, is ox Japanese schistosomiasis antibody test test strips of the present invention.

1.7.2 packing

1.7.3 the packing of test strips

The test strips of 80mm * 3mm packs with aluminium foil bag by the specification of 10/bag, 20/bag or 50/bag.

1.7.4 store

Test strips is 2～8 ℃ of storages, and the term of validity is 12 months.15～30 ℃ of room temperature storages, the term of validity are 6 months.

Embodiment 2:(detects the preparation 2 of ox Japanese schistosomiasis antibody test strips)

The making of 1 test strips

⑴ immunological probe dry plate preparation is with among the embodiment 1 1.1, but has 5 places different:

1. use the blue latex beads of 300nm.

2. the anti-yellowing buffalo IgG of used rabbit protein content is 2mg/ml when the latex beads mark.

3. the confining liquid preparation takes by weighing casein 0.500g, sucrose 10.000g, NaN ₃0.020g all put into 100ml pH7.20.05M Tris-HCl damping fluid, 37～40 ℃ of heating water baths are to fully dissolving.Namely be mixed with casein containing protein 0.5%, sucrose 10%, NaN ₃The confining liquid that 0.02% pH7.20.05M Tris-HCl damping fluid consists of.

4. the polyester film treating fluid is prepared 0.2M Tris 125ml, 0.1N HCl 225ml, casein 2.500g, sucrose 50.000g, NaN ₃0.100g mix, 37 ℃ of water-baths dissolvings add Tween-20 1.5ml again and stir, adding distil water mixes to 500ml.Namely be mixed with casein containing protein 0.5%, sucrose 10%, NaN ₃0.02%, the polyester film treating fluid that the pH7.20.05M Tris-HCl damping fluid of Tween-20 0.3% consists of.

When 5. being sprayed on the anti-yellowing buffalo IgG of latex beads mark rabbit bond on the polyester film of having processed, the flow velocity of drawing the setting of film metal spraying machine is 4 μ l/cm.

⑵ the preparation of chromatographic film is with among the embodiment 1 1.2, but has the number place different:

1. the damping fluid that dilutes antigen and goat anti-rabbit igg antibody is pH8.50.05M TBS damping fluid, and compound method is: 0.2mol Tris solution 25ml and 0.1N HCl 15ml mix, add NaCl 0.850g, and dissolving adds to 100ml with distilled water again.

2. line is 3mg/ml with the protein content of japonice ovum antigen.

3. line is 0.1mg/ml with the protein content of goat anti-rabbit igg antibody.

4. the film flow velocity of drawing of japonice ovum antigen and goat anti-rabbit igg antibody is 2 μ l/cm.

5. the preparation 0.2M Tris 100ml of sample pad treating fluid, 0.1N HCl 180ml, casein 0.250g, sucrose 50.000g, NaN ₃0.1g mix, 37 ℃ of water-baths dissolvings add the 1.0ml Tween-20 again and stir, adding distil water mixes to 500ml.Namely be mixed with casein containing protein 0.5%, sucrose 10%, NaN ₃0.02%, the sample pad treating fluid that the pH7.20.04M Tris-HCl damping fluid of Tween-20 0.2% consists of.

All the other are all identical with embodiment 1 such as making steps such as the assembling of sample pad preparation, thieving paper preparation, test strips kilocalorie and slittings.

Embodiment 3:(detects the preparation 3 of ox Japanese schistosomiasis antibody test strips)

The making of 1 test strips

⑴ immunological probe dry plate preparation is with among the embodiment 1 1.1, but has 4 places different:

1. the anti-yellowing buffalo IgG of used rabbit protein content is 3mg/ml when the latex beads mark.

2. the confining liquid preparation takes by weighing casein 0.300g, sucrose 5.000g, NaN ₃0.030g all put into 100ml pH7.20.05M Tris-HCl damping fluid, 37～40 ℃ of heating water baths are to fully dissolving.Namely be mixed with casein containing protein 0.3%, sucrose 5%, NaN ₃The confining liquid that 0.03% pH7.20.05M Tris-HCl damping fluid consists of.

3. the polyester film treating fluid is prepared 0.2M Tris.125ml, 0.1N HCl 225ml, casein 1.500g, sucrose 25.000g, NaN ₃0.150g mix, 37 ℃ of water-baths dissolvings add Tween-20 2.0ml again and stir, adding distil water mixes to 500ml.Namely be mixed with casein containing protein 0.3%, sucrose 5%, NaN ₃0.03%, the polyester film treating fluid that the pH7.20.05M Tris-HCl damping fluid of Tween-20 0.4% consists of.

When 4. being sprayed on the anti-yellowing buffalo IgG of latex beads mark rabbit bond on the polyester film of having processed, the flow velocity of drawing the setting of film metal spraying machine is 3 μ l/cm.

1. the damping fluid that dilutes antigen and goat anti-rabbit igg antibody is pH8.50.05M TBS damping fluid, and compound method is seen embodiment 2.

2. line is 5mg/ml with the protein content of japonice ovum antigen.

3. line is 1mg/ml with the protein content of goat anti-rabbit igg antibody.

4. the film flow velocity of drawing of japonice ovum antigen and goat anti-rabbit igg antibody is 1 μ l/cm.

5. the preparation 0.2M Tris 100ml of sample pad treating fluid, 0.1N HCl 180ml, casein 0.150g, sucrose 25.000g, NaN ₃0.250g mix, 37 ℃ of water-baths dissolvings add the 2.0ml Tween-20 again and stir, adding distil water mixes to 500ml.Namely be mixed with casein containing protein 0.03%, sucrose 5%, NaN ₃0.05%, the sample pad treating fluid that the pH7.20.04M Tris-HCl damping fluid of Tween-20 0.4% consists of.

Embodiment 4:(detects the preparation 4 of ox Japanese schistosomiasis antibody test strips)

The making of 1 test strips

1. use the blue latex beads of 200nm.

2. the anti-yellowing buffalo IgG of used rabbit protein content is 4mg/ml when the latex beads mark.

3. the confining liquid preparation takes by weighing casein 0.200g, sucrose 10.000g, NaN ₃0.050g all put into 100ml pH7.20.05M Tris-HCl damping fluid, 37～40 ℃ of heating water baths are to fully dissolving.Namely be mixed with casein containing protein 0.2%, sucrose 10%, NaN ₃The confining liquid that 0.05% pH7.20.05M Tris-HCl damping fluid consists of.

4. the polyester film treating fluid is prepared 0.2M Tris 125ml, 0.1N HCl 225ml, casein 1.000g, sucrose 50.000g, NaN ₃0.250g mix, 37 ℃ of water-baths dissolvings add Tween-20 2.5ml again and stir, adding distil water mixes to 500ml.Namely be mixed with casein containing protein 0.2%, sucrose 10%, NaN ₃0.05%, the polyester film treating fluid that the pH7.20.05M Tris-HCl damping fluid of Tween-20 0.5% consists of.。

When 5. being sprayed on the anti-yellowing buffalo IgG of latex beads mark rabbit bond on the polyester film of having processed, the flow velocity of drawing the setting of film metal spraying machine is 2 μ l/cm.

2. line is 7mg/ml with the protein content of japonice ovum antigen.

3. line is 2mg/ml with the protein content of goat anti-rabbit igg antibody.

5. the preparation 0.2M Tris 100ml of sample pad treating fluid, 0.1N HCl 180ml, casein 0.050g, sucrose 50.000g, NaN ₃0.250g mix, 37 ℃ of water-baths dissolvings add the 2.5ml Tween-20 again and stir, adding distil water mixes to 500ml.Namely be mixed with casein containing protein 0.01%, sucrose 10%, NaN ₃0.05%, the sample pad treating fluid that the pH7.20.04M Tris-HCl damping fluid of Tween-20 0.5% consists of.

Embodiment 5:(uses the method for ELISA test strip ox Japanese schistosomiasis antibody)

1 serum to be checked

1.1 1000～1500 of the positive cow's serum experimental infection of blood fluke blood fluke cercarias infect 6 parts of 2 cow's serums of 35～49d, natural infection blood fluke in Hubei also incubates 4 parts of the cow's serums made a definite diagnosis through excrement.

1.2 the negative cow's serum of blood fluke picks up from 10 parts of the healthy cow's serums of blood fluke Pest-or disease-free area Jinhua, Zhejiang, 5 parts of experimental infection trypanosome cow's serums, 5 parts of natural infection fascioliasis cow's serums.

2 test strips are originally tested the test strips that used test strips is the preparation of embodiment 2 technological parameters.

3 detecting steps

3.1 the preparation of serum dilution is with Na ₂HPO ₄, KH ₂PO ₄, NaCl and distilled water volume 0.199g: 0.082g: 1g by weight: the 200ml ratio is mixed, after 37～40 ℃ of heating water baths dissolvings, the cooling serum dilution;

(2) dilution of cow's serum to be checked: get 60 of 1.5ml plastic centrifuge tubes, numbering, wherein 30 every pipes of centrifuge tube add serum dilution 0.05ml, 30 arms add serum dilution 0.1ml in addition, add again serum 0.05ml to be checked in every centrifuge tube, cow's serum to be checked and serum dilution are distinguished by volume 1:1 and 1:2 ratio mixed diluting;

(3) detect operation: get 60 of test strips, lie on the operator's console, the serum 0.05ml to be checked that draws after diluting drops on the sample pad of test strips;

(4) result judges: observations behind 5～15min, no matter be cow's serum to be checked and serum dilution by volume 1:1 or 1:2 dilution proportion, 6 parts of experimental infection snail fever cow's serums and 4 parts of natural infection cow's serums, the detection T line of test strips and Quality Control C line all present blue band, are judged to the positive; And 10 parts of healthy cow's serums of Pest-or disease-free area, 5 parts of trypanosomiasis cow's serums and 5 parts of fascioliasis cow's serums only present 1 blue band at the Quality Control C of test strips line position, therefore the feminine gender of being judged to.The test strips of this experimental test detects and judged result according to the using method of test strips, can correctly detect the positive cow's serum of snail fever, and having no with trypanosomiasis, fascioliasis cow's serum has cross reaction.

The sensitivity tests of test example 1:(test strips)

1 test strips is originally tested the test strips that used test strips is the preparation of embodiment 3 technological parameters.

2 blood serum samples to be checked are used 10 healthy oxes of infection by cercariae of schistosoma, and every is infected 500～1000 of cercarias, and different time is taken a blood sample after (0d) and the infection before infecting, preparation serum.

3 detect operation serum to be checked with 2 times of dilutions of serum dilution (prescription is seen " method of using ELISA test strip ox Japanese schistosomiasis antibody "), draw the 0.05ml dilute serum and are added drop-wise on the sample pad of test strips, carry out the immunochromatography reaction.5～15min judges yin and yang attribute according to the color that T line and C line position manifest, and 2 red lines are declared the positive, and 1 red line is declared feminine gender.

4 testing results see Table 1.

The sensitivity Detection information slip of table 1 test strips

As a result, ELISA test strip infects front 10 cow's serums, whole negative reactions.28d cow's serum after check is infected has 2 cow's serums to detect positive reaction.10 cow's serums of experimental infection 35d～49d all detect schistosome antibody (recall rate 100%).According to other test, the IHA method can detect experimental infection blood fluke 35d and the above blood sample of 35d.The susceptibility that test strips is described is similar to the IHA method or slightly high.But the IHA method detects a sample and approximately needs 1～2hr, and uses ELISA test strip, operates simplyr, just can obtain a result in 5～15 minutes.

The specific test of test example 2:(test strips)

1 test strips is originally tested the test strips that used test strips is the preparation of embodiment 1 technological parameter.

20 parts of the healthy cow's serums of 2 blood serum sample Pest-or disease-free areas to be checked; 40 parts of the positive cow's serums of snail fever, wherein experimental infection blood fluke cercaria 35～49d cow's serum is 20 parts, and the natural infection blood fluke also incubates the miracidium method through excrement and confirms 20 parts of positive cow's serums; Fluke is finished in natural infection east, and 20 parts of cow's serums of making a definite diagnosis through the excrement inspection; The natural infection Fasciola hepatica, 15 parts of the cow serums that confirms through excrement inspection worm's ovum method; 15 parts of experimental infection trypanosome cow's serums.

3 detect operation judges see " method of using ELISA test strip ox Japanese schistosomiasis antibody " with the result.

4 testing results see Table 2.

The specific detection information slip of table 2 test strips

With ELISA test strip experimental infection and 40 cow's serums of natural infection snail fever, be positive reaction.Detect east and finish the serum of fluke, Fasciola hepatica, trypanosomiasis ox and healthy ox, be negative reaction.Illustrate that this test strips has stronger specificity, having no with other parasitic diseases in the sample that detects has cross reaction.Repeatable test between criticizing in test example 3:(test strips is criticized)

What 1 test strips was originally tested used test strips batch is: 20091103,20091105,20091109,20091111,20091113.Prepare according to embodiment 2 technological parameters.Just make the schistosome antigen of T line, front 2 batches of used blood fluke soluble antigens are to begin preparation by 5% worm's ovum suspension, and then 3 batches of soluble antigens are to begin preparation by 3% worm's ovum suspension.Concentration when being used for line all is diluted to and contains albumen 3mg/ml.Other preparation conditions are all identical.

50 parts of (30 parts of cow's serums of 500～1000 35～49d of experimental infection blood fluke cercaria of 2 serum snail fever positive serums to be checked, 20 parts of the epidemic-stricken area such as Hubei, Sichuan natural infection blood fluke and the cow's serums that confirm through the excrement method of incubating), 80 parts of the negative cow's serums of snail fever (60 parts of the ground cow's serums such as Jinhua, Zhejiang, Quzhou pick up from the Fuyang, Zhejiang slaughterhouse and economize 20 parts of ox serum from Jilin, Shandong etc.).

3 detect operation serum to be checked repeats to do 2 times of dilutions with serum dilution for front 4 times, and the 5th repeats serum dilution and does 3 times of dilutions, and the application of sample amount is 0.05ml.The test strips duplicate detection of each batch 5 times, relatively testing result is observed the repeatability that test strips is criticized testing result between interior batch.

4 testing result measurement results are summarised in the table 3.

Table 3 test strips batch between and batch in repeated measurement result information slip

The measurement result that every batch of front and back are 5 times is all identical, measurement result between each batch test strips is also identical, the initial concentration of worm's ovum suspension can be ignored the repeatability impact of test strips when the preparation egg antigen was described, serum is done 2 times of dilutions and done 3 times of dilutions to not obviously impact of testing result.This test strips batch between and batch in repeatability all better, be suitable in batches standardized production.

The storage life test of test example 4:(test strips)

The used test strips of 5 batches of test strips of 1 test strips and test example 3 is identical.In this test example, every batch test strips is divided into two parts preserves, a part is put 2～8 ℃ of Refrigerator stores, and another part is put 15～35 ℃ of room temperature preservation.

20 parts of the positive cow's serums of 2 Serum experiments infection snail fever to be checked, 20 parts of healthy cow's serums.

3 detection methods after storage life after on-test 1st month and on-test at quarterly intervals, get the test strips of preserving with distinct methods each 20 parts of the positive cow's serum of experimental infection snail fever and healthy cow's serums are detected, detect the serum dilution of operation and judge all identical with test example 3 with amount of serum and result.

4 testing results see Table 4.It still is 100% that test strips is put 15 months positive rate of 4～8 ℃ of Refrigerator stores, and the test strips positive rate of preserving 18 months is 80%.And test strips to put 6 months positive rates of room temperature preservation still be 100%, preserve and just only had 70% in 9 months.Therefore, the preservation condition of test strips is: 4～8 ℃ of Refrigerator stores, the term of validity are decided to be 12 months should be no problem.

The measurement result table of preserving under the table 4 bos sinicus diagnosis test paper condition of different temperatures

The serum antibody growth and decline situation of experimental infection snail fever ox before and after test example 5:(treats with ELISA test strip)

1 test strips is originally tested the test strips that used test strips is the preparation of embodiment 4 technological parameters.

2 serum to be checked are selected 5 of healthy oxes, before the experimental infection blood fluke cercaria, and the bovine jugular vein blood sampling, the serum of preparation is as 0w serum.1000～1500 of every cattle infected blood fluke cercarias, ox 1w to the 10w after infect, weekly blood sampling once, separation of serum ,-20 ℃ of preservations.11w treats with praziquantel behind the cattle infected, every the 7d treatment once, treats continuously 3 times.The 1st～3w takes a blood sample weekly 1 time after the treatment, and the per two week blood samplings of the 4th～24w are 1 time after the treatment, separation of serum, and-20 ℃ of preservations are to be checked.

3 detect operation serum to be checked does 2 times of dilutions with serum dilution, draws the 0.05ml dilute serum and is added drop-wise on the sample pad of test strips, carries out the immunochromatography reaction.5～15min judges yin and yang attribute according to the color that T line and C line position manifest.T line and C line red stripes all occurs and are judged to the positive.Antibody horizontal in the serum is higher, and the T line color is darker, can be judged to according to the T line color depth: presenting strong red stripes is strong positive, with " +++" expression, present apparent red stripes, positive, with " ++ " expression, presenting the light red band is the weak positive, represents with "+".Only have the C line high-visible red stripes to occur and be judged to feminine gender, represent with "-".The apparent red line of color appears in the C line, and a very slight color appears in the T line, if suspicious if hidden aobvious red zone is judged to, represents with " ± ".

4 testing results see Table 5.

The serum antibody growth and decline situation of experimental infection snail fever ox before and after table 5 treatment

As seen from the above table, detected antibody in the cow's serum of the 4th～5w behind the infection by Schistosoma, after infection after the 8th～10w and the treatment during the 1st～4w, antibody horizontal reaches the peak, then from 6w after treating, antibody horizontal descends gradually, 1,2,3,4 and No. 5 ox successively after treatment the 24th, 16,22,20 and 20w antibody turn out cloudy.Its result hints that this test strips has certain early diagnosis and efficacy assessment is worth.

Test example 6:(test strips and excrement are incubated the coincidence rate test of miracidium method, IHA method and DIFA method)

Also the do not go through schistosomiasis diagnosis reagent that goes on the market of China so far.Detection method commonly used has excrement to incubate miracidium method, IHA method and DIFA method at present.

1 detects reagent

1.1 the test strips of this test of test strips usefulness is the test strips of embodiment 3 technological parameters preparation, lot number is: 20091115.

1.2 solidifying antigen diagnose reagent is by the production of Jiangxi Province's preventing and treating verminosis research institute between the solidifying used blood fluke of antigen diagnose reagent IHA method between blood fluke, lot number is: 20091023.

1.3 the used Dot Immunogold Filtration Assay reagent of Dot Immunogold Filtration Assay reagent D IFA method is by the trial-production of academy of agricultural sciences, Zhejiang Province animal and veterinary research institute, lot number is: 20091015.

2 serum to be checked are in the heavy epidemic-stricken area in Hubei and Sichuan, detect with the excrement method of incubating and gather and process respectively 50 and 70 cow's serums in the positive ox.Incubate the serum that gathers 200 and 300 oxen the negative ox from excrement.Gather 500 cow's serums from blood fluke Pest-or disease-free area Jinhua, Zhejiang and other places.Each 5 parts of the cow's serums of experimental infection blood fluke cercaria 28d, 35d, 42d.

2 detection methods

2.1 test strips method (immunochromatographic method) serum to be checked is done 2 times of dilutions with serum dilution, draws 0.05ml dilute serum to be checked and drips on the sample pad of test strips.5～15min result of determination: 2 blue lines occur and be judged to the positive, 1 blue line only occurs and be judged to feminine gender.

2.2IHA every antigen adding distil water of method 1ml dissolving, mixing uses.Use U-shaped hemagglutination plate, drip 0.85%NaCl 4 the second holes in hemagglutination plate first row the first hole with quantitative dropper (or 25 μ l pipettors) and add 1.Inhale serum 1 to be checked with quantitative dropper (or 25 μ l pipettors) and be added dropwise in first row the first hole, sucking-off 1 is added dropwise in the second hole behind abundant mixing, discards 1 behind the mixing, and making serum dilution is 1:10.Draw mixing antigen liquid 1 with quantitative dropper (or 25 μ l pipettors) and be added dropwise in the second hole, rotate immediately approximately 1min clock (or oscillator shakes up approximately 0.5min) of jolting.Leave standstill the 1hr observations under the room temperature.Red blood cell all sinks at the bottom of the hole, and naked eyes see that the dot of the smooth of the edge, densification is negative.Red blood cell forms the thin layer aggegation, and the edge presents irregular gauffer, be judged to " ++ ++ " positive; Red blood cell forms the thin layer aggegation, is full of at the bottom of the whole hole, be judged to " +++" positive; Red blood cell forms the thin layer aggegation, and area is " +++" little person, is judged to " ++ " positive; Most of heavy the combining at the bottom of the hole of red blood cell forms a round dot, and naked eyes are seen the fuzzy or middle comparatively significantly blank spot that occurs of periphery, are judged to "+" positive.

2.3DIFA method is used the Dot Immunogold Filtration Assay kit.Serum to be checked is done 80 times of dilutions with 0.85%NaCl solution first.Begin point sample from the first hole, the reaction plate left side, dip dilute serum with glass capillary, light 0.5s, 2 samples of every hole point, 10 samples of every deblocking reaction plate point, continuous point sample 5 deblocking reaction plates in limit of pasting on the nitrocellulose filter of reaction plate aperture.3min clock behind the point sample drips first liquid 0.05ml from reaction plate first hole of point sample the earliest, and interval 5～7s drips the second hole again, and the rest may be inferred.Treat that first liquid infilters in the reaction plate, every hole drips second liquid 0.05ml, and after second liquid infiltrated, every hole dripped the third liquid 0.05ml.The point sample place manifests punctation, is judged to the positive, yellow spotting or the colourless feminine gender that is judged to.

2.4 the method stool for hatching schistosoma miracidium method of incubating excrement adopts the quick ight soil miracidium hatching method of an excrement three inspections.Excrement is searched fresh cow dung 50g each time, directly drops into to eluriate to water clearly in the 260 order nylon mesh pockets, and the excrement slag is put into the 500ml flask, hatches under 25～26 ℃.Observed miracidium once in 1,3,5 hour after the hatching, be no less than 1 minute each observing time.Just be judged to the positive if observe miracidium.Do not observe miracidium and be judged to feminine gender.To note simultaneously after front twice observation, flask should be stirred or the jolting several, in order to allow the minority worm's ovum that overstocks in excrement slag bottom hatch miracidium.

4 results

4.1 test strips and excrement are incubated the coincidence rate of miracidium method

50 parts and 70 parts of the positive cow's serums made a definite diagnosis are detected with the excrement method of incubating in heavy epidemic-stricken area in Hubei and Sichuan, 200 parts and 300 parts of negative cow's serums, and the excrement of blood fluke Pest-or disease-free area Zhejiang collection is incubated 500 parts of negative cow's serums.Detect with test strips.Relatively test strips method and excrement are incubated the coincidence rate of miracidium method.Result's (seeing Table 6) is incubated positive cow's serum for excrement, and the positive coincidence rate that test bar method and excrement are incubated method is 100%, and excrement is incubated negative cow's serum, and the negative match-rate that test bar method and excrement are incubated method is 99.6%.

Table 6 test strips and excrement are incubated the check of the coincidence rate that is diagnosed as yin and yang attribute

Excrement is incubated the method complex operation, needs nearly 1 day time just to go out the result, and is easily undetected in the low density situation of worm's ovum.But positive findings reliably is its advantage.Often be used to contrast in investigation schistosomiasis diagnosis method reliability.The test strips method can all detect the serum antibody that excrement is incubated positive ox, illustrates that the ELISA test strip method has higher reliability.

4.2 the coincidence rate of test strips and IHA method and DIFA method

Infect each 5 parts of blood fluke cercaria 28d, 35d, 42d cow's serums with test strips, IHA method and DIFA method difference test experience, excrement ovum method is made a definite diagnosis 60 parts of natural infection snail fever cow's serums, 60 parts of the healthy cow's serums of Pest-or disease-free area.Testing result sees Table 7.

The coincidence rate of table 7 ELISA test strip method and IHA method and DIFA method

As seen from the above table, the cow's serum of experimental infection 28d only has the DIFA method all to detect, and except the cow's serum of experimental infection 28d, for all the other serum, the testing result of test strips, IHA and three kinds of methods of DIFA is in full accord.The serological method coincidence rate that test strips and two kinds are commonly used is 100%.But IHA method operation steps is more, and detection speed is slower, and finishing one-time detection needs 1～2 hour.DIFA method detection speed is very fast, only needs 5 minutes kinds can survey 40～50 samples.But its reagent making influence factor is many, difficult realization sequencing, batch production production.Its reagent is liquid, can not be by aviation and post office mailing.Test strips method operation is the simplest, only needs 1 step an of application of sample, goes out the result in 5～15 minutes.Test strips is solid, posts unrestricted.Sample of every test strips test uses very flexible.Its preparation technology also is suitable for mass production.

From above embodiment and test example as seen, bos sinicus diagnosis test paper of the present invention has the susceptibility suitable with the IHA method, more outstanding specificity and repeatability, and easy to use storage life reached more than 1 year flexibly, was suitable in batches batch production production.Can be widely used in the efficacy assessment of diagnosis, generaI investigation, quarantine and the medicine of bos sinicus, have a good application prospect.

Claims

1. a test strips that detects ox Japanese schistosomiasis antibody is characterized in that this test strips is combined by parts sample pad, immunological probe dry plate, chromatographic film, thieving paper, PVC substrate and arrow label glued membrane and handle label glued membrane; Wherein, the immunological probe dry plate is the carboxyl with red or other color carboxylic polystyrene latex beads, forms behind the covalent bonds thing with the amido of the anti-yellowing buffalo IgG of rabbit antibody protein and makes after detection reagent solution as immunological probe is adsorbed in the polyester film; Chromatographic film is take nitrocellulose filter as the film base, schistosome ovum soluble antigen and goat anti-rabbit igg antibody are drawn at this film respectively detects the T line and Quality Control C line forms with drawing the film metal spraying machine; First with overlapping bonding being assemblied on the PVC substrate of above-mentioned front four parts head portion, again with the arrow label glue-film stickup on the immunological probe dry plate, in addition with handle label glue-film stickup after on the thieving paper, with cutting cutter by after setting width and cutting ox Japanese schistosomiasis antibody test test strips.

2. method for preparing the described ox Japanese schistosomiasis of claim 1 antibody test test strips is characterized in that carrying out according to the following steps:

(1) preparation of immunological probe dry plate: comprise

1) preparation of the anti-yellowing buffalo IgG of rabbit:

1. the preparation of yellow water ox IgG: gather healthy ox, buffalo blood, getting respectively each 5ml of serum behind the separation of serum mixes, add equivalent 0.85% NaCl, extract yellow water ox IgG with 30% ammonium sulfate precipitation method, the yellow water ox IgG of precipitation dissolves with 0.85% NaCl of 2 times of amounts of former serum volume, repeat to extract 2 times with 35% ammonium sulfate precipitation method again, at last with the yellow water ox IgG of 5ml 0.85% NaCl dissolving centrifugation, remove NH in the solution with dialysis ₄ ⁺And SO ₄ ^2-After, put into the concentrated method of PEG with distilled water diluting or with bag filter again, regulate protein concentration to 10mg/ml, for subsequent use in-20 ℃ of sealed type storages;

3. the preparation of the anti-yellowing buffalo IgG of rabbit antibody: get the anti-yellowing buffalo IgG of rabbit serum 10ml, extract the anti-yellowing buffalo IgG of rabbit antibody with 35% ammonium sulfate precipitation method, with 2 times to the anti-yellowing buffalo IgG of rabbit of 0.85% NaCl dissolution precipitation of serum amount antibody, after so repeatedly extracting 3 times, the centrifugal sediment of the anti-yellowing buffalo IgG of the rabbit antibody of the 3rd sulphoxylic acid ammonium precipitation with 5ml 0.85% NaCl dissolving, is removed NH in the solution with dialysis ₄ ⁺And SO ₄ ^2-After the protein determination instrument is measured the anti-yellowing buffalo IgG of rabbit antibody protein content, put into the method that PEG concentrates with the borate buffer dilution of pH7.20 0.1M or with bag filter, protein concentration is adjusted to 4mg/ml after, for subsequent use in-20 ℃ of sealed type storages;

2) washing of latex beads: with the polystyrene latex microballoon liquid of diameter 200nm redness or other color, at 10000 rpm, centrifugal 10min under 4 ℃ of conditions, remove supernatant, sediment is reduced to original volume with high purity water, after disperseing the latex beads of precipitation to become unit for uniform suspension with ultrasound wave, use again above-mentioned identical method repeated centrifugation, resuspension 1～2 time;

3) activation of latex beads carboxyl: with pH6.5 0.1mol MES, 10% suspension with the look latex beads, the EDCHCl of 20mg/mL and the NHS of 20mg/mL, by volume 2hr is mixed, reacted to the ratio of 9:2:1:0.75; Then centrifugal 25min under 4 ℃ of conditions of 11000 rpm removes supernatant, and sediment is resuspended with the borate buffer of used pH7.20 0.1M with 5 times of amounts of look latex volume, impacts with ultrasound wave and is reduced into uniform suspending liquid; With vertical mixed instrument mixing 15min, at 11000 rpm, centrifugal 25min removes supernatant under 4 ℃ of conditions, sediment is resuspended with the borate buffer of used pH7.20 0.1 M with 2.5 times of amounts of look latex volume, impact with ultrasound wave and to be reduced into uniform suspending liquid, be the latex beads of activated carboxylic;

4) the anti-yellowing buffalo IgG of latex beads mark rabbit antibody: add equal-volume in the centrifuge tube that activated carboxylic latex beads suspending liquid is housed and be diluted to protein concentration as the anti-yellowing buffalo IgG of the rabbit of 1～4mg/mL antibody take the borate buffer of pH7.20 0.1M, on vertical mixed instrument more than the hybrid reaction 2hr; Add the 20%BSA oscillating reactions 1hr of cumulative volume 1/10th amounts, reacted potpourri is removed supernatant with 4 ℃ of centrifugal 25min of 11000 rpm again, and sediment is with casein containing protein 0.2%～0.5%, sucrose 5%～10%, NaN ₃The confining liquid original volume that 0.02%～0.05% pH7.2 0.05M Tris-HCl damping fluid consists of is resuspended, ultrasound wave impacts and is reduced into uniform suspending liquid, at vertical mixed instrument mixing 15min, again with 4 ℃ of centrifugal 25min of 11000rpm, remove supernatant, sediment is resuspended with the same confining liquid that is equivalent to the anti-yellowing buffalo IgG of latex beads suspending liquid and rabbit antibody cumulative volume 75%, ultrasound wave impacts and is reduced into uniform suspending liquid, and the immunological probe that this liquid is redness or other color detects reagent solution;

5) preparation of immunological probe dry plate: above-mentioned immunological probe is detected reagent solution be sprayed on and pass through casein containing protein 0.2%～0.5%, sucrose 5%～10%, NaN with drawing the flow velocity of film metal spraying machine with 2～5 μ l/cm ₃On the polyester film of the pretreated long 300mm of polyester film treating fluid that the pH7.2 0.05M Tris-HCl damping fluid of 0.02%～0.05%, Tween-20 0.2%～0.5% consists of * wide 8.5mm, through 37 ℃, the 1hr oven dry is the immunological probe dry plate;

(2) preparation of chromatographic film:

1) preparation of schistosome ovum soluble antigen: by the healthy new zealand rabbit of body weight 2.5～3kg, inoculate 2000 of blood fluke cercarias for every, the experimental infection rabbit was butchered in inoculation and gets liver in rear 45 days, separate and the purifying japonice ovum, grind fragmentation after freeze drying, be made into 3%～5% suspending liquid with 0.85%NaCl solution, 2～8 ℃ were soaked 3, multigelation is 10 times again, the broken 30min of ice-bath ultrasonic ripple, the centrifugal 1hr of 10000r/min gets supernatant, the dress bag filter, with pH7.0 0.01M PB dialysis 16 hours, changed dislysate 1 time every 4 hours, after dialysis finishes, with the centrifugal 60min of 10000rp, supernatant is the schistosome ovum soluble antigen; After detecting its protein content, be diluted to 1～7mg/mL concentration with pH8.2～8.5 0.05M TBS, namely can be used for detecting on the chromatographic film drafting of T line;

2) preparation of goat anti-rabbit igg:

1. the preparation of rabbit igg: the blood that gathers healthy new zealand rabbit with the heart blood collection method, separation of serum, get rabbit anteserum 10ml, add again 10ml 0.85% NaCl mixing, extract rabbit igg with 35% ammonium sulfate precipitation method, repeated precipitation is extracted 3 times, after sediment dissolves with 5ml 0.85% NaCl, remove ammonium sulfate with dialysis, and measure the protein content of rabbit igg at the protein determination instrument;

Separate goat anti-rabbit igg antibody and extract thick goat anti-rabbit igg antibody once with 30% ammonium sulfate precipitation method first, extract 2 times with 45% ammonium sulfate precipitation method, centrifugal sediment usefulness 5ml 0.85% NaCl dissolves, and removes NH in the solution with dialysis ₄ ⁺And SO ₄ ^2-, the centrifugal 30min of 3000rpm behind the detection supernatant protein content, is diluted to supernatant the concentration of 0.1～2mg/mL with pH8.2～8.5 0.05M TBS, namely can be used for the drafting of Quality Control C line on the chromatographic film;

(3) preparation of sample pad: with 23mm * 300mm glass fibre membrane by casein containing protein 0.01%～0.05%, sucrose 5%～10%, NaN ₃0.02%～0.05% and the pH7.2 0.04M Tris-HCl damping fluid of Tween-20 0.2%～0.5% sample pad treating fluid that consists of in soak 1hr, take out 37 ℃ of oven dry;

(7) assembling of each parts of test strips, slitting, packing and storage:

3. application rights requires the method for 1 described ELISA test strip ox Japanese schistosomiasis antibody, it is characterized in that carrying out according to the following steps:

(1) preparation of serum dilution: with Na ₂HPO _4,KH ₂PO _4,NaCl and distilled water is volume 0.199g: 0.082g: 1g by weight: the 200ml ratio is mixed, after 37～40 ℃ of heating water baths dissolvings, the cooling serum dilution;