CN105606820A - Porcine reproductive and respiratory syndrome antibody detecting card and preparation method thereof - Google Patents
Porcine reproductive and respiratory syndrome antibody detecting card and preparation method thereof Download PDFInfo
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- CN105606820A CN105606820A CN201610040981.1A CN201610040981A CN105606820A CN 105606820 A CN105606820 A CN 105606820A CN 201610040981 A CN201610040981 A CN 201610040981A CN 105606820 A CN105606820 A CN 105606820A
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/68—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids
- G01N33/6854—Immunoglobulins
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
- G01N33/531—Production of immunochemical test materials
- G01N33/532—Production of labelled immunochemicals
- G01N33/533—Production of labelled immunochemicals with fluorescent label
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- G—PHYSICS
- G01—MEASURING; TESTING
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- G01N33/558—Immunoassay; Biospecific binding assay; Materials therefor using diffusion or migration of antigen or antibody
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
- G01N33/569—Immunoassay; Biospecific binding assay; Materials therefor for microorganisms, e.g. protozoa, bacteria, viruses
- G01N33/56983—Viruses
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Abstract
The invention discloses a porcine reproductive and respiratory syndrome antibody detecting card and a preparation method thereof. The porcine reproductive and respiratory syndrome antibody detecting card comprises a detecting card body assembled in a shell, wherein the detecting card body comprises a bottom plate; a sample pad, a label pad, a nitrocellulose film and absorbent paper are sequentially arranged on the bottom plate; the nitrocellulose film is coated with porcine reproductive and respiratory syndrome antigens serving as a detection line, and coated with goat anti-chicken antibodies serving as a quality control line; a sample adding hole is formed in the shell and corresponds to the sample pad; and window holes are formed and correspond to the detection line and the quality control line on the nitrocellulose film. The porcine reproductive and respiratory syndrome antibody detecting card is high in sensitivity, small in detection intra-batch difference and inter-batch difference, good in repeatability and stable in performance, can quickly detect porcine reproductive and respiratory syndrome antibodies in situ, and has high practical value.
Description
Technical field
The present invention relates to a kind of test card, particularly relate to a kind of test card for Quantitative detection pig serum pig blue-ear disease poison antibody and preparation method thereof.
Background technology
Pig blue-ear disease claims again porcine reproductive and respiratory syndrome (Porcinereproductiveandrespiratorysyndrome, PRRS) come into vogue from phase later 1980s, World Organization for Animal Health's definite designation in 1992, main infection pig, especially sow, this disease has a strong impact on its reproductive function, clinical principal character is miscarriage, produces stillborn foetus, mummy tire, weak tire, expiratory dyspnea, in pathogenic process, there will be two ear skin cyanosis of transience, therefore be called again blue otopathy. Pig blue-ear disease be by pig blue-ear disease poison pack up with in-pig breeding and various age pig, a kind of serious infectiousness epidemic disease that particularly breathing problem of piglet is feature, and the countries and regions of mainly raising pigs that have been found everywhere through the world, become one of main epidemic disease threatening pig industry sound development.
Vaccine is one of important means of controlling epidemic disease, and daily monitoring vaccine produces antibody and just given higher expectation by people. For plant, the daily monitoring of swinery is the important means of selecting vaccine, assessment immune programme for children reasonability, grasping swinery health status, also can reflect from the side whether pig farm management is reasonable, is also the Main Basis of in good time vaccinate simultaneously.
Have on the market pig blue-ear disease poison antibody colloidal gold rapid detection card, by the detection to sample, can judge roughly in pig body it is the blue ear antibody content of pig, animal doctor judges whether to inject the blue ear vaccine of pig according to testing result.
On same market, also a large amount of use detected the blue ear enzyme-labeled antibody of pig kit, it is ELISA Kit, ELISA kit needs ELIASA, incubation reaction condition, washes slat element, operation element environment and professional and technical personnel, detect in addition sample and also need complicated pre-treatment, as gather blood, separation of serum etc.; Although and collaurum is easy to detect fast, do not need professional and technical personnel and working environment, the sensitivity of its detection is low, and can only be qualitative, detection range is narrow. And the object of patent of the present invention is exactly the shortcoming separately that overcomes above two class kits, by shirtsleeve operation, fast, accurately, quantitatively (sxemiquantitative) analyzed with sensitivity, realize at the Site Detection of raising pigs at scene.
Summary of the invention
Object of the present invention is just to provide a kind of pig blue-ear disease poison antibody test card and preparation method thereof, can solve above-mentioned the deficiencies in the prior art part completely.
Object of the present invention realizes by following technical proposals:
A kind of pig blue-ear disease poison antibody test card, comprise assembling test card body in the enclosure, described test card body comprises base plate, on base plate, set gradually sample pad, label pad, nitrocellulose filter and blotting paper, on described nitrocellulose filter, the blue ear antigen of coated pig is detection line, coated goat-anti chicken antibody is nature controlling line, and on shell, counter sample pad is offered well, and the detection line on corresponding nitrocellulose filter and nature controlling line are offered form hole.
As preferably, described base plate is PVC base plate, on PVC base plate, paste successively from left to right sample pad, label pad, nitrocellulose filter and blotting paper, and the right-hand member of sample pad rides on the left end of label pad, the right-hand member of label pad rides on the left end of nitrocellulose filter, and blotting paper left end rides on the right-hand member of nitrocellulose filter.
As preferably, described label pad is made up of carrier substrate and label, label is in carrier substrate, to spray the skim that group of the lanthanides fluoroscopic examination microballoon becomes with group of the lanthanides fluorescence Quality Control microspheroidal, and detecting microballoon is the microballoon that is marked with the blue ear antigen of pig, and Quality Control microballoon is the Quality Control microballoon that is marked with chicken lgY or rabbit lgG.
As preferably, the blue ear antigen of pig adopting in the detection line on described nitrocellulose filter and label pad is deactivation pig blue-ear disease poison antigen, the blue ear vaccine of pig or the blue ear recombination structure of pig albumen M albumen, N albumen and GP5; The goat-anti chicken antibody that on described nitrocellulose filter, nature controlling line adopts is lgG or the goat-anti rabbit lgG of goat-anti chicken lgY.
As preferably, described detection microballoon and Quality Control microballoon are surface with hydroxyl or amino silica gel microball or polystyrene microsphere.
As preferably, described in be marked at the group of the lanthanides fluorescent microsphere on biological raw material group of the lanthanides fluorescence refer to the fluorescence that lanthanide series Eu, Sm or Tb and beta-diketone ligand produce. Its luminous efficiency is high, absorption spectrum is wide, emission spectrum is very narrow, this Tekes displacement is wide, makes the sensitivity of its spectrofluorimetry very high.
As preferably, described sample pad is to detect the sample pad of serum or blood plasma, or for detecting the sample pad of whole blood.
As preferably, this test card utilizes on the label of pig blue-ear disease poison antibody in pig blood sample, pig blue-ear disease poison antigen and nitrocellulose filter coated pig blue-ear disease poison antigen to detect pig blood sample pig blue-ear disease poison antibody in this by dual-antigen sandwich method.
As preferably, this test card is quantitative chromatography test card, it is to detect the detection line of test card after chromatography and the fluorescence signal that nature controlling line sends by detector, then the calibration curve storing according to the IC-card of this batch of test card, then calculated the concentration of thing to be checked from calibration curve by the fluorescence signal of detection line and nature controlling line.
A preparation method for pig blue-ear disease poison antibody test card, its feature exists, and comprises the following steps:
Step 1, nitrocellulose filter is sticked on PVC base plate, and the lgG that spraying has been diluted to the goat-anti chicken lgY of 0.2～0.5mg/ml on nitrocellulose filter forms nature controlling line and be diluted to the blue ear antigen formation of the pig detection line of 0.3～1.0mg/ml, smoke rear for subsequent use;
Step 2, with confining liquid sealing nitrocellulose filter 10～30 minutes, cleans nitrocellulose filter 10～30 minutes with 10mM phosphate buffer, for subsequent use after dry;
Step 3, preparation is marked with the group of the lanthanides fluorescent microsphere of chicken lgY and is marked with the group of the lanthanides fluorescent microsphere of the blue ear antigen of pig;
Step 4, being marked with chicken lgY and being marked with the group of the lanthanides fluorescent microsphere of the blue ear antigen of pig and being diluted to respectively concentration 0.05～0.4 μ g/ml and 1～8 μ g/ml, and is sprayed in carrier substrate and is formed label pad, smokes rear for subsequent use;
Step 5, is bonded at sample pad, label pad, blotting paper on PVC base plate successively, is assembled into kilocalorie, then cuts into test strips with cutter, is assemblied in test card shell, obtains pig blue-ear disease poison antibody test card.
Explanation of nouns:
The lgG of goat-anti chicken lgY: the immunoglobulin G of goat-anti chicken lgY;
Goat-anti rabbit lgG: the immunoglobulin G of goat-anti rabbit lgG;
Chicken lgY: chicken yolk antibody;
Rabbit lgG: rabbit immunoglobulin G;
Anti-pig RBC: anti-swine erythrocyte surface protein antibody.
Compared with prior art, beneficial effect of the present invention is: highly sensitive, batch interpolation and the difference between batch that detect are little, there is good repeatability, stable performance, both can detect whole blood sample, also can detect serum and plasma sample, with BVDV (bovine viral diarrhea virus), BDV border disease virus, the reproduction of PRRSV(pig and breath syndrome virus), PCV pig circular ring virus, PPV(pig parvoviral), PRV(porcine rotavirus) etc. antibody there is no cross reaction. Can realize pig blue-ear disease poison antibody field quick detection, there is higher practical value.
Brief description of the drawings
Fig. 1 is structural representation of the present invention;
Fig. 2 is the structural representation of test card body in Fig. 1;
Fig. 3 is the top view of Fig. 2;
Fig. 4 is pig blue-ear disease poison antibody test calibration curve.
Detailed description of the invention
Below in conjunction with specific embodiments and the drawings, the present invention is further illustrated.
As shown in Figures 1 to 4, a kind of pig blue-ear disease poison antibody test card, comprise the test card body being assemblied in shell 10, described test card body comprises PVC base plate 1, on PVC base plate 1, paste successively from left to right sample pad 2, label pad 3, nitrocellulose filter 4 and blotting paper 5, and the right-hand member of sample pad 2 rides on the left end of label pad 3, the right-hand member of label pad 3 rides on the left end of nitrocellulose filter 4, and blotting paper 5 left ends ride on the right-hand member of nitrocellulose filter 4. On described nitrocellulose filter 4, the blue ear antigen of coated pig is detection line 6, and coated goat-anti chicken antibody is nature controlling line 7. On shell 10, counter sample pad 2 is offered well 11, and detection line 6 and nature controlling line 7 on corresponding nitrocellulose filter 4 are offered form hole 12.
The goat-anti chicken antibody that on described nitrocellulose filter 4, nature controlling line 7 adopts is lgG or the goat-anti rabbit lgG of goat-anti chicken lgY.
Described label pad 3 is made up of carrier substrate and label, label is in carrier substrate, to spray the skim that group of the lanthanides fluoroscopic examination microballoon becomes with group of the lanthanides fluorescence Quality Control microspheroidal, and detecting microballoon is the microballoon that is marked with the blue ear antigen of pig, and Quality Control microballoon is the Quality Control microballoon that is marked with chicken lgY or rabbit lgG. Described detection microballoon and Quality Control microballoon are surface with hydroxyl or amino silica gel microball or polystyrene microsphere. And the particle diameter that detects microballoon and Quality Control microballoon is 50～500nm.
The blue ear antigen of pig adopting in detection line 6 on described nitrocellulose filter 4 and label pad 3 is deactivation pig blue-ear disease poison antigen, the blue ear vaccine of pig or the blue ear recombination structure of pig albumen M albumen, N albumen and GP5.
The sample pad that described sample pad 2 is detection serum or blood plasma, or for detecting the sample pad of whole blood. The sample pad that detects whole blood can or add the sample pad that has anti-pig RBC for hemofiltration film.
In described pig blood sample, on the label of pig blue-ear disease poison antibody, pig blue-ear disease poison antigen and nitrocellulose filter, coated pig blue-ear disease poison antigen is to detect pig blood sample pig blue-ear disease poison antibody in this by dual-antigen sandwich method.
The group of the lanthanides fluorescent microsphere of spray in label pad 3, its group of the lanthanides fluorescence refers to the fluorescence that lanthanide series and beta-diketone ligand produce, and the group of the lanthanides fluorescent material that is marked at the group of the lanthanides fluorescent microsphere on biological raw material refers to the fluorescence that lanthanide series Eu, Sm and Tb and beta-diketone ligand produce. Adopt detector to detect unique Fluorescence Characteristic of lanthanide series and complex.
Preparation stores the IC-card of pig blue-ear disease poison antibody test calibration curve: 6 parts of the calibration solutions that configuration contains pig blue-ear disease poison antibody, concentration is respectively 0,5,15,45,100,200ng/ml. The calibration solution of above-mentioned variable concentrations is added respectively in the well of the test card assembling, chromatography, after 15 minutes, detects by detector, and the testing result linear regression obtaining for 6 times is obtained to calibration curve, finally this curve data is deposited in IC-card. Every a collection of test card needs an IC-card of corresponding configuration.
The using method of this test card: sample to be checked (taking blood plasma as example) and sample diluting liquid are pressed to 1:40 dilution proportion, the sample 80ul having diluted is added in well 11, under the effect of blotting paper 5, sample moves to blotting paper 5 directions from sample pad 2. Chromatography 15min in the situation that avoiding strong illumination, then at detector, test card is detected, detector obtains the fluorescence signal of detection line 6 and nature controlling line 7, and criticize therewith the calibration curve storing in the IC-card of test card and contrast, calculate the concentration of sample to be checked by calibration curve, referring to the calibration curve of Fig. 4.
The preparation method of above-mentioned test card is as follows:
Step 1, nitrocellulose filter is sticked on PVC base plate, the lgG that adopts some film metal spraying special purpose machinery to spray the goat-anti chicken lgY that has been diluted to 0.5mg/ml on nitrocellulose filter forms nature controlling line and has been diluted to the blue ear inactivation of viruses formation of the pig detection line of 1.0mg/ml, discharge rate is 1 μ l/cm, then at the temperature of 37 DEG C, smokes 8 hours;
Step 2, with the PVA(polyvinyl alcohol of confining liquid (the BSA(bovine serum albumin(BSA) that consists of 5g/L of this confining liquid), mass concentration 1%), the phosphate buffer of the 10mM of pH7.5) seal nitrocellulose filter 10～30 minutes, clean nitrocellulose filter 30 minutes with 10mM phosphate buffer, dry for subsequent use after 2 hours;
Step 3, preparation is marked with the group of the lanthanides fluorescent microsphere of chicken lgY and is marked with the group of the lanthanides fluorescent microsphere of the blue ear recombination structure of pig Protein G P5 recombinant antigen, the group of the lanthanides fluorescent microsphere of 1ml is joined to the MES(2-(N-morpholine of 50mg) ethyl sulfonic acid) buffer solution (0.1M, pH7.0) in, add again 10mg carbodiimide (EDC) and 10mgN-HOSu NHS sulfonate sodium stirring and dissolving, room temperature reaction carries out centrifugally operated after 30 minutes, centrifugal sediment is redissolved with 50mM borate buffer (pH8.2), the chicken lgY that adds 2mg to dialyse, stirring reaction 24 hours at ambient temperature, then centrifugal, after sealing, in dilution, preserve again (preserving environment temperature is 2～8 DEG C), must be marked with the group of the lanthanides fluorescent microsphere of chicken lgY, adopt the group of the lanthanides fluorescent microsphere of the blue ear recombination structure of above-mentioned same method mark pig Protein G P5 recombinant antigen,
Step 4, being marked with chicken lgY and being marked with the group of the lanthanides fluorescent microsphere of the blue ear recombination structure of pig Protein G P5 recombinant antigen and being diluted to respectively concentration 0.1 μ g/ml and 3 μ g/ml, adopt a some film metal spraying machine to be sprayed on and in carrier substrate, form label pad, discharge rate is 2.5 μ l/cm, then at the temperature of 37 DEG C, smokes 8 hours;
Step 5, is bonded at sample pad, label pad, blotting paper on PVC base plate successively, is assembled into kilocalorie, then cuts into the wide test strips of 5mm with cutter, is assemblied in test card shell, obtains this pig blue-ear disease poison antibody test card.
The foregoing is only preferred embodiment of the present invention, not in order to limit the present invention, all any amendments of doing within the spirit and principles in the present invention, be equal to and replace and improvement etc., within all should being included in protection scope of the present invention.
Claims (9)
1. a pig blue-ear disease poison antibody test card, comprise assembling test card body in the enclosure, it is characterized in that: described test card body comprises base plate, on base plate, set gradually sample pad, label pad, nitrocellulose filter and blotting paper, on described nitrocellulose filter, the blue ear antigen of coated pig is detection line, coated goat-anti chicken antibody is nature controlling line, and on shell, counter sample pad is offered well, and the detection line on corresponding nitrocellulose filter and nature controlling line are offered form hole.
2. pig blue-ear disease poison antibody test card according to claim 1, it is characterized in that: described base plate is PVC base plate, on PVC base plate, paste successively from left to right sample pad, label pad, nitrocellulose filter and blotting paper, and the right-hand member of sample pad rides on the left end of label pad, the right-hand member of label pad rides on the left end of nitrocellulose filter, and blotting paper left end rides on the right-hand member of nitrocellulose filter.
3. pig blue-ear disease poison antibody test card according to claim 1, it is characterized in that: described label pad is made up of carrier substrate and label, label is in carrier substrate, to spray the skim that group of the lanthanides fluoroscopic examination microballoon becomes with group of the lanthanides fluorescence Quality Control microspheroidal, and detecting microballoon is the microballoon that is marked with the blue ear antigen of pig, and Quality Control microballoon is the Quality Control microballoon that is marked with chicken lgY or rabbit lgG.
4. pig blue-ear disease poison antibody test card according to claim 3, is characterized in that: the blue ear antigen of pig adopting in the detection line on described nitrocellulose filter and label pad is deactivation pig blue-ear disease poison antigen, the blue ear vaccine of pig or the blue ear recombination structure of pig albumen M albumen, N albumen and GP5; The goat-anti chicken antibody that on described nitrocellulose filter, nature controlling line adopts is lgG or the goat-anti rabbit lgG of goat-anti chicken lgY.
5. pig blue-ear disease according to claim 3 poison antibody test card, is characterized in that: described in be marked at the group of the lanthanides fluorescent microsphere on biological raw material group of the lanthanides fluorescence refer to the fluorescence of lanthanide series Eu, Sm or Tb and beta-diketone ligand generation.
6. pig blue-ear disease poison antibody test card according to claim 1, is characterized in that: described sample pad is to detect the sample pad of serum or blood plasma, or for detecting the sample pad of whole blood.
7. pig blue-ear disease according to claim 1 poison antibody test card, is characterized in that: this test card utilizes on the label of pig blue-ear disease poison antibody in pig blood sample, pig blue-ear disease poison antigen and nitrocellulose filter coated pig blue-ear disease poison antigen to detect pig blood sample pig blue-ear disease poison antibody in this by dual-antigen sandwich method.
8. pig blue-ear disease poison antibody test card according to claim 1, it is characterized in that: this test card is quantitative chromatography test card, it is to detect the detection line of test card after chromatography and the fluorescence signal that nature controlling line sends by detector, then the calibration curve storing according to the IC-card of this batch of test card, then calculated the concentration of thing to be checked from calibration curve by the fluorescence signal of detection line and nature controlling line.
9. a preparation method for pig blue-ear disease poison antibody test card, its feature exists, and comprises the following steps:
Step 1, nitrocellulose filter is sticked on PVC base plate, and the lgG that spraying has been diluted to the goat-anti chicken lgY of 0.2～0.5mg/ml on nitrocellulose filter forms nature controlling line and be diluted to the blue ear antigen formation of the pig detection line of 0.3～1.0mg/ml, smoke rear for subsequent use;
Step 2, with confining liquid sealing nitrocellulose filter 10～30 minutes, cleans nitrocellulose filter 10～30 minutes with 10mM phosphate buffer, for subsequent use after dry;
Step 3, preparation is marked with the group of the lanthanides fluorescent microsphere of chicken lgY and is marked with the group of the lanthanides fluorescent microsphere of the blue ear antigen of pig;
Step 4, being marked with chicken lgY and being marked with the group of the lanthanides fluorescent microsphere of the blue ear antigen of pig and being diluted to respectively concentration 0.05～0.4 μ g/ml and 1～8 μ g/ml, and is sprayed in carrier substrate and is formed label pad, smokes rear for subsequent use;
Step 5, is bonded at sample pad, label pad, blotting paper on PVC base plate successively, is assembled into kilocalorie, then cuts into test strips with cutter, is assemblied in test card shell, obtains pig blue-ear disease poison antibody test card.
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| CN107486715A (en) * | 2017-08-18 | 2017-12-19 | 湖南匡楚科技有限公司 | Pig feed raw material detects the assembly method of card automatic assembling machine |
| CN110894243A (en) * | 2019-12-16 | 2020-03-20 | 中国农业大学 | Porcine reproductive and respiratory syndrome virus chimeric antigen and colloidal gold immunochromatographic test strip for detecting porcine reproductive and respiratory syndrome virus antibody |
| CN111537729A (en) * | 2020-04-22 | 2020-08-14 | 中国海洋大学 | Paralichthys rhabdovirus disease rapid diagnosis test paper and preparation method thereof |
| CN111579795A (en) * | 2020-05-15 | 2020-08-25 | 安徽中起生物科技有限公司 | Immunofluorescence test paper for detecting porcine reproductive and respiratory syndrome virus GP5 protein IgY antibody, preparation method and application thereof |
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| Publication number | Priority date | Publication date | Assignee | Title |
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| CN111579795A (en) * | 2020-05-15 | 2020-08-25 | 安徽中起生物科技有限公司 | Immunofluorescence test paper for detecting porcine reproductive and respiratory syndrome virus GP5 protein IgY antibody, preparation method and application thereof |
| CN113504368A (en) * | 2021-07-14 | 2021-10-15 | 广东工业大学 | Test strip for detecting salmonella and preparation method thereof |
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Application publication date: 20160525 |