CN105527429A - Porcine circovirus antibody detection card and preparation method thereof - Google Patents
Porcine circovirus antibody detection card and preparation method thereof Download PDFInfo
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- CN105527429A CN105527429A CN201610039823.4A CN201610039823A CN105527429A CN 105527429 A CN105527429 A CN 105527429A CN 201610039823 A CN201610039823 A CN 201610039823A CN 105527429 A CN105527429 A CN 105527429A
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- G—PHYSICS
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- 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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- 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
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- 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
- G01N33/558—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
- G01N2333/00—Assays involving biological materials from specific organisms or of a specific nature
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Abstract
The invention discloses a porcine circovirus antibody detection card and a preparation method thereof. The porcine circovirus antibody detection card comprises a detection card body assembled in a shell, wherein the detection card body comprises a bottom plate; the bottom plate is provided with a sample cushion, a marking cushion, a nitrocellulose film and water-absorbing paper in sequence; the nitrocellulose film is wrapped with a porcine circovirus antigen serving as a detection line, and is wrapped with goat anti-chicken antibody serving as a quality control line; the shell is provided with a sample adding hole corresponding to the sample cushion; a window hole is formed corresponding to the detection line and the quality control line on the nitrocellulose film. The porcine circovirus antibody detection card has the advantages of high sensitivity, small intra-batch difference and inter-batch difference, high repeatability, stable performance, capability of realizing field rapid detection of the porcine circovirus antibody, and 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 pig circular ring virus antibody in Quantitative detection Swine serum and preparation method thereof.
Background technology
Porcine circovirus 2 type (Porcinecircovirustype2, PCV2) is the nineties in 20th century, and in world wide, people detect a kind of Novel pig little ring-type sample virus (Allan and Ellis, 2000) sick pig and some without in obvious clinical symptoms pig body.This virus is different from the known PCV be separated by PK-15 pollution of cell culture, and someone advises original PCV called after PCV-II I type (PCV-1), by the emerging PCV called after circovirus type II (PCV-2) relevant to clinical disease.PCV-2 there will be pmws (PMWS), the scorching nephrotic syndrome (PNDS) of pigskin, respiratory diseases in pigs mixing disease, breeding difficulty syndrome etc. after infecting.Now the various disease conditions caused by PCV2 is all named " Porcine circovirus desease ".In PCV2, the harm that PMWS causes pig industry is the most serious, and it mainly causes pig gradually to become thin, suffers from diarrhoea, respiratory symptom, ochrodermia or occur that jaundice, production performance reduce.PCV2 also encroaches on the immune system of pig in addition, suppresses the immunologic function of pig, can also interact, increase the weight of the harm of these diseases with the epidemic disease cause of disease such as pig blue-ear disease, influenza, Eaton agent pneumonia, haemophilus parasuis and streptococcus.Huge economic loss is caused to pig industry.
Vaccine immunity is the Main Means of China's pig annulus prevention and control, gather the rear Swine serum of immunity, and to detect tiring of pig annulus 2 antiviral antibody in serum be the main method of monitoring pig annulus, namely evaluating the resistivity of pig annulus immune effect of vaccine, immune programme for children and monitoring pig circular ring virus, is also the Main Basis of in good time vaccinate.
Have pig circular ring virus antibody colloidal gold rapid detection card on the market, by the detection to sample, can judge roughly in pig body it is pig annulus antibody content, animal doctor judges whether to need injection pig annulus vaccine according to testing result.
On same market, also a large amount of use detects pig circular ring virus enzyme-labeled antibody kit, i.e. ELISA Kit, ELISA kit needs microplate reader, incubation reaction condition, washes slat element, operation element environment and professional and technical personnel, detect the sample pre-treatment that also needs are complicated in addition, as gathered blood, separation of serum etc.; Although and collaurum is easy to detect fast, do not need professional and technical personnel and working environment, its sensitivity detected is low, and can only be qualitative, and sensing range is narrow.And this patent pig annulus antibody kit just overcomes the respective shortcoming of above two class kits, simple to operate, realize scene, fast, accurately, with sensitivity quantitatively (sxemiquantitative) detects.
Summary of the invention
Object of the present invention is just to provide a kind of pig circular ring virus antibody test card and preparation method thereof, can solve above-mentioned the deficiencies in the prior art part completely.
Object of the present invention is realized by following technical proposals:
A kind of pig circular ring virus antibody test card, comprise assembling test card body in the enclosure, described test card body comprises base plate, base plate pastes sample pad from left to right successively, label pad, nitrocellulose filter and thieving 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, thieving paper left end rides on the right-hand member of nitrocellulose filter, described label pad is made up of carrier substrate and label, label is carrier substrate sprays the skim that lanthanide fluoro detects microballoon and the formation of lanthanide fluoro Quality Control microballoon, described detection microballoon and Quality Control microballoon are surface with the silica gel microball of hydroxyl or amino or polystyrene microsphere, described nitrocellulose filter wrapping by pig annulus antigen is detection line, bag is nature controlling line by goat-anti chicken antibody, on shell, well offered by counter sample pad, detection line on corresponding nitrocellulose filter and nature controlling line offer form hole.
As preferably, described detection microballoon is the microballoon being marked with pig annulus antigen, and Quality Control microballoon is the Quality Control microballoon being marked with chicken lgY or rabbit lgG.
As preferably, the pig annulus antigen that the detection line on described nitrocellulose filter and label pad adopt is deactivation pig circular ring virus antigen, pig annulus vaccine, pig circular ring virus 2 virus capsid protein PCV2-Cap or 2 porcine circovirus polypeptide; 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 the silica gel microball of hydroxyl or amino or polystyrene microsphere.
As preferably, described in the lanthanide fluoro of lanthanide fluoro microballoon that is marked on biological raw material refer to the fluorescence that lanthanide series Eu, Sm or Tb and beta-diketone ligand produce.Its luminescence efficiency is high, absorption spectrum is wide, emission spectrum is very narrow, this Tekes displacement is wide, makes the sensitivity of its fluorescent spectroscopy very high.
As preferably, described sample pad is detect the sample pad of serum or blood plasma, or for detecting the sample pad of whole blood.
As preferably, this test card utilizes the label of pig circular ring virus antibody in pig blood sample, pig circular ring virus antigen and nitrocellulose filter wrap and is detected pig circular ring virus antibody in pig blood sample by pig circular ring virus antigen by dual-antigen sandwich method.
As preferably, this test card is quantitative chromatography test card, it is the fluorescence signal sent by detection line and the nature controlling line of test card after detector detection chromatography, then according to the typical curve that the IC-card of this batch of test card stores, then the concentration of thing to be checked is calculated from typical curve by the fluorescence signal of detection line and nature controlling line.
A preparation method for pig circular ring virus antibody test card, its feature exists, and comprises the following steps:
Step one, nitrocellulose filter is pasted onto on PVC base plate, and the lgG spraying the goat-anti chicken lgY being diluted to 0.2 ~ 0.5mg/ml on nitrocellulose filter forms nature controlling line and has been diluted to the pig annulus antigen formation detection line of 0.3 ~ 1.0mg/ml, smokes rear for subsequent use;
Step 2, closes nitrocellulose filter 10 ~ 30 minutes with confining liquid, cleans nitrocellulose filter 10 ~ 30 minutes with 10mM phosphate buffer, for subsequent use after dry;
Step 3, preparation is marked with the lanthanide fluoro microballoon of chicken lgY and is marked with the lanthanide fluoro microballoon of pig annulus antigen;
Step 4, being marked with chicken lgY and being marked with the lanthanide fluoro microballoon of pig annulus antigen and being diluted to concentration 0.05 ~ 0.4 μ g/ml and 1 ~ 8 μ g/ml respectively, and is sprayed in carrier substrate and is formed label pad, smoked rear for subsequent use;
Step 5, is bonded at sample pad, label pad, thieving paper successively on PVC base plate, is assembled into kilocalorie, then cut into test strips with shearer, is assemblied in test card shell, namely obtains pig circular ring virus 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 detected and difference between batch little, there is good repeatability, stable performance, both can detect whole blood sample, also can detect serum and plasma sample, with the reproduction of PRRSV(pig and breath syndrome virus), CSFV CSFV, PPV(pig parvoviral), PRV(porcine rotavirus) etc. antibody there is no cross reaction.Pig circular ring virus antibody field quick detection can be realized, there is higher practical value.
Accompanying drawing explanation
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 vertical view of Fig. 2;
Fig. 4 is pig circular ring virus antibody test typical curve.
Embodiment
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 circular ring virus antibody test card, comprise the test card body be assemblied in shell 10, described test card body comprises PVC base plate 1, PVC base plate 1 pastes sample pad 2, label pad 3, nitrocellulose filter 4 and thieving paper 5 from left to right successively, 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 thieving paper 5 left end rides on the right-hand member of nitrocellulose filter 4.Described nitrocellulose filter 4 wrapping by pig annulus antigen is detection line 6, and wrapping by goat-anti chicken antibody is nature controlling line 7.On shell 10, well 11 offered by counter sample pad 2, and the detection line 6 on corresponding nitrocellulose filter 4 and nature controlling line 7 offer form hole 12.
The goat-anti chicken antibody that on described nitrocellulose filter 4, nature controlling line 4 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 carrier substrate sprays the skim that lanthanide fluoro detects microballoon and the formation of lanthanide fluoro Quality Control microballoon, and detection microballoon is the microballoon being marked with pig annulus antigen, Quality Control microballoon is the Quality Control microballoon being marked with chicken lgY or rabbit lgG.Described detection microballoon and Quality Control microballoon are surface with the silica gel microball of hydroxyl or amino or polystyrene microsphere.Further, the particle diameter detecting microballoon and Quality Control microballoon is 50 ~ 500nm.
The pig annulus antigen that detection line 6 on described nitrocellulose filter 4 and label pad 3 adopt is deactivation 2 porcine circovirus antigen, pig annulus vaccine, pig circular ring virus 2 virus capsid protein PCV2-Cap or pig annulus 2 polypeptide.
Described sample pad 2 is detect the sample pad of serum or blood plasma, or for detecting the sample pad of whole blood.The sample pad detecting whole blood for hemofiltration film or can add the sample pad having anti-pig RBC.
In described pig blood sample, the label of pig circular ring virus antibody, pig circular ring virus antigen and nitrocellulose filter wrapping by pig circular ring virus antigen is detect pig circular ring virus antibody in pig blood sample by dual-antigen sandwich method.
The lanthanide fluoro microballoon of spray in label pad 3, its lanthanide fluoro refers to the fluorescence that lanthanide series and beta-diketone ligand produce, and the lanthanide fluoro material being marked at the lanthanide fluoro microballoon on biological raw material refers to the fluorescence that lanthanide series Eu, Sm and Tb and beta-diketone ligand produce.Detector is adopted to detect unique Fluorescence Characteristic of lanthanide series and complex.
Preparation stores the IC-card of pig circular ring virus antibody test typical curve: the calibration solution 6 part of configuration containing pig circular ring virus antibody, concentration is respectively 0,5,15,45,100,200ng/ml.Added respectively by the calibration solution of above-mentioned variable concentrations in the well of the test card assembled, chromatography, after 15 minutes, is detected by detector, and the testing result linear regression obtained 6 times obtains typical curve, finally by this curve data stored in IC-card.Every a collection of test card needs a corresponding configuration IC-card.
The using method of this test card: measuring samples (for blood plasma) and sample diluting liquid are pressed 1:40 dilution proportion, and add in well 11 by the sample 80ul diluted, under the effect of thieving paper 5, sample moves from sample pad 2 to thieving paper 5 direction.The chromatography 15min when avoiding strong illumination, then with detector, test card is detected, detector obtains the fluorescence signal of detection line 6 and nature controlling line 7, and criticize the typical curve stored in the IC-card of test card therewith and contrast, the concentration of sample to be checked is calculated, see the typical curve of Fig. 4 by typical curve.
The preparation method of above-mentioned test card is as follows:
Step one, nitrocellulose filter is pasted onto on PVC base plate, the lgG adopting some film metal spraying custom-built machine to spray the goat-anti chicken lgY being diluted to 0.3mg/ml on nitrocellulose filter forms nature controlling line and has been diluted to the 2 porcine circovirus recombination structure albumen formation detection line of 0.5mg/ml, discharge rate is 1 μ l/cm, then at the temperature of 37 DEG C, smokes 8 hours;
Step 2, PVA(polyvinyl alcohol (PVA) by confining liquid (the BSA(bovine serum albumin(BSA) consisting of 5g/L of this confining liquid), mass concentration 1%), the phosphate buffer of the 10mM of pH7.5) close nitrocellulose filter 30 minutes, nitrocellulose filter is cleaned 30 minutes with 10mM phosphate buffer, for subsequent use after dry 2 hours;
Step 3, preparation is marked with the lanthanide fluoro microballoon of chicken lgY and is marked with the lanthanide fluoro microballoon of 2 porcine circovirus recombinant antigen, the lanthanide fluoro microballoon of 50mg is joined the MES(2-(N-morpholine of 50mg) ethyl sulfonic acid) damping fluid (0.1M, pH7.0) in, add 10mg carbodiimide (EDC) and 10mgN-N-Hydroxysuccinimide sulfonate sodium stirring and dissolving again, room temperature reaction carried out centrifugally operated after 30 minutes, centrifugal sediment 50mM borate buffer (pH8.2) is redissolved, add the chicken lgY that 2mg dialysed, stirring reaction 24 hours at ambient temperature, then centrifugal, after closing, (Conservation environment temperature is 2 ~ 8 DEG C) is preserved again in dilution, the lanthanide fluoro microballoon of chicken lgY must be marked with, above-mentioned same method is adopted to mark the lanthanide fluoro microballoon of 2 porcine circovirus recombinant antigen,
Step 4, being marked with chicken lgY and being marked with the lanthanide fluoro microballoon of 2 porcine circovirus recombinant antigen and being diluted to concentration 0.1 μ g/ml and 3 μ g/ml respectively, adopt some film metal spraying machine to be sprayed in carrier substrate and 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, thieving paper successively on PVC base plate, is assembled into kilocalorie, then cuts into the wide test strips of 5mm with shearer, is assemblied in test card shell, namely obtains this pig circular ring virus antibody test card.
The foregoing is only preferred embodiment of the present invention, not in order to limit the present invention, all any amendments done within the spirit and principles in the present invention, equivalent replacement and improvement etc., all should be included within protection scope of the present invention.
Claims (8)
1. a pig circular ring virus antibody test card, comprise assembling test card body in the enclosure, it is characterized in that: described test card body comprises base plate, base plate pastes sample pad from left to right successively, label pad, nitrocellulose filter and thieving 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, thieving paper left end rides on the right-hand member of nitrocellulose filter, described label pad is made up of carrier substrate and label, label is carrier substrate sprays the skim that lanthanide fluoro detects microballoon and the formation of lanthanide fluoro Quality Control microballoon, described detection microballoon and Quality Control microballoon are surface with the silica gel microball of hydroxyl or amino or polystyrene microsphere, described nitrocellulose filter wrapping by pig annulus antigen is detection line, bag is nature controlling line by goat-anti chicken antibody, on shell, well offered by counter sample pad, detection line on corresponding nitrocellulose filter and nature controlling line offer form hole.
2. pig circular ring virus antibody test card according to claim 1, is characterized in that: described detection microballoon is the microballoon being marked with pig annulus antigen, Quality Control microballoon is the Quality Control microballoon being marked with chicken lgY or rabbit lgG.
3. pig circular ring virus antibody test card according to claim 2, is characterized in that: the pig annulus antigen that the detection line on described nitrocellulose filter and label pad adopt is deactivation pig circular ring virus antigen, pig annulus vaccine, pig circular ring virus 2 virus capsid protein PCV2-Cap or 2 porcine circovirus polypeptide; 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.
4. pig circular ring virus antibody test card according to claim 2, is characterized in that: described in the lanthanide fluoro of lanthanide fluoro microballoon that is marked on biological raw material refer to the fluorescence that lanthanide series Eu, Sm or Tb and beta-diketone ligand produce.
5. pig circular ring virus antibody test card according to claim 1, is characterized in that: described sample pad is detect the sample pad of serum or blood plasma, or for detecting the sample pad of whole blood.
6. pig circular ring virus antibody test card according to claim 1, is characterized in that: this test card utilizes the label of pig circular ring virus antibody in pig blood sample, pig circular ring virus antigen and nitrocellulose filter wrap and detected pig circular ring virus antibody in pig blood sample by pig circular ring virus antigen by dual-antigen sandwich method.
7. pig circular ring virus antibody test card according to claim 1, it is characterized in that: this test card is quantitative chromatography test card, it is the fluorescence signal sent by detection line and the nature controlling line of test card after detector detection chromatography, then according to the typical curve that the IC-card of this batch of test card stores, then the concentration of thing to be checked is calculated from typical curve by the fluorescence signal of detection line and nature controlling line.
8. a preparation method for pig circular ring virus antibody test card, its feature exists, and comprises the following steps:
Step one, nitrocellulose filter is pasted onto on PVC base plate, and the lgG spraying the goat-anti chicken lgY being diluted to 0.2 ~ 0.5mg/ml on nitrocellulose filter forms nature controlling line and has been diluted to the pig circular ring virus antigen formation detection line of 0.3 ~ 1.0mg/ml, smokes rear for subsequent use;
Step 2, closes nitrocellulose filter 10 ~ 30 minutes with confining liquid, cleans nitrocellulose filter 10 ~ 30 minutes with 10mM phosphate buffer, for subsequent use after dry;
Step 3, preparation is marked with the lanthanide fluoro microballoon of chicken lgY and is marked with the lanthanide fluoro microballoon of pig annulus antigen;
Step 4, being marked with chicken lgY and being marked with the lanthanide fluoro microballoon of pig annulus antigen and being diluted to concentration 0.05 ~ 0.4 μ g/ml and 1 ~ 8 μ g/ml respectively, and is sprayed in carrier substrate and is formed label pad, smoked rear for subsequent use;
Step 5, is bonded at sample pad, label pad, thieving paper successively on PVC base plate, is assembled into kilocalorie, then cut into test strips with shearer, is assemblied in test card shell, namely obtains pig circular ring virus antibody test card.
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| CN106053827A (en) * | 2016-05-17 | 2016-10-26 | 上海凯璟生物科技有限公司 | Irritable bowel syndrome marker Vinculin antibody detection kit and preparation method thereof |
| CN107037211A (en) * | 2016-11-04 | 2017-08-11 | 上海海茉生物科技有限公司 | A kind of test-strips of quantitative detection compound and the detection method of lateral immunochromatography |
| CN108226514A (en) * | 2017-08-07 | 2018-06-29 | 华中农业大学 | A kind of newcastle disease virus antibody rapid quantitative detection reagent box and its application |
| CN108828220A (en) * | 2018-03-29 | 2018-11-16 | 杭州泰熙生物技术有限公司 | The detection card and preparation method thereof of Benzodiazepine substance in a kind of detection health care product |
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