CN1584599A - Reagent strip for determining antibody and antigen of swine circular virus II - Google Patents
Reagent strip for determining antibody and antigen of swine circular virus II Download PDFInfo
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- CN1584599A CN1584599A CN 200410025288 CN200410025288A CN1584599A CN 1584599 A CN1584599 A CN 1584599A CN 200410025288 CN200410025288 CN 200410025288 CN 200410025288 A CN200410025288 A CN 200410025288A CN 1584599 A CN1584599 A CN 1584599A
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Abstract
A PCV2 antigen or antibody test paper consists of support layer which is water proof thin sheet layer and reaction reagent carrier absorption layer which comprises fibre layer, gold mark fibre layer and cellulose film layer in sequence from sample end as well as handle end being layer of water absorption material.
Description
Technical field
The present invention relates to a kind of disease of domestic animals quick diagnosis utensil, particularly relate to a kind of porcine circovirus 2 type (PCV2) antigen or antibody test test strips.
Background technology
Pig circular ring virus (porcine circovirus PCV) is the animal virus of the present minimum of finding, the about 17nm of virion diameter, and for covalence closed, ring-type, Single-stranded DNA virus, PCV have two kinds of genotype: PCV1 and PCV2.The PCV1 no pathogenicity, but extensively be present in the pig body and pig source continuous cell line, PCV2 has pathogenic to pig, cause that mainly piglet is disconnected
Milk back multisystem exhaustion syndrome (post-weaning multisystemic wasting syndrome, PMWS).PMWS is a kind of new infectious disease, finds in Canada first in 1991, mainly betides 5-12 weanling pig in age in week, shows as progressive emaciation, expiratory dyspnea, ochrodermia, diarrhoea, jaundice.Subsequently, the U.S., Britain, Germany, France, Ireland, Czech, Spain, TaiWan, China, Holland, Switzerland, Argentina, Mexico, Japan, Denmark, Italy, Korea S, Hungary, states such as Thailand have reported in succession should disease, epidemiology survey shows that this disease also is widely current in China, and causes suitable serious economy loss.PCV2 is except causing PMWS, also can cause growing and fattening pigs dermatitis and nephrotic syndrome (porcinedermatitis and nephropathy syndrome, PDNS), and with the breeding difficulty of farrowing sow, the congenital battle array of newborn piglet is quivered, and newborn piglet diarrhoea and hyperplasia necrotizing pneumonia are in close relations.According to the epidemiology of PMWS, clinical symptoms and cut open the inspection pathological change and can infect PCV2 and make tentative diagnosis still need be with laboratory diagnosis technology for detection PCV2 antigen and antibody thereof but make a definite diagnosis.To swinery PCV2 antibody horizontal, particularly the maternal antibody level is monitored; can understand pig body immune level really, hold the suitableeest immunity of eqpidemic disease opportunity, guarantee to obtain good immune effect; effectively the extensive damage that therefore causes is avoided in invasion and attack and the propagation of prevention and control PCV2.
At present, the PCV2 detection technique that adopts both at home and abroad mainly contains: viral separation and Culture, indirect immunofluorescence (IFA), immuno-enzymatic monolayer assay (IMPA), in situ hybridization (ISH), PCR (PCR) or the like.Though above-mentioned technology and method can detect PCV2 and antibody horizontal thereof, also obtain certain effect in practice, all there is the test operation complexity, length consuming time, need specific professional skill and instrument and equipment etc., often be limited to and carry out in the laboratory, be difficult in basic unit and popularize and promote.Therefore, development is applicable to quick, the easy PCV2 and the antibody horizontal appliance thereof of culturing basic unit, swinery PCV2 immune level is monitored in real time, set up science, swinery eqpidemic disease immune programme for children flexibly, to the prevention of eqpidemic disease with control significant.
Summary of the invention
The purpose of this invention is to provide a kind of porcine circovirus 2 type (PCV2) antigen or antibody test test strips.
It has supporting layer, reaction reagent carrier absorption layer, supporting layer is the lamella that do not absorb water, adsorbed layer is fixed on the supporting layer, and adsorbed layer is followed successively by fibrage from the sample end, gold mark fibrage, the cellulose rete, handle end is an absorbent material layer, is printed with contrast trace " | " and detects trace " | " on the cellulose rete, and its permutation and combination is " || ".The supporting layer lamella hard plastic slip that does not absorb water; or the cardboard bar that does not absorb water; the fibrage glass wool; absorbent material layer thieving paper; cellulose rete nitrocellulose filter; gold mark fibrage is coated with diaphragm with the glass wool of ADSORPTION OF GOLD mark albumen on glass wool and gold mark glass wool and absorbent paper layer, be partial to glass wool one side 0.5cm place at the glass wool diaphragm corresponding with gold mark glass wool intersection and print the sample mark line.
The good effect that the present invention is useful is: PCV2 antigen or antibody test test strips are simple to operate, everybody can operate, can better satisfy different levels personnel's needs, arrive individual breed etc. as eqpidemic disease monitoring, customs quarantine control, health and epidemic prevention, intensive culture, be easy to apply on a large scale, have vast market prospect and bigger economical, societal benefits.PCV2 antigen or antibody test test strips have following advantage:
(1) high specificity, the susceptibility height.PCV2 antigen or antibody test test strips are that the basis is prepared from monoclonal antibody or recombinant expressed dCap albumen; Monoclonal antibody is the anti-PCV2 dCap specificity homogeneous antibody of high-affinity, and specific recognition PCV2 Cap capsid protein is with the PCV1 no cross reaction; Reorganization dCap albumen is that non-totivirus antigen, security are good, does not contain irrelevant foreign protein, has good resistance originality, therefore has very high specificity and susceptibility.
(2) easy and simple to handle quick.When using PCV2 antigen or antibody test test strips to detect PCV2 antigen or antibody, need not to join in addition other reagent, was the decidable testing result in 2-10 minute.
(3) show testing result image, accurately directly perceived.PCV2 antigen or antibody test test strips with rufous " | " and " || " as the feminine gender and the positive mark that detect, show on cellulose membrane that promptly a brownish red " | " trace is illustrated in the detected sample liquid PCV2 virus or antibody is negative, article two, brownish red " || " trace be illustrated in the test sample PCV2 virus or antibody positive, the result judges image, directly perceived, accurate, simple and clear, be not prone to the erroneous judgement of false negative and false positive.
(4) cost is low, small investment.PCV2 antigen or antibody test test strips can detect on-the-spot complete operation, and detection settles at one go, and is with low cost, small investment, instant effect.
Description of drawings
Fig. 1 is PCV2 antigen and antibody test test strips side-looking structural representation;
Fig. 2 is PCV2 antigen and antibody test test strips plan structure synoptic diagram, among the figure: supporting layer 1, sample end fibrage 2. gold mark glass wool 3;. cellulose rete 4, handle end absorbent material layer 5, contrast trace 6 detects trace 7; sample end diaphragm 8, mark line 9, handle end diaphragm 10.
Fig. 3 is the antigen active qualification result of reorganization PCV2 dCap albumen, among the figure:
A is that the SDS-PAGE of recombinant protein analyzes, B is that the Western Blot of recombinant protein identifies, 1 is e. coli bl21,2 for containing the thalline of inducing of empty carrier pGEX-4T-1,3 do not induce thalline for containing recombinant plasmid, 4. induce the 4h thalline for containing recombinant plasmid, 5. are the reorganization GST-dCap fusion of purifying, 6 is the reorganization dCap albumen after the fibrin ferment cutting, and M. is protein Marker.
Fig. 4 is anti-PCV2 dCap monoclonal antibody specificity qualification result, among the figure:
1 is anti-PCV2 dCap monoclonal antibody mAb1 specific recognition GST-dCap fusion, 2 is anti-PCV2 dCap monoclonal antibody mAb2 specific recognition GST-dCap fusion, 3 is the natural Cap albumen of mAb1 specific recognition PCV2 virus, 4 is the natural Cap albumen of mAb2 specific recognition PCV2 virus, 5 is mAb1 nonrecognition PCV1 recombinant C ap albumen, and 6 is mAb2 nonrecognition PCV1 recombinant C ap albumen.
Embodiment
PCV2 antigen or antibody test test strips have supporting layer 1, reaction reagent carrier absorption layer, supporting layer is the lamella that do not absorb water, adsorbed layer is fixed on the supporting layer, and adsorbed layer is followed successively by fibrage 2 from the sample end, gold mark fibrage 3, cellulose rete 4, handle end is an absorbent material layer 5, is printed with contrast trace " | " 7 and detects trace " | " 6 on cellulose rete 4, and its permutation and combination is " || ".The supporting layer lamella hard plastic slip that does not absorb water; or the cardboard bar that does not absorb water; the fibrage glass wool; absorbent material layer thieving paper; cellulose rete nitrocellulose filter; gold mark fibrage is coated with diaphragm with the glass wool of ADSORPTION OF GOLD mark albumen on glass wool and gold mark glass wool and absorbent paper layer, be partial to glass wool one side 0.5cm place at the glass wool diaphragm corresponding with gold mark glass wool intersection and print the sample mark line.
The gold mark albumen of PCV2 Detection of antigen test strips is the anti-PCV2 stoning of colloid gold label localization signal peptide capsid protein (dCap) monoclonal antibody mAb1, contrast trace (7) on the cellulose rete is the goat anti-mouse IgG polyclonal antibody, and detecting trace (6) is anti-PCV2 dCap protein monoclonal antibody mAb2; The gold mark albumen of antibody test test strips is colloid gold label reorganization PCV2 dCap albumen, and the contrast trace (7) on the cellulose rete is anti-PCV2 dCap protein monoclonal antibody mAb1, and detecting trace (6) is the anti-pig IgG polyclonal antibody of goat.Reorganization PCV2 dCap albumen is to utilize the protokaryon efficient expression system to express and the genetic engineering recombinant protein of purifying, can with PCV2 positive serum specific bond.Anti-PCV2 dCap protein monoclonal antibody specific recognition PCV2 Cap albumen with pig circular ring virus 1 type (PCV1) Cap albumen no cross reaction, can effectively be distinguished PCV1 and PCV2 virus.
(1) preparation of reorganization PCV2 dCap albumen
Efficiently express PCV2 stoning localization signal peptide capsid protein (dCap) with technique for gene engineering, preparation reorganization PCV2dCap albumen.According to PCV2 gene order design Cap Auele Specific Primer (upstream primer: 5 '-GC
GGATCCAATGGCATCT TCAACAC-3 '; Downstream primer: 5 '-CCG
CTCGAGTTAAGGGTTAAGTGGG-3 '), increasing from pig PCV2 genome with PCR does not contain the Cap gene of nuclear localization signal, and it is subcloned on protokaryon efficient expression vector pGEX-4T-1, merges with GST at the N end, makes up the pGEX-dORF2 recombinant expression carrier.Behind 0.1mM IPTG abduction delivering, use the ultrasonic treatment bacterium, the fusion (GST-dCap) that utilizes GST affinity column purifying to express, remove the GST label with the fibrin ferment enzymolysis again, obtain reorganization PCV2 dCap albumen (Fig. 3), measure GST-dCap fusion and the active and protein concentration (10-20mg/ml) of reorganization dCap proteantigen respectively, be used to prepare the gold mark glass wool of anti-PCV2 dCap monoclonal antibody or PCV2 antibody test test strips.
(2) anti-PCV2 dCap MONOCLONAL ANTIBODIES SPECIFIC FOR
To recombinate PCV2 dCap albumen and immunologic adjuvant mixed in equal amounts, fully emulsified, be mouse 3 times with the only immune BALB/c of 50-100 μ g/, each 15-30 days at interval; Behind the 3rd booster immunization 3-4 days, with the bloodletting of immune mouse eyeball, anesthesia caused death, and in 75% alcohol-pickled 5-10min, aseptic its splenocyte of getting shreds and through 100 order nylon net filters, the centrifugal 10min of 1000r/min collects splenocyte; With 1 * 10
8Splenocyte and 2-5 * 10
7SP2/0 myeloma cell mix, the centrifugal 10min of 1000r/min abandons supernatant, 40%-50%PEG4000 (pH8.5-9.0) with 0.7-1ml in 37 ℃ water-bath slowly adds cell, behind the incubation 1min, slowly add serum-free 1640 nutrient culture media 15ml, to stop the effect of PEG, 37 ℃ of water-bath 5-10min, the centrifugal 10min of 1000r/min abandons supernatant, cell is resuspended in HAT selects in the nutrient culture media, and add 96 well culture plates (100 μ l-200 μ l/ hole), put 37 ℃ of 5%CO
2Cultivate in the incubator.Cultivate after 7-10 days, by 96 hole ELISA Plate, detect the culture supernatant of hybridoma, picking strong positive cell clone (OD with ELISA with the PCV2 dCap recombinant protein bag of 5-10 μ g/ml
492More than=0.8), through continuous 3 times limiting dilution assay cloning, prepare anti-PCV2 dCap monoclonal antibody, the chromosome number of the hybridoma cell strain of setting up is 92-98, the natural capsid protein (Cap) of the monoclonal antibody specific recognition reorganization PCV2 dCap albumen of its secretion and the PCV2 virus of purifying, with other albumen, particularly cross reaction (Fig. 4) does not take place in PCV1 Cap albumen, and affinity constant reaches 10
-9, light chain subtype is κ, and the heavy chain hypotype is IgG1, and prepared anti-PCV2 dCap monoclonal antibody is used to prepare the gold mark glass wool of PCV2 Detection of antigen test strips or the printing that the antibody test test strips contrasts trace.
(3) goat anti-mouse or pig IgG Polyclonal Antibody Preparation
Take mouse or pig blood, separation of serum prepares mouse or porcine blood serum IgG albumen with saturated ammonium sulfate, get 1 portion of mouse or porcine blood serum and add 2 parts of physiological saline mixings, add equal-volume saturated ammonium sulfate mixing, put 4 ℃ of refrigerator 3h, 4 ℃ of centrifugal 15min of 4000r/min abandon supernatant; With an amount of physiological saline solution precipitation, add saturated ammonium sulfate to final concentration 33%, put 4 ℃ of refrigerator 3h, 4 ℃ of centrifugal 15min of 4000r/min abandon supernatant; With a small amount of physiological saline solution precipitation, put in 4 ℃ of refrigerators and cross the liquid dialysis with physiological saline, change liquid 2～3 times, 4 ℃ of centrifugal 15min of 4000r/min collect supernatant, measure protein concentration (10-20mg/ml).With the mouse of 50～100 μ g/kg body weight or pig IgG albumen with immunologic adjuvant through subcutaneous and intramuscular injection immune health goat 3～4 times, the last immunity is after 10 days, venous blood collection, measure its serum antibody titer at 1: 2000 when above with ELISA, hyper-immune serum is collected in heart blood sampling or arteria carotis bloodletting; With saturated ammonium sulfate method purifying goat anti-mouse or pig IgG antibody, do not repeat, again through DEAE cellulose ion-exchange chromatography purifying.Prepared goat anti-mouse IgG polyclonal antibody is used for the printing of PCV2 Detection of antigen test strips contrast trace, and the anti-pig IgG polyclonal antibody of goat is used for the printing that PCV2 antibody test test strips detects trace.
(4) preparation of gold mark glass wool
Prepare aurosol with the sodium citrate reducing process, promptly in the 50-100ml 0.01-0.05% aqueous solution of chloraurate of boiling, add the 0.5-2% citric acid three sodium solution of 2-4ml, obtain the collaurum about diameter 15nm.With 0.1mol/L K
2CO
3Transfer collaurum pH to 8.5-9.5, with 1: 1000-1: 1300 mark is than anti-PCV2 dCap monoclonal antibody to be marked or PCV2 dCap recombinant protein are added in the pH8.5-9.5 aurosol, behind the mark 10min, add 20%PEG10000 to final concentration 0.05%, 4 ℃ of centrifugal 20min of 1500-3000r/min, remove unconjugated gold grain, 4 ℃ of centrifugal 1h of 12000r/min, abandon supernatant, after obtaining preliminary purification gold mark protein mixture, with propylene glucosan S-400 column chromatography, separation and purification gold mark albumen obtains the anti-PCV2 dCap monoclonal antibody of colloid gold label or the PCV2 dCap albumen of recombinating.With 1: 100-1: the colloid gold label protein adsorption of 1500 dilutions is in the processed glass cotton, and 4 ℃ of low-temperature vacuum dryings prepare gold mark glass wool.
(5) the enforcement structure of PCV2 antigen or antibody test test strips
PCV2 antigen or antibody test test strips are implemented structure referring to Fig. 1, Fig. 2, among the figure, 1 for supporting layer with the strip of foil that do not absorb water, can adopt the plastic slice bar in the enforcement or adopt the hard paper sheet material that does not absorb water, reaction reagent carrier absorption layer is by 2,3, and 4,5, combine, from sample end 2, above handle end 5 sticks on supporting layer 1 successively; Wherein 2 is sample end fibrage, can use glass fibre cotton to be called for short glass wool in the enforcement, 3 fibrages for ADSORPTION OF GOLD mark albumen, can adopt the processed glass cellucotton, abbreviate gold mark glass wool as, 4 is the cellulose rete, can adopt nitrocellulose filter in the enforcement, 5 is the handle end absorbent material layer, adopt thieving paper, all can as filter paper or other thieving paper, test strips length overall 8cm, width 0.4cm, 6 is the contrast trace of printing on nitrocellulose filter with goat anti-mouse IgG polyclonal antibody or anti-PCV2 dCap monoclonal antibody mAb1 " | ", 7 is the detection trace of printing on nitrocellulose filter with anti-PCV2 dCap monoclonal antibody mAb2 or the anti-pig IgG polyclonal antibody of goat " | ", and contrast trace on the cellulose rete and detection trace assembled arrangement are " || ".8 is diaphragm; the diaphragm that covers glass wool and gold mark glass wool is 8; on the diaphragm at deflection glass wool one side 0.5cm place on the white diaphragm of glass wool and gold mark glass wool intersection correspondence position, be printed on a mark line 9; be printed on arrow and Max printed words on mark line 9 the right, the diaphragm that covers on the water accepting layer filter paper is 10, and diaphragm can be used yellow or other color; 2; the intersection fiber infiltration that crosses one another each other of 3,4,5 each layers.
(6) PCV2 antigen or antibody test test strips are implemented the detection reaction principle
PCV2 Detection of antigen test strips detection reaction principle is: after PCV2 Detection of antigen test strips sample end inserts detected sample solution, solution to be checked drives antigen to be checked by siphon and spreads to nitrocellulose filter with gold mark albumen, and finally be penetrated in the filter paper layer, antigen to be checked combines with gold mark albumen in diffusion process, form gold mark albumen-antigenic compound, this compound can combine with the anti-PCV2 dCap monoclonal antibody mAb2 in detecting trace, generate rufous " | " mark, part can not combine and continue diffusion with the gold mark albumen of antibodies with the detection trace, on tunica fibrosa, combine with the goat anti-mouse IgG polyclonal antibody of contrast in the trace, generate rufous mark " | ", two kinds of marker combination stacks, form two rufous positive marks " || ", otherwise have only gold mark albumen to combine with the contrast trace, do not have gold mark albumen-antigenic compound to form, can not combine, then generate negative marker " | " with detecting trace.If do not have the rufous mark to show on the cellulose membrane, show that then test strips lost efficacy.
PCV2 antibody test test strips detection reaction principle is: after PCV2 antibody test test strips sample end inserts detected sample solution, solution to be checked drives antibody to be checked by siphon and spreads to nitrocellulose filter with gold mark albumen, and finally be penetrated in the filter paper layer, antibody to be checked combines with gold mark albumen in diffusion process, form gold mark albumen-antibody complex, this compound can combine with the anti-pig IgG polyclonal antibody of goat in detecting trace, generate rufous " | " mark, part can not combine and continue diffusion with the gold mark albumen of antibodies with the detection trace, on tunica fibrosa, combine with the anti-PCV2 Cap monoclonal antibody mAb1 of contrast in the trace, generate rufous mark " | ", two kinds of marker combination stacks, form two rufous positive marks " || ", otherwise have only gold mark albumen to combine with the contrast trace, do not have gold mark protein antibodies compound to form, can not combine, then generate negative marker " | " with detecting trace.If do not have the rufous mark to show on the cellulose membrane, show that then test strips lost efficacy.
(7) PCV2 antigen or antibody test test strips detect the example operation method
The preparation of test sample solution: gather pig lymph node to be checked, spleen, lung or serum, with sample diluting liquid or physiological saline 1: 5,1: 10,1: 20 doubling dilution, preparation gradient dilution sample solution.
PCV2 antigen or antibody test test strips detect the example operation method: PCV2 antigen or antibody test test strips sample end are inserted respectively in the gradient dilution detected sample liquid, insertion depth is no more than mark line 9, take out test strip after about 10 seconds, about 2 minutes of horizontal positioned, observations, manifesting two rufous marks " || " expression testing result on cellulose membrane is positive, it is negative to manifest a mark " | ", the sample greatest dilution that test strip is positive is the antigen or the antibody titer of sample to be checked, if test strip all is negative at each dilutability of sample to be checked, then show and do not contain PCV2 virus or antibody in the sample to be checked, if test strip does not have mark to show, show that then test strips lost efficacy.
Embodiment 1 PCV2 Detection of antigen test strips
Referring to Fig. 1, Fig. 2, press (1) method step preparation reorganization PCV2 dCap albumen among the embodiment, prepare anti-PCV2 dCap monoclonal antibody mAb1 and mAb2 by (2) method step among the embodiment, prepare the goat anti-mouse IgG polyclonal antibody by (3) method step among the embodiment, press (4) method step preparation gold mark glass wool among the embodiment, at last, various compositions are assembled into PCV2 Detection of antigen test strips, among the figure, 1 is supporting layer, make with the plastic slice bar, 2 is the fibrage of sample end, make with glass wool, 3 mark the fibrage of anti-PCV2 dCap monoclonal antibody for ADSORPTION OF GOLD, promptly be adsorbed with the glass wool of gold mark albumen, be called for short golden labeling antibody cotton, 4 is the cellulose rete, present embodiment adopts nitrocellulose filter, 5 for absorbent material layer is that handle end is made with filter paper, will number 2,3,4,5 each layers stick on the plastic slice bar 1 from left to right, on cellulose nitrate rete 4, and the 6 contrast traces of on nitrocellulose filter, printing for the goat anti-mouse IgG polyclonal antibody " | ", 7 is the detection trace of printing on nitrocellulose filter with anti-PCV2dCap monoclonal antibody mAb2 " | ", and two traces form combination trace " || " side by side.8 is white diaphragm; cover glass wool 2 and be adsorbed with the BT gold and mark on the glass wool; on the corresponding diaphragm 8-1 of 2 and 3 intersections position, be partial to glass wool 2 one side 0.5cm places and be printed on mark line 9; 9 right-hand member is printed on arrow and max printed words, and filter paper layer 5 is to be coated with yellow diaphragm 10 on the water accepting layer (handle end).
When using PCV2 Detection of antigen test strips to detect pig PCV2 infection, gather pork sample to be checked, after fully grinding, with physiological saline 1: 10 or dilution in 1: 20, prepare sample solution to be checked, PCV2 Detection of antigen test strips sample end is inserted in the detected sample liquid, and insertion depth is no more than mark line 9, takes out test strip after about 10 seconds, about 2 minutes of horizontal positioned, observations, the sample that manifests two rufous marks " || " on cellulose membrane is the PCV2 positive, shows that sample to be checked infects PCV2, manifest a mark " | " and be the PCV2 feminine gender, show that then sample to be checked does not have PCV2 to infect,, show that then test strips lost efficacy if test strip does not have mark to show.
Embodiment 2 PCV2 antibody test test strips
Referring to Fig. 1, Fig. 2, preparation reorganization PCV2 dCap albumen, anti-PCV2 dCap monoclonal antibody mAb1, with embodiment one roughly the same, do not repeat.Prepare the anti-pig IgG polyclonal antibody of goat by (3) method step among the embodiment, press (4) method step preparation gold mark glass wool among the embodiment, last, various compositions are assembled into PCV2 antibody test test strips, among the figure, 1,2,4,5,8,9,10 labels do not repeat with embodiment three.3,6,7 is different with embodiment three.In the present embodiment, 3 glass wools for ADSORPTION OF GOLD mark PCV2 dCap recombinant protein, 6 is anti-PCV2 dCap monoclonal antibody mAb1 contrast trace, 7 is that the anti-pig IgG polyclonal antibody of goat detects trace.
When using PCV2 antibody test test strips to detect porcine blood serum PCV2 antibody horizontal, gather pig blood to be checked, separation of serum, with physiological saline 1: 10,1: 20,1: 40 doubling dilution, preparation gradient dilution sample solution, PCV2 antibody test test strips sample end is inserted respectively in the gradient dilution detected sample liquid, insertion depth is no more than mark line 9, take out test strip after about 10 seconds, about 2 minutes of horizontal positioned, observations, the sample that manifests two rufous marks " || " on cellulose membrane is the PCV2 antibody positive, manifesting a mark " | " is the PCV2 negative antibody, the sample greatest dilution of the PCV2 positive is the PCV2 antibody titer of blood serum sample to be checked, if test strip all is negative at each dilutability of serum to be checked, shows that then serum to be checked does not contain PCV2 antibody, if test strip does not have mark to show, show that then test strips lost efficacy.
Claims (6)
1. porcine circovirus 2 type antigen or antibody test test strips, it is characterized in that: it has supporting layer (1), reaction reagent carrier absorption layer, supporting layer is fixed on the supporting layer for the lamella that do not absorb water, adsorbed layer, adsorbed layer is followed successively by fibrage (2) from the sample end, gold mark fibrage (3), cellulose rete (4), handle end are absorbent material layer (5), be printed with contrast trace " | " (7) and detection trace " | " (6) on cellulose rete (4), its permutation and combination is " | | ".
2. according to described a kind of porcine circovirus 2 type antigen of claim 1 or antibody test test strips; it is characterized in that: the said supporting layer lamella hard plastic slip that does not absorb water; or the cardboard bar that does not absorb water; the fibrage glass wool; absorbent material layer thieving paper; cellulose rete nitrocellulose filter; gold mark fibrage is marked the glass wool of albumen with ADSORPTION OF GOLD; be coated with diaphragm glass wool and golden marking on glass wool and the absorbent paper layer, at glass wool and the corresponding diaphragm deflection glass wool one side 0.5cm place printing sample mark line of gold mark glass wool intersection.
3. a kind of porcine circovirus 2 type antigen according to claim 1 and 2 or antibody test test strips, it is characterized in that: the gold mark albumen of said Detection of antigen test strips is colloid gold label resisting porcine circovirus 2 type stoning localization signal peptide capsid protein monoclonal antibody mAb1, contrast trace (7) on the cellulose rete is the goat anti-mouse IgG polyclonal antibody, and detecting trace (6) is resisting porcine circovirus 2 type stoning localization signal peptide capsid protein monoclonal antibody mAb2.
4. a kind of porcine circovirus 2 type antigen according to claim 1 and 2 or antibody test test strips, it is characterized in that: the gold mark albumen of said antibody test test strips is colloid gold label reorganization porcine circovirus 2 type stoning localization signal peptide capsid protein, contrast trace (7) on the cellulose rete is resisting porcine circovirus 2 type stoning localization signal peptide capsid protein monoclonal antibody mAb1, and detecting trace (6) is the anti-pig IgG polyclonal antibody of goat.
5. according to claim 3 or 4 described a kind of porcine circovirus 2 type antigen or antibody test test strips, it is characterized in that: said reorganization porcine circovirus 2 type stoning localization signal peptide capsid protein is the genetic engineering recombinant protein that utilizes expression of protokaryon efficient expression system and purifying, with porcine circovirus 2 type positive serum specific bond.
6. according to claim 3 or 4 described a kind of porcine circovirus 2 type antigen or antibody test test strips, it is characterized in that: said resisting porcine circovirus 2 type stoning localization signal peptide capsid protein monoclonal antibody specific recognition porcine circovirus 2 type capsid proteins, with pig circular ring virus 1 type capsid protein no cross reaction.
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| CN 200410025288 CN1584599A (en) | 2004-06-15 | 2004-06-15 | Reagent strip for determining antibody and antigen of swine circular virus II |
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| CN 200410025288 CN1584599A (en) | 2004-06-15 | 2004-06-15 | Reagent strip for determining antibody and antigen of swine circular virus II |
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| CN101650366A (en) * | 2008-08-11 | 2010-02-17 | 万志静 | Quick test paper for detecting enterovirus and method for preparing same |
| CN101768218A (en) * | 2010-03-02 | 2010-07-07 | 福建省农业科学院畜牧兽医研究所 | Preparation method for PCV-II Cap protein monoclonal antibody, antibody and application |
| CN102735680A (en) * | 2012-07-05 | 2012-10-17 | 北京大北农科技集团股份有限公司 | Test strip for quickly detecting porcine circovirus 2 (PCV2) antibody by adopting colloidal gold |
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| CN101650366A (en) * | 2008-08-11 | 2010-02-17 | 万志静 | Quick test paper for detecting enterovirus and method for preparing same |
| CN101650366B (en) * | 2008-08-11 | 2014-04-02 | 万志静 | Quick test paper for detecting enterovirus and method for preparing same |
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| CN101768218B (en) * | 2010-03-02 | 2013-05-01 | 福建省农业科学院畜牧兽医研究所 | Preparation method for PCV-II Cap protein monoclonal antibody, antibody and application |
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| CN105717293A (en) * | 2014-12-03 | 2016-06-29 | 洛阳普莱柯万泰生物技术有限公司 | Kit for detecting porcine circovirus type 2 |
| CN105675873A (en) * | 2016-01-04 | 2016-06-15 | 洛阳普莱柯万泰生物技术有限公司 | Detection kit and application thereof |
| CN105675873B (en) * | 2016-01-04 | 2017-11-10 | 洛阳普莱柯万泰生物技术有限公司 | A kind of detection kit and its application |
| CN105527429A (en) * | 2016-01-21 | 2016-04-27 | 成都微瑞生物科技有限公司 | Porcine circovirus antibody detection card and preparation method thereof |
| CN107340398A (en) * | 2017-06-06 | 2017-11-10 | 北京热景生物技术股份有限公司 | Turn the anti-Miao Le Shi pipes hormone quantitative determination reagent and method of luminescence method based on |
| CN107340398B (en) * | 2017-06-06 | 2019-07-26 | 北京热景生物技术股份有限公司 | Turn the anti-Miao Le Shi pipe hormone quantitative determination reagent and method of luminescence method based on |
| CN110687302A (en) * | 2019-10-30 | 2020-01-14 | 浙江省医学科学院 | Test paper strip and method for rapidly detecting porcine circovirus type 2 antigen or antibody colloidal gold |
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