CN103743901A - Colloidal gold test strip for detecting canine parvovirus - Google Patents
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Abstract
本发明涉及生物检测领域，具体提供了一种用于检测犬细小病毒的胶体金试纸条，所述胶体金试剂条包含：1）包被犬细小病毒抗原的多克隆抗体和二抗IgG两个条带的硝酸纤维膜；2）含有胶体金标记犬细小病毒抗原的单克隆抗体的玻璃纤维膜。本发明胶体金试纸条采用双抗体夹心方法及免疫层析技术检测犬细小病毒抗原，具有特异性好、灵敏度高、操作简单、检测快速准确、不需要复杂的设备等优点，可以满足临床检验的要求。
The present invention relates to the field of biological detection, and specifically provides a colloidal gold test strip for detecting canine parvovirus. The colloidal gold test strip comprises: 1) a polyclonal antibody and a secondary antibody IgG coated nitrocellulose membrane with 2 bands; 2) glass fiber membrane containing monoclonal antibody to canine parvovirus antigen labeled with colloidal gold. The colloidal gold test strip of the present invention adopts double-antibody sandwich method and immune chromatography technology to detect canine parvovirus antigen, has the advantages of good specificity, high sensitivity, simple operation, fast and accurate detection, and does not require complex equipment, etc., and can meet clinical testing requirements. requirements.
Description
Technical field
The present invention relates to field of biological detection, be specifically related to a kind of colloidal gold strip for detection of canine parvovirus and application thereof.
Background technology
Colloidal gold immunochromatographimethod technology (Gold Immunochromatographic assay, GICA) be a kind of mark diagnostic technology based on antigen and antibody specific reaction, within 1971, Faulk introduces immunochemistry collaurum, thereby has been born immune colloidal gold technique.Risen again the eighties in 20th century and take the immune colloid gold detection technique that NC film is solid phase carrier, this technology is a kind of quick and simple novel diagnostic techniques growing up in the technical foundation such as McAb, ELISA.The nineteen ninety-fives such as Liu Yukai are made early pregnancy quick diagnosis by immunochromatographic method and measure, and have occurred subsequently the widespread use of people's early pregnancy test strips.Hao Guilan etc. utilize immune colloid gold test, in the dove cotton swab sample 30min that takes to die of illness, complete the rapid differential diagnosis to a morbidity dove group.The gaggle that the use immune colloid gold test diagnosis such as Wang Changmei go out to suffer from I type poultry paramyxovirus disease.Xu Gelin etc. have prepared and have been suitable for the diagnosis test paper that field is detected Rabies Virus Antigen in animal brain or saliva.Zheng Ming etc. use dual-antigen sandwich method in domestic model antibody against swine fever virus detect immuno-chromatographic test paper strip.Liu Chunlong etc. have set up the Colloidal Gold of detection Ig G in bovine Colostrum (IgG) content.Li Kemei etc. are the envelope antigen as this check-out console with cloth Shandong bacterium purified protein derivative (PPD), has set up the new method that detects the anti-cloth Shandong bacteria antibody in people and different animals blood sample by indirect method.
Collaurum (Colloidal gold) is the hydrosol of gold chloride (HAuCl4), and gold chloride, under the effect of reductive agent, aggregates into the gold grain of specific size, and because electrostatic interaction becomes a kind of stable colloidal state.Collaurum is electronegative under alkali condition, borrows electrostatic attraction and forms strong bonded with the positive charge group of protein molecule.
The principle of GICA is to using collaurum as trace labelling thing, take microporous barrier as solid phase carrier, coated known antigens or antibody, add after test sample, transudation or capillary siphoning effect through microporous barrier are combined antibody or antigen coated antigen or antibody on film in sample, then are reacted with it and formed red visible result by colloid gold label thing.The most frequently used is double antibodies sandwich method detectable antigens.
During mensuration, test strips lower end is immersed in liquid sample, lower end absorbent material is that imbitition moves to upper end, makes the colloidal gold composite on dry plate redissolve, and drive it to film bar, to ooze and move while flowing through C place.Specific antigen to be measured in sample, can be combined by the antibody in colloidal gold composite, and this antigen antibody complex flow to test section and by insolubilized antibody, caught, and shows red reaction lines (T) on film.Superfluous colloidal gold composite continues to move ahead, and to being combined with the anti-mouse IgG of solid phase (monoclonal antibody in colloidal gold composite is mouse IgG) with reference to district, and shows red Quality Control lines (R).' negative ' specimens is reactionless lines, and only show Quality Control lines
At present, the most often the method for the detection canine parvovirus (CPV) of application is blood clotting (HA) and hemagglutination-inhibition test (HI) and ELISA method.Wherein HA test is the most economical, but also existent defect obtains the fresh red blood cell that involves great expense more difficult on the one hand, and on the other hand, the method is only applicable to CPV and infects later stage pattern detection, the requirement of inadaptable early stage, fast detecting in recent years.ELISA method is applied more, but operation requirements is higher, and needs microplate reader result of determination, is therefore unsuitable for basic unit and uses.
Summary of the invention
In order to solve problems of the prior art, the object of this invention is to provide a kind of CPV of being applicable to and infect early stage detection reagent strip, utilize this reagent strip to detect canine parvovirus, method is simple, detection time is short, cost is low.
In order to realize the object of the invention, first the present invention provides a kind of colloidal gold strip for detection of canine parvovirus, and described colloid gold reagent bar comprises:
1) the coated polyclonal antibody of canine parvovirus antigen and the nitrocellulose membrane of two bands of two anti-IgG;
2) glass fibre membrane of the monoclonal antibody that contains colloid gold label canine parvovirus antigen.
Wherein, two described anti-IgG are sheep anti-mouse igg antibody.
Further, in described nitrocellulose membrane, the concentration of the polyclonal antibody of canine parvovirus antigen is 1mg/mL, and the concentration of described sheep anti-mouse igg antibody is 1mg/mL.
As preferably, in described glass fibre membrane, the mark pH value of the monoclonal antibody of the canine parvovirus antigen of colloid gold label is 8.5, and monoclonal antibody is combined concentration with collaurum be 40 μ g/mL.
As preferably, described colloid gold particle mean diameter is 25nm.
Further, described colloidal gold strip can detect the minimum HA of canine parvovirus and tires as 1:80.
Aforementioned colloidal gold strip, also comprises base plate, sample pad and thieving paper, on described base plate, pastes nitrocellulose membrane, and thieving paper is pasted in nitrocellulose membrane top, and nitrocellulose membrane below overlaps sticking glass tunica fibrosa and sample pad successively mutually.
The present invention also provides a kind of method of preparing aforementioned colloidal gold strip, comprises the steps:
1) prepare the polyclonal antibody of canine parvovirus antigen;
2) preparation of nitrocellulose membrane: the polyclonal antibody of canine parvovirus antigen is diluted to 1mg/mL, sheep anti-mouse igg antibody is diluted to 1mg/mL; Nitrocellulose membrane is affixed on after base plate, draws polyclonal antibody and the sheep anti-mouse igg antibody of above-mentioned canine parvovirus antigen with three-dimensional stroke film instrument on nitrocellulose membrane, form respectively detection line and nature controlling line, room temperature is coated spends the night, standby;
3) preparation of glass fibre membrane: it is 8.5 that collaurum is adjusted to antibody pH value, monoclonal antibody is combined concentration with collaurum be 40 μ g/mL, after stabilizer treatment, the colloidal gold solution of the good monoclonal antibody of mark is evenly applied on the glass fibre element film of having handled well, freeze drying, standby;
4) finally in step 2) top of the nitrocellulose membrane that makes pastes thieving paper, mutually overlaps successively sticking glass tunica fibrosa and sample pad below nitrocellulose membrane.
The present invention further provides the application of aforementioned colloidal gold strip in detecting canine parvovirus.
Beneficial effect of the present invention is:
Colloid gold test paper strip adoption double-antibody sandwich method of the present invention and immunochromatography technique detect canine parvovirus antigen, compare with other detection techniques, and it detects sample does not need special processing, and reagent and sample consumption are minimum, and sample size can be low to moderate 1 μ L～2 μ L; Both can be used for antigen and detected, also can be used for antibody test.And can greatly shorten detection time, not need to use the expensive instruments such as fluorescent microscope, enzyme mark detector, just can detect qualitatively canine parvovirus antigen, testing result is clear is easy to judgement, and test findings can be preserved for a long time.So colloidal gold strip of the present invention has, and specificity is good, highly sensitive, simple to operate, detection quick and precisely, does not need the complicated advantages such as equipment, can meet the requirement of clinical examination, also be applicable to very much the clinical sample detection uses such as place that field condition, outpatient service, household person and experiment condition do not possess.
Accompanying drawing explanation
Fig. 1 is anti-many antivenom purifications of CPV rabbit result in the embodiment of the present invention; Wherein: 1 dyes Marker in advance for high-molecular-weight protein, 2 is sample solution, and 3 is that how anti-purifying is.
Fig. 2 is anti-CPV monoclonal antibody purification result in the embodiment of the present invention; Wherein: 1 dyes Marker in advance for high-molecular-weight protein, 2 is sample solution, and 3 is purified monoclonal antibody.
Fig. 3 is the colloidal gold solution of preparing in the embodiment of the present invention.
Fig. 4 is the microplate reader scanning result of the colloidal gold solution prepared in the embodiment of the present invention.
Fig. 5 is monoclonal antibody and the combination of collaurum under different pH values in the embodiment of the present invention; From left to right for not being: control group, pH7, pH8, pH8.5.
Fig. 6 is monoclonal antibody and the combination of collaurum under variable concentrations in the embodiment of the present invention.
Fig. 7 is test strips assembly model figure of the present invention; Wherein 1 is thieving paper, and 2 is nitrocellulose filter, and 3 is glass fibre membrane, and 4 is sample pad, and 5 is base plate.
Fig. 8 is ELISA test strip result of the present invention; Be respectively from left to right: CPV cells and supernatant, DMEM, PBST.
Embodiment
Following examples are used for illustrating the present invention, but are not used for limiting the scope of the invention.
Embodiment
Canine parvovirus (Canine Parvovirus, CPV) can cause canine parvovirus disease, be the deadly infectious disease of a kind of height contact of dog, it is principal character that clinical symptoms be take hemorrhagic enteritis or apyetous myocarditis, is that one of epidemic disease that dog industry is the most serious is supported by current harm China.In canine parvovirus disease, canine distemper, the sick three kinds of canine viral diseases of canine coronavirus, the most serious with canine parvovirus disease, infection rate can reach 100%, and mortality ratio reaches 10%～50%.CPV belongs to Parvoviridae, and parvovirus belongs to, wire minus strand Single-stranded DNA virus, gene group leader 5323bp.
1. the preparation of polyclonal antibody and purifying
Virus culture: in 50mL cell bottle, cultivate F81 cell, after cultivating for 4～5 generations, select growth form good and be paved with bottle at the bottom of 80% cell, outwell culture supernatant, add 9.5mL to contain the DMEM nutrient solution of 2% hyclone, synchronously add CPV500 μ L.CO2 incubator is cultivated 4～5d, when cell is while drawing in the net shape, cell bottle is put to multigelation in refrigerator-freezer and receive poison 3 times, and-80 ℃ save backup.
CPV viral purification: CPV supernatant adds PEG20000 and NaCl, the then centrifugal precipitation of staying.The resuspended rear dialysis of PBS, concentrated with sucrose, after packing, save backup.
Antibody preparation and purification: new zealand white rabbit is according to 500 μ L//time immunity, and immunity is 3 times altogether, three exempt from rear indirect ELISA detects serum titer, records it and tires when 1:20000, rabbit heart blood sampling, centrifugal collection supernatant.Warp
chromatograph antibody purification, after sample solution and purifying, sample is through SDS-PAGE electrophoresis.Result shows, before purifying, foreign protein is more, and after purifying, destination protein purity is higher, at 50kDa and 25kDa, occurs object band.
The preparation of 2.CPV monoclonal antibody and purifying
Cell is cultivated: recovery cell, place CO
237 ℃ of cultivations of incubator.When cell covers with individual layer, go down to posterity, outwell culture supernatant, complete medium is blown down monoclonal antibody cell gently, and half cell liquid of sucking-off proceeds in new cell bottle, supplement two bottles in nutrient culture media to 10mL, continue cultivation.
Mouse ascites preparation:
1) 0.5mL/ sterilizing paraffin of the female mouse lumbar injection of Balb/c multiparity;
2) hybridoma suspending in exponential phase, the centrifugal 3min of 1000r/min, abandons supernatant, the full nutrient culture media re-suspended cell precipitation of toing many or too much for use;
3) get 50 μ L cell suspensions, add 50 μ L0.5% platforms to expect that blue dye liquor mixes, and uses blood counting chamber counting cells.
4) adjust cell density to 2-3 * 10
6individual/mL, every mouse peritoneal injection 1 * 10
6individual cell;
5), after 7～10d, when mouse web portion obviously expands, No. 16 syringe needles thrust abdominal cavity and gather ascites;
6) by the centrifugal 10min of ascites 2000r/min, collect supernatant, low temperature is preserved;
7) CPV1:320 coated elisa plate, monoclonal antibody starts to do 10 times of dilutions from 1:100, measures tiring of odd contradictive hydroperitoneum.
The purifying of antibody is also identified: warp
chromatograph antibody purification, after sample solution and purifying, sample is through SDS-PAGE electrophoresis.Result shows, before purifying, foreign protein is more, and after purifying, destination protein purity is higher, at 50kDa and 25kDa, occurs object band.
3. prepare collaurum
In 250mL conical flask, add 99mL pure water and the heating of 1mL1% gold chloride, after boiling, add fast 1.5mL1% trisodium citrate aqueous solution, do not stop to stir, the solution colour of observing in conical flask changes simultaneously, in 3min by light yellow to grey to atropurpureus, finally become aubergine.Add again thermal agitation 15min, add pure water after cooling to return to original volume, be colloidal gold solution.
Reducing process is prepared colloidal gold solution, and colloidal gold solution is claret, bright in colour, bright, and inclusion-free can see that in face of daylight light belt occurs.Get two hole ELISA Plate, every hole adds collaurum 200 μ L, and microplate reader 400-600nm full wavelength scanner, is spaced apart 2nm.Interpretation of result, the colloidal-gold curve gradient of preparation is mild, and absorption peak width is little, uniform particles; Maximum absorption band, at wavelength 522nm place, according to the relation between the gold nano particulate particle diameter of bibliographical information, shape and ultraviolet-ray visible absorbing spectrum, shows that colloid gold particle mean diameter is in 25nm left and right.
4. monoclonal antibody is combined optimal pH and is determined with collaurum
Get 3 1.5mL EP pipes, add respectively the 1mL collaurum of having prepared, use 0.01mol/L K2CO3 by the pH difference furnishing 7,8,9 of collaurum.Get 50 μ L1mg/mL monoclonal antibody IgGs and add in the colloidal gold solution of modulated good pH, after concussion 20min, the static 10min of room temperature.Every pipe adds 200 μ L10%NaCl solution, and concussion mixes after 10min, the static 30min of room temperature.Observing colloid gold change color, collaurum keeps red minimum pH value 8.5 to be defined as optimum value.
5. monoclonal antibody is combined optium concentration and is determined with collaurum
Get 10 1.5mL centrifuge tubes, add respectively 1mL collaurum, 0.01M sal tartari 350 μ L adjust best pH8.5, and the collaurum 1mL that taking-up mixes up pH value is in new 1.5mL centrifuge tube; Each pipe adds 1mg/mL monoclonal antibody 0 μ L, 5 μ L, 10 μ L, 15 μ L successively, 20 μ L, 25 μ L, 30 μ L, 35 μ L, 40 μ L, 45 μ L, make its concentration contain antibody 0 μ g, 5 μ g in reaching every milliliter, 10 μ g, 15 μ g, 20 μ g, 25 μ g, 30 μ g, 35 μ g, 40 μ g, 45 μ g, place 10min under room temperature after concussion mixing 20min; Then every pipe adds 200 μ L10%NaCl, after concussion mixing 10min, under room temperature, places 10min.Colloidal gold solution is measured OD value at 580nm wavelength, take OD value as ordinate, and protein consumption is that horizontal ordinate is made a curve, and protein concentration corresponding to point that curve is close with transverse axis is at first 35 μ g/mL, determine that concentration is the 120%-130% of Cmin, so the best is 40 μ g/mL.
6. the preparation of gold mark monoclonal antibody and purifying
Get collaurum 1mL in 1.5mL centrifuge tube, 2000r/min4 ℃ of centrifugal 20min, draws supernatant, abandons precipitation.With 0.01M sal tartari, regulate optimal pH; Get collaurum that 1mL mixes up pH value in new 5mL1.5mL centrifuge tube, slowly add appropriate 1mg/mL monoclonal antibody, concussion 20min, standing 10min; Add stabilizing agent 10%BSA solution 100 μ L, making final concentration is 1%, concussion 15min, standing 10min.Adopt differential centrifugation purifying colloidal gold probe: by the colloidal gold probe 4000r/min tentatively making, centrifugal 20min, collects supernatant above; Supernatant 12000r/min, 4 ℃ of centrifugal 45min, abandon supernatant, and the pipe end is the loose shape precipitation of kermesinus; To precipitate with 200 μ L collaurum washing lotion (1%BSA, 0.02%NaN3,2mM Tris damping fluids; PH8.2) resuspended, and collect in a centrifuge tube, be total to 1mL; 12000r/min, 4 ℃ of centrifugal 45min, abandon supernatant; Repeated centrifugation washing is 1 time again, altogether washed twice; The resuspended damping fluid of 600 μ L (1%BSA, 1% sucrose, 0.02%NaN3,0.3%Tween-20,20mM Tris damping fluid for precipitation; PH8.2) resuspended, put 4 ℃ of Refrigerator stores standby.
7. the preparation of test strips
The colloidal gold solution of the good monoclonal antibody of mark is evenly applied to metal spraying on the glass fibre element film of having handled well; 37 ℃ of dried overnight.To three-dimensional, draw rabbit how anti-, the 1mg/mL sheep anti-mouse igg antibody that adds respectively 1mg/mL purifying in the sample cell 1 of film instrument and sample 3, NC film is affixed on after base plate, with the three-dimensional film instrument of drawing, draw detection line (T) and nature controlling line (C) on NC film, room temperature is coated with spends the night; Sticky note thieving paper above base plate NC film, sticking glass tunica fibrosa and sample pad successively below NC film, as shown in the figure.With cutting bar machine, test paper plate is cut into the wide test strips of 0.4cm.
8. ELISA test strip
Draw respectively CPV cells and supernatant, each 1mL of DMEM, PBST adds in 7mL centrifuge tube; The test strips assembling is put into respectively three centrifuge tubes, after 2min, takes out, and puts into plate and observes, and continues 20min; Nature controlling line does not develop the color, and test strips is invalid; Nature controlling line is red, and detection line does not develop the color negative, without canine parvovirus, detects.Nature controlling line and detection line all take on a red color positive, have canine parvovirus to detect.
By detecting after CPV antigen doubling dilution, determine test strips sensitivity.ELISA test strip rabies viruses (RV) and CDV (CDV), determine its specificity.By U.S. CPV antigen measuring ELISA kit and test strips, detect 80 parts of CPV fecal specimens simultaneously, calculate both coincidence rates, TP(true positive, true positives), TN(true negative, true negative), FN(false negative, false negative), FP(false positive, false positive), coincidence rate (%)=(TP+TN)/(TP+TN+FP+FN) * 100.
Known canine parvovirus HA is tired as the antigen doubling dilution of 1:5120, by test strips, measure, result is as shown in table 1, can detect the minimum HA of CPV and tire as 1:80.ELISA test strip rabies viruses (RV) and CDV (CDV), be negative findings, shows that test strips specificity is good.By commercialization ELISA kit and test strips, detect 80 parts of CPV fecal specimens simultaneously, calculate both coincidence rates, TP(true positive, true positives), TN(true negative, true negative), FN(false negative, false negative), FP(false positive, false positive), coincidence rate (%)=(TP+TN)/(TP+TN+FP+FN) * 100, both coincidence rates 92.5%, as shown in table 2.
The mensuration of table 1 test strips sensitivity
Table 2 test strips and the comparison of ELISA testing result
Although above the present invention is described in detail with a general description of the specific embodiments, on basis of the present invention, can make some modifications or improvements it, this will be apparent to those skilled in the art.Therefore, these modifications or improvements, all belong to the scope of protection of present invention without departing from theon the basis of the spirit of the present invention.
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| CN105486871A (en) * | 2015-11-25 | 2016-04-13 | 北京世纪元亨动物防疫技术有限公司 | Colloidal gold test strip for rapidly detecting hemagglutination inhibition titer of canine parvovirus antibody, and kit and detection method thereof |
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