CN102879573A - Immune colloidal gold test paper capable of simultaneously detecting canine distemper and canine parvo of foxes, raccoon dogs and minks and a method for preparing immune colloidal gold test paper - Google Patents
Immune colloidal gold test paper capable of simultaneously detecting canine distemper and canine parvo of foxes, raccoon dogs and minks and a method for preparing immune colloidal gold test paper Download PDFInfo
- Publication number
- CN102879573A CN102879573A CN2012103791547A CN201210379154A CN102879573A CN 102879573 A CN102879573 A CN 102879573A CN 2012103791547 A CN2012103791547 A CN 2012103791547A CN 201210379154 A CN201210379154 A CN 201210379154A CN 102879573 A CN102879573 A CN 102879573A
- Authority
- CN
- China
- Prior art keywords
- parvovirus
- ermine
- cdv
- test paper
- pad
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Granted
Links
Images
Abstract
The invention discloses immune colloidal gold test paper capable of simultaneously detecting canine distemper and canine parvo of foxes, raccoon dogs and minks and a method for preparing the immune colloidal gold test paper. The test paper consists of a test paper box and a test paper tape, and the test paper tape is made by sequentially bonding a hard polyvinyl chloride lining plate, a nitrocellulose membrane, a long colloidal gold combination pad, a short colloidal gold combination pad, a sample pad and an absorption pad. VP2 gene monoclonal antibody- colloidal gold biomarkers resistant to mink-source canine distemper viruses and parvoviruses are sprayed on the long and short colloidal gold combination pads respectively. Two detection lines formed by rabbit anti-mink source canine distemper virus and parvovirus antibodies and a quality control line formed by a rabbit antimouse antibody are sprayed on the nitrocellulose membrane. The test paper is applicable to quick detection of pestilences of foxes, raccoon dogs and minks, and detection of two pestilences including canine distemper and canine parvo of foxes, raccoon dogs and minks can be simultaneously completed by the same test paper, and detection is simple, convenient, quick and accurate.
Description
Technical field
The present invention relates to the detection utensil of cultivated animals epidemic disease, be specifically related to a kind ofly can examine simultaneously the canine distemper of fox, racoon dog, ermine and the immune colloid gold test paper of parvovirus.The invention still further relates to the preparation method of this test paper.
Background technology
Canine distemper (Canine distemper, CD) is a kind of height contagious disease that many animals comprise fox, recoon dog and mink in the dog that caused by CDV (Canine distemper virus, CDV) and the carnivorous order; Parvovirus (Canine Parvo, CP) is a kind of acute infectious disease that is comprised fox, recoon dog and mink by the dog class animal that parvovirus (Canine Parvovirus, CPV) causes.These two kinds of infectious diseases often cause the animal morbidities such as large quantities of foxes, recoon dog and mink, and case fatality rate is generally between 30%～80%.Along with increasing considerably of the numbers of animals raised of the fur-bearing animals such as China fox, recoon dog and mink, and increasing of exchanging of strange land, the incidence of disease and fatal rate the trend that rising all arranged of these two kinds of infectious diseases in the fur-bearing animals such as China fox, recoon dog and mink, be at present the Fur Animal Feedings such as China fox, recoon dog and mink already to be endangered maximum epidemic disease, be subject to paying attention to widely.The methods such as at present, viral separation, electron microscopy, neutralization test, enzyme linked immunosorbent assay (ELISA), PCR (PCR) have been brought into play effect in the diagnosis of canine distemper, parvovirus.But these methods respectively have drawback, and conventional Virus Isolation and neutralization test process are loaded down with trivial details, consuming time longer; Electron microscopy is difficult to apply in basic unit; Protocols in Molecular Biology such as PCR etc. are responsive, special, and technology and equipment requires high, and these detection methods are difficult in the on-the-spot use of veterinary clinic.
The immunochromatography colloidal gold technique has easy to be quick, high specificity, and susceptibility is high, the naked eyes interpretation, experimental result is easily preserved, and need not the advantages such as special instruments and equipment and reagent, has been widely used in veterinary clinic disease detection or the diagnosis.At present, the numerous epidemic diseases that detect dog have the colloid gold test paper of its corresponding epidemic disease, and are not exclusively used in the colloid gold test paper that detects fox, recoon dog and mink epidemic disease.If clinically fox, recoon dog and mink generation canine distemper or (with) parvovirus, can only use the canine distemper that detects dog or (with) the parvovirus colloid gold test paper, and the canine distemper of preparation dog or (with) the required canine parvovirus prevention of parvovirus colloid gold test paper or (with) antigen of the monoclonal antibody of parvovirus all separates from dog, be used on fox, recoon dog, the mink and may cause false positive or false negative result, poor accuracy.So far, the pathogen of also not separating from mink is made the immunochromatography colloid gold test paper as the monoclonal antibody of antigen preparation, does not more have to detect simultaneously the canine distemper of fox, recoon dog, mink and the test paper of two kinds of epidemic diseases of parvovirus.
Summary of the invention
The object of the invention is to for the problems referred to above, provide a kind of and can detect simultaneously the canine distemper of fox, racoon dog, ermine and the immune colloid gold test paper of parvovirus, this test paper is easy to use, simple to operate, detect fast, accurately, and the enough same test paper of energy detect canine distemper and the parvovirus of fox, racoon dog, ermine simultaneously.The present invention also aims to provide the preparation method of the immune colloid gold test paper of a kind of canine distemper that can detect simultaneously fox, racoon dog, ermine and parvovirus.
The technical scheme that realizes above-mentioned purpose is: a kind ofly can detect simultaneously the canine distemper of fox, racoon dog, ermine and the immune colloid gold test paper of parvovirus, the test strips cartridge of well and colour developing district opening is set above being included in, and is arranged on the test strips in this test strips cartridge; Described test strips includes the rigid polyvinyl chloride liner plate, nitrocellulose filter, sample pad and absorption pad, described rigid polyvinyl chloride liner plate is arranged on the box bottom surface of described test strips cartridge, described nitrocellulose filter place described rigid polyvinyl chloride liner plate above, end in described test strips cartridge is provided with a long collaurum pad that places above the described nitrocellulose filter and places the short collaurum pad of one above the described long collaurum pad, described sample pad place described short collaurum pad above, top and the described well of this sample pad is corresponding, described absorption pad place the other end of described test strips cartridge be positioned at described nitrocellulose filter above; Be coated with respectively the VP2 gene monoclonal antibody of anti-ermine source CDV and parvovirus-colloid gold label thing on described long collaurum pad and the short collaurum pad, the VP2 gene monoclonal antibody of this anti-ermine source CDV and parvovirus is the VP2 gene protein immunity Balb/c mouse with histidine-tagged parvovirus with the Yeast expression of the ermine source CDV of purifying and restructuring, through Fusion of Cells, the VP2 gene monoclonal antibody that screening obtains anti-ermine source CDV and parvovirus is that hybridoma cell line is secreted, the nature controlling line that two detection lines that are coated with respectively on the described nitrocellulose filter that the anti-ermine of rabbit source CDV and parvovirus antibody consist of and rabbit anti-mouse antibody consist of, these two detection lines are corresponding with described colour developing district opening with nature controlling line.
The preparation method of the immune colloid gold test paper of the described canine distemper that can detect simultaneously fox, racoon dog, ermine and parvovirus, it may further comprise the steps:
1, the VP2 gene protein with histidine-tagged parvovirus of the Yeast expression of the ermine source CDV of preparation purifying and restructuring;
2, respectively with the VP2 gene protein immunity Balb/c mouse with histidine-tagged parvovirus of the Yeast expression of the ermine source CDV of purifying and restructuring, through Fusion of Cells, screening obtains secreting the hybridoma cell line of the VP2 gene monoclonal antibody of anti-ermine source CDV and parvovirus;
3, the VP2 gene monoclonal antibody of preparation anti-ermine source CDV and parvovirus;
4, with trisodium citrate and gold chloride reaction preparation collaurum;
5, the anti-ermine source CDV that step 3 is made and the VP2 gene monoclonal antibody of parvovirus add respectively in the collaurum that step 4 prepares, and obtain respectively the VP2 gene monoclonal antibody of anti-ermine source CDV and parvovirus-colloid gold label thing;
6, will resist the VP2 gene monoclonal antibody of ermine source CDV and parvovirus-colloid gold label thing to be sprayed on respectively on long collaurum pad and the short collaurum pad;
7, preparation rabbit anti-ermine source CDV and parvovirus antibody, preparation rabbit anti-mouse antibody;
8, the antibody with the anti-ermine of rabbit source CDV and parvovirus is sprayed on two detection lines of formation on the nitrocellulose filter, and the rabbit anti-mouse antibody is sprayed on nature controlling line of formation on the nitrocellulose filter;
9, rigid polyvinyl chloride liner plate, nitrocellulose filter, long collaurum pad, short collaurum pad, sample pad, absorption pad are bonded together the composition test strips in order, and this test strips fixedly packed in the test strips cartridge on request, obtain describedly detecting simultaneously the canine distemper of fox, racoon dog, ermine and the immune colloid gold test paper of parvovirus.
The present invention is by the immunochromatography reaction of colloid gold label colour developing, can detect simultaneously the canine distemper of fox, racoon dog, ermine and the immune colloid gold test paper of parvovirus with preparation, structure is more reasonable, use rabbit anti-ermine source CDV and parvovirus antibody as detection line, set up in the competition immunochromatography fast detecting testing sample whether contain CDV and parvovirus.
Compared with prior art, the present invention has following outstanding advantages:
1, is applicable to the fast detecting of canine distemper and the parvovirus of fox, racoon dog, ermine, and can detects simultaneously CDV and parvovirus with same test paper.
2, detection paper of the present invention high specificity as a result is highly sensitive.CDV or parvovirus cell median infective dose (TCID
50) reach 9.4 * 10
5Being diluted to 1:512 still can detect.
3, test paper of the present invention does not need any instrument and equipment, and is easy to carry, and testing cost is low.
4, test paper of the present invention easy control simple to operate need not professional's operation.
5, strip of the present invention is preserved conveniently, good stability.
Description of drawings
Accompanying drawing is the structural representation that the present invention can detect the immune colloid gold test paper of the canine distemper of fox, racoon dog, ermine and parvovirus simultaneously.
Embodiment
The invention will be further described below in conjunction with drawings and Examples.
Such as accompanying drawing, the present invention can detect the canine distemper of fox, racoon dog, ermine simultaneously and the immune colloid gold test paper of parvovirus includes test strips and test strips cartridge 3, and test strips is fixed in the test strips cartridge 3.Test strips cartridge 3 has box body and loam cake two parts, offers well 1 in the keep left position of side of loam cake, offers colour developing district opening 2 at the loam cake medium position.Test strips is to be combined into one by bonding by rigid polyvinyl chloride liner plate 4, nitrocellulose filter 5, sample pad 6, absorption pad 7, long collaurum pad 8 and short collaurum pad 9 to consist of, its concrete unitized construction: nitrocellulose filter 5 places above the rigid polyvinyl chloride liner plate 4, the side that keeps left on nitrocellulose filter 5 sets gradually long collaurum pad 8, short collaurum pad 9 and sample pad 6 from the bottom to top, and absorption pad 7 is arranged on above the nitrocellulose filter 5 position on the right side.Test strips can adopt, and mode bonding, that draw-in groove is fixed, the box body loam cake compresses is fixed in the test strips cartridge.Be coated with two detection lines 10 and a nature controlling line 11 at the middle part of nitrocellulose filter 5.Long collaurum pad 8 and short collaurum pad 9 are made by glass fibre element film, a long collaurum pad 8 and a short collaurum pad 9 are one group, spray respectively CDV monoclonal antibody and the parvovirus VP2 gene protein monoclonal antibody of the anti-mink of colloid gold label on this two collaurums pad, the VP2 gene monoclonal antibody of described anti-ermine source CDV and parvovirus is the VP2 gene protein immunity Balb/c mouse with histidine-tagged parvovirus with the Yeast expression of the ermine source CDV of purifying and restructuring, through Fusion of Cells, the VP2 gene monoclonal antibody that screening obtains anti-ermine source CDV and parvovirus is that hybridoma cell line is secreted.Nitrocellulose filter 5 is the detection film of test strips, and what the above kept left side is two detection lines 10, and nature controlling line 11 is positioned at the right side, and two detection lines 10 spray respectively the anti-ermine of rabbit source CDV and parvovirus antibody, nature controlling line 11 spraying rabbit anti-mouse antibodies.When containing corresponding virus in the test sample, virus can be distinguished first and golden mark anti-ermine source CDV monoclonal antibody and parvovirus VP2 gene monoclonal antibody is combined, then be combined and form macroscopic colour band after the enrichment at the antibody on corresponding detection line under the chromatography effect, and then judge according to the colour developing result.
The concrete preparation method that the present invention can detect the immunochromatography colloid gold test paper of the canine distemper of fox, racoon dog, ermine and parvovirus simultaneously is:
1, the preparation of anti-ermine source CDV monoclonal antibody: the CDV Hebei strain culture purified that will from mink, separate, CDV Hebei, ermine source strain immunity Balb/c mouse with culture purified, the 1st abdominal part hypodermic complete Freund's adjuvant+ermine source CDV antigen (1:1) 100 μ L, by the amount of 50 a μ g albumen/mouse, the Balb/c female mice of 4 no-special pathogens of initial immunity (SPF).The afterwards subcutaneous first time in one week, booster immunization was injected incomplete Freund's adjuvant+CDV antigen (1:1) 100 μ L, and the antigen amount is 50 μ g; Behind 14d, lumbar injection incomplete Freund's adjuvant+ermine source CDV antigen (1:1) 200 μ L, the antigen amount is 100 μ g, interval 14d, tail vein blood is measured immune effect.Above-mentioned, after antibody titer reached requirement, 3d (being separated by more than 2 weeks with immunity last time) with non-emulsification antigen booster immunization once before merging.Behind the 3d, the aseptic spleen of getting merges.Through Fusion of Cells, cell conditioned medium screens with antigen coated ELISA Plate, establishes simultaneously the positive and negative contrast.The positive colony that obtains is through repeated detection, and secretory antibody is stable.Behind the limiting dilution assay clone 3 times, positive rate reaches 100%.Be total to get 14 strain positive colony cells in accordance with the law, use again the ermine source CDV coated elisa plate of purifying, make compound detection, cell line with the 14 strain positives, again use ermine source CDV wrapper sheet, adopt enzyme-linked immunologic adsorption test method, do programmed screening, obtain 3 strain positive hybridoma cell strains and be numbered: 4-1E7,6-2H10,7-1C6.Subgroup identification is carried out in the 3 strain positive cell strains that screen, obtain the positive hybridoma cell strain of 3 strain Immunoglobulin IgG1 types.Its titer of ascites can reach 1:51200 and 1:204800, and cell culture supernatant is tired and can be reached 1:256 and 1:512.The centrifugal 15min of ascites 12 000 * g with collecting discards the top layer grease, supernatant caprylic acid-ammonium purifying.Through sad-ammonium sulfate salting-out process purifying a small amount of assorted band is arranged still, the monoclonal antibody sample behind G-albumen affinity purification, almost occurs without any assorted band again, and antibody purity is very high.The monoclonal antibody that obtains only with CDV generation specific reaction, and do not have cross reaction with other virus such as parvovirus and canine infectious hepatitis virus-I type (CAV-I) and canine infectious hepatitis virus-II type (CAV-II).
2, the preparation of the monoclonal antibody of anti-Yeast expression ermine source parvovirus VP2 gene protein: ermine source parvovirus VP2 gene is increased with polymerase chain reaction method, success is increased and has been arrived parvovirus VP2 gene, with parvovirus VP2 Gene cloning in Pichia pastoris secretion expression carrier pPICZAA, make up recombinant eukaryotic expression plasmid pPICZAA-VP2, after this recombinant plasmid linearization, transform among the pichia pastoris phaff X-33, methanol induction is expressed parvovirus VP2 gene, and polyacrylamide gel electrophoresis and Western blot are identified expressing protein.Result's parvovirus VP2 gene that successfully increased has made up recombinant eukaryotic expression plasmid pPICZAA-VP2, gives expression to about 68 kD albumen in pichia pastoris phaff.After Western blot was identified, expressing protein was destination protein gene VP2.Expression strain enlarges expression system to be cultivated in nutrient culture media, and supernatant is concentrated with 4 ℃ of precipitations of 70% ammonium persulfate, adopt His select nickel-affinity chromatography column separating purification obtain the Yeast expression of recombinating with histidine-tagged VP2 gene protein, the about 3mg/L of expression.With the recombinant expressed parvovirus VP2 gene protein immunity Balb/c mouse of the yeast of purifying, the recombinant VP 2 gene protein after filtering is strengthened primary immune response with complete Freund's adjuvant every two weeks, use the antigen of incomplete Freund's adjuvant emulsification instead.Generally 3 exempt to exempt from 4 after 10d detect the Balb/c mouse antibodies and tire, when antibody titer reach tire reach 1: 5000～1: 10000 when above for fusion.Get immune Balb/c mouse spleen and be prepared into splenocyte, take polyglycol PEG1500 as fusion agent and the myeloma cell merge.Choose cell monoclonal, be incubated at 96 porocyte culture plates.Use the immunogene wrapper sheet, the clone who selects is adopted enzyme-linked immunologic adsorption test method, do for the first time screening, obtain 27 strain positive hybridoma cell strains.With the positive hole of limiting dilution assay clone purification, with the cell line of the 27 strain positives, use again wrapper sheet of ermine source parvovirus, adopt enzyme-linked immunologic adsorption test method, do programmed screening, obtain 11 strain positive hybridoma cell strains.By the enzyme-linked immunologic adsorption test method screening, obtain the monoclonal antibody hybridoma cell strain of the anti-parvovirus VP2 of 4 strains energy stably excreting gene protein.In the 4 strain monoclonal antibodies, 2 strains belong to Immunoglobulin IgG2 b subclass, and 2 strains belong to the Immunoglobulin IgG1 subclass, and its titer of ascites can reach 1:51200 and 1:204800, and cell culture supernatant is tired and can be reached 1:256,1:512.The monoclonal antibody that obtains only with parvovirus and parvovirus VP2 gene protein generation specific reaction thereof, and do not have cross reaction with other virus such as CDV and canine infectious hepatitis virus-I type and canine infectious hepatitis virus-II type; Fluorescence immunization coloration virus detects and shows that further the specificity of monoclonal antibody is effective.
3, the preparation of collaurum: adopt trisodium citrate reduction method to prepare collaurum.Measure three of 100mL with volumetric flask and boil off ionized water, pour in the flat bottom flask that specification is 500.0mL, flask is placed in the heating jacket of magnetic force heating stirrer, put into magnetic stir bar, open and stir knob to suitable speed, open the heating knob, be heated to boiling.Add 1.0mL 1% gold chloride (HAuC14) solution, continue heating 2min, then press 100.0mL 0.01 % gold chloride; Add 1.8mL 1% citric acid three sodium solution, continue heating.Lurid aqueous solution of chloraurate after trisodium citrate adds in the 2min gradually by the xanthochromia ash again blackening redden at last or be orange red.Until solution become shiny red or orange red after, continue again to add thermal agitation 15min.It is limpid transparent orange red that the collaurum outward appearance of preparation is, and scanning it by spectrophotometer, to produce the corresponding wavelength X max of absorption maximum in absorption spectrum be 522nm, and by electron microscopic observation, even particle size, mean diameter are 18.5nm.
4, ermine source CDV monoclonal antibody (1C6) and parvovirus VP2 gene protein monoclonal antibody (2B3) colloid gold label and purifying.Get a series of small test tubes, add respectively 5 mL collaurums, with 0.1 mol/L sal tartari (K
2CO
3) the pH value of regulating collaurum is adjusted to respectively 6,6.5,7,7.5,8,8.5,9,9.5,10; Then CDV monoclonal antibody (1C6) the 100.0 μ L that add respectively 1.0mg/mL in the collaurum of every kind of different pH values with sealed membrane closed test tube mouth, leave standstill 15min, 4 ℃ of centrifugal l0 min of 2000r/min.Get supernatant and scan with ultraviolet one visible spectrophotometer (400～600 nm) in visible-range, obtain the collaurum visible absorption spectrum, measure maximum absorption wavelength, light absorption value.Collaurum pH value corresponding when maximum absorption band occurring is as optimum mark pH value.The optimal pH of CDV monoclonal antibody (1C6) colloid gold label is 8.0, and when the monoclonal antibody addition was 10 μ g in the 1mL collaurum, corresponding absorption peak was maximum, adds 20% on this basis again, and namely the suitableeest labelled amount is 12 μ g/mL collaurums.Because be the mouse resource monoclonal antibody all, parvovirus VP2 gene protein monoclonal antibody (2B3) is identical with the suitableeest mark of parvovirus monoclonal antibody (1C6).The collaurum that in the small beaker of 50.0mL, adds 20.0mL, transfer pH to optimum mark pH value, slowly add the monoclonal antibody of the good 1mg/mL of a certain amount of dilution under the state that stirs, stir 30min, adding 10% bovine serum albumin(BSA) (BSA) to final concentration is 1%, continue to stir 30 min, behind 4 ℃ of placement 2h, above-mentioned colloid gold label thing is sub-packed in the centrifuge tube of 7.0mL, with the centrifugal 15min of 2 000r/min, the sucking-off supernatant is abandoned precipitation; With centrifugal 45 min of 8 700r/min, supernatant discarded night, add the mark cleansing solution to original volume; Again with the centrifugal 30min of 10 000r/min, supernatant discarded night, add the mark cleansing solution to original volume; With centrifugal 30 min of 10 000r/min, supernatant discarded night, precipitation is resuspended with 1.00mL mark cleansing solution, and it is for subsequent use to put 4 ℃ of refrigerators.
5, nitrocellulose filter and collaurum pad are coated: 0.01 mol/L pH, 7.2 phosphate buffers (PBS) of 6% methyl alcohol are coated damping fluid, 0.22um membrane filtration mistake, put 4 ℃ for subsequent use.Confining liquid adopts 2% bovine serum albumin(BSA) (BSA), 1% polysorbas20,0.5% tygon to adjoin pyrrolidone K30 (PVP K30), and 2.5% sucrose, 0.02% nitrine are received (NaN
3), 0.01 mol/L pH7.4 PBS solution, 0.22 μ m miillpore filter filters, put 4 ℃ for subsequent use.The detection film is nitrocellulose filter, selects Millipore HF135, and the flow velocity of film is 135 scholars, 34 s/cm, and the aperture is approximately 0.45 μ m.First with the anti-ermine of rabbit source CDV and parvovirus antibody, rabbit anti-mouse antibody with described coated damping fluid dilution after, be sprayed at respectively on the described nitrocellulose filter Millipore HF135, as detection line 10(hundstaupe hot line and parvovirus line) and nature controlling line 11, being sprayed on the anti-ermine of the rabbit source CDV antibody concentration that detects hundstaupe hot line on the film nitrocellulose filter detection line 10 is 1.5mg/mL, and quantity for spray is 2-3 μ L/cm; Being sprayed on the anti-ermine of the rabbit source parvovirus antibody concentration that detects parvovirus line on the film nitrocellulose filter detection line 10 is 1.0mg/mL, and quantity for spray is 3-4 μ L/cm; Be sprayed on and detect that rabbit anti-mouse antibody concentration is 0.8mg/mL on the film nitrocellulose filter nature controlling line 11, quantity for spray is 1-3 μ L/cm, in 37 ℃ of incubator dry for standby.The material of long and short collaurum pad is glass fibre membrane, after the anti-ermine source CDV monoclonal antibody and the dilution of parvovirus VP2 gene protein monoclonal antibody with colloid gold label, be sprayed at respectively on long collaurum pad 8 and the short collaurum pad 9 37 ℃ of dry for standby.The anti-ermine of the colloid gold label source CDV monoclonal antibody (1.5mg/mL) and the short collaurum pad anti-ermine source parvovirus VP2 gene protein monoclonal antibody (1.5mg/mL) that are sprayed on long collaurum pad are done 5 times of dilutions, and quantity for spray is respectively 50-60 μ L/cm.
6, the assembling of test paper: with the sample pad 6 of handling well, be coated with the long collaurum pad 8 of anti-ermine source CDV and parvovirus VP2 gene protein monoclonal antibody-colloid gold label thing, short collaurum pad 9, be coated with the anti-ermine of rabbit source CDV and parvovirus antibody as detection line 10 and be coated with the rabbit anti-mouse antibody as the nitrocellulose filter 5 of nature controlling line 11, absorption pad 7 orders stick on the rigid polyvinyl chloride liner plate 4 successively, form test strips, have above this test strips is installed in the test strips cartridge 3 of well 1 and colour developing district opening 2, Vacuum Package, normal temperature is preserved, the term of validity 6 months.
The present invention can detect the application of the immunochromatography colloid gold test paper of the canine distemper of fox, racoon dog, ermine and parvovirus simultaneously:
1, the pre-service of fox, racoon dog, ermine sample: the fox of the doubtful canine distemper of collecting, racoon dog, ermine sample such as sick fox, racoon dog, the tears of ermine, nose is wiped away and the fox of parvovirus infections, racoon dog, ermine ight soil or the fox that dies of illness, racoon dog, ermine internal organs sample, respectively has negative sample to compare.The pathological material of disease of doubtful CDV fox, racoon dog, ermine such as tears, nose wipe away, die of illness every part in internal organs sample is got about 0.5g(mL) about, add the about 0.5mL of physiological saline, make suspension.Every part in the ight soil of the fox of doubtful parvovirus infections, racoon dog, ermine is got about about 0.5g, adds the about 0.5mL of physiological saline, leaves standstill 5min (or centrifugal) after fully shaking.
2, detect: get respectively supernatant 100 μ L and drop on the test paper of the present invention observations behind the 10min.Get respectively simultaneously 100 μ L 0.01M PBS and known TCID
50Be 9.4 * 10
5CDV, parvovirus cell culture fluid (1:512 dilution) in the well of other two test paper, do negative blank and positive criteria product control test.
3, the result judges: when nature controlling line 11 presents red line, the fox, racoon dog, the ermine sample that in well 1, add doubtful canine distemper, at opening 2 places, colour developing district, red line appears in detection line 10, positive for canine distemper, namely contain CDV in the sample, red line does not appear, negative for canine distemper, namely do not contain CDV in the sample; The sample of the fox of the doubtful parvovirus infections of adding, racoon dog, ermine in well 1, red line appears in detection line 10, is that parvovirus is positive, be to contain parvovirus in the sample, red line does not appear, and negative for parvovirus, namely do not contain parvovirus in the sample.If red line does not appear in nature controlling line 11, then test paper is invalid.
Effect of the present invention is given an example:
1, specific test: CDV and parvovirus cell culture fluid that oneself is known carry out cross matching, establish simultaneously the PBS negative control.The TCID that oneself is known with 0.01M PBS
50Be 9.4 * 10
5CDV and parvovirus cell culture fluid make doubling dilution to 1:512, get respectively 100 μ L and drop in well, use detection paper of the present invention, carry out sensitivity tests.When containing simultaneously CDV and parvovirus in the sample, in colour developing district opening 2, two red detection lines of detection line 10() (having 2 red stripes to occur) all is positive, and when only containing CDV or parvovirus in the sample, then a detection line in correspondence presents positive reaction (being equipped with 1 red line in corresponding positions occurs), the reaction (all without band occur) that all is negative of PBS negative control (detection line occurs without any band) and canine infectious hepatitis virus-I type and canine infectious hepatitis virus-other viruses such as II type, come to the same thing after repeatedly repeating 5 times, illustrate that the method has the specificity of height.
2, sensitivity tests: with PBS to known CDV or parvovirus (cell median infective dose TCID
50Be 9.4 * 10
5) make doubling dilution to 1:512, to get respectively 100 μ L and drop in well, test paper still is positive (detection line has 2 red stripes to occur), proves that the susceptibility of test paper of the present invention is strong.
3, stability test: 5 batches of test paper of the present invention (batch number 120516,120518,120519,120528,120530) are placed 7d in 37 ℃ of constant incubators after, detect simultaneously 10 parts of CDVs and parvovirus positive and 10 parts of CDVs and parvovirus negative sample with same batch of test paper preserving 4 ℃, the result shows: 37 ℃ of effect 7d without destruction, proves that test paper at high temperature stablizes to test paper of the present invention.
4, storage life test: will be with the test paper of the present invention of a collection of preparation, respectively 4 ℃ and room temperature preservation 1 to 6 months, took out in 0 month, 3 months, 6 months, detect CDV and the parvovirus positive of 1 part of doubling dilution and carry out the storage life sensitivity tests, detect 10 parts of known CDVs and parvovirus positive and CDV and parvovirus negative sample and carry out the storage life specific test, observation period susceptibility, specificity and stability are to determine the storage life of test paper.The result shows: test paper of the present invention does not all change through 6 months phases of room temperature preservation specificity, susceptibility.
5, compare with enzyme linked immunosorbent assay, hemagglutination test method: the canine distemper of fox, racoon dog, ermine is made a definite diagnosis enzyme linked immunosorbent assay detection commonly used clinically, and the parvovirus of fox, racoon dog, ermine is made a definite diagnosis hemagglutination test method commonly used and detected.In the tears of 120 parts of tested canine distemper suspected case foxes, racoon dog, ermine (nose wipe away, the die of illness internal organs) sample, detecting the positive with enzyme linked immunosorbent assay is 47 parts, feminine gender is 73 parts, it is 49 parts with the detection paper positive of the present invention, feminine gender is 71 parts, the result shows: canine distemper detects Positive rate 39.16% height (test findings detects with polymerase chain reaction method simultaneously, conforms to) with test paper method Positive rate 40.83% of the present invention than enzyme linked immunosorbent assay; In ight soil (internal organs of the dying of illness) sample of 120 parts of tested parvovirus suspected case foxes, racoon dog, ermine, detecting the positive with the blood coagulation tests method is 50 parts, feminine gender is 70 parts, it is 53 parts with the detection paper positive of the present invention, feminine gender is 67 parts, the result shows: parvovirus detects Positive rate 71.42% height (test findings detects with polymerase chain reaction method simultaneously, conforms to) with test paper method Positive rate 79.10% of the present invention than the hemagglutination test experimental technique.
Claims (2)
1. one kind can be detected the canine distemper of fox, racoon dog, ermine and the immune colloid gold test paper of parvovirus simultaneously, and the test strips cartridge of well and colour developing district opening is set above being included in, and is arranged on the test strips in this test strips cartridge; Described test strips includes the rigid polyvinyl chloride liner plate, nitrocellulose filter, sample pad and absorption pad, it is characterized in that: described rigid polyvinyl chloride liner plate is arranged on the box bottom surface of described test strips cartridge, described nitrocellulose filter place described rigid polyvinyl chloride liner plate above, end in described test strips cartridge is provided with a long collaurum pad that places above the described nitrocellulose filter and places the short collaurum pad of one above the described long collaurum pad, described sample pad place described short collaurum pad above, top and the described well of this sample pad is corresponding, described absorption pad place the other end of described test strips cartridge be positioned at described nitrocellulose filter above; Be coated with respectively the VP2 gene monoclonal antibody of anti-ermine source CDV and parvovirus-colloid gold label thing on described long collaurum pad and the short collaurum pad, the VP2 gene monoclonal antibody of described anti-ermine source CDV and parvovirus is the VP2 gene protein immunity Balb/c mouse with histidine-tagged parvovirus with the Yeast expression of the ermine source CDV of purifying and restructuring, through Fusion of Cells, the VP2 gene monoclonal antibody that screening obtains anti-ermine source CDV and parvovirus is that hybridoma cell line is secreted, the nature controlling line that two detection lines that are coated with respectively on the described nitrocellulose filter that the anti-ermine of rabbit source CDV and parvovirus antibody consist of and rabbit anti-mouse antibody consist of, these two detection lines are corresponding with described colour developing district opening with nature controlling line.
2. the preparation method of the immune colloid gold test paper of the canine distemper that can detect simultaneously fox, racoon dog, ermine claimed in claim 1 and parvovirus, it is characterized in that: it may further comprise the steps:
(1) the VP2 gene protein with histidine-tagged parvovirus of the Yeast expression of the ermine source CDV of preparation purifying and restructuring;
(2) respectively with the VP2 gene protein immunity Balb/c mouse with histidine-tagged parvovirus of the Yeast expression of the ermine source CDV of purifying and restructuring, through Fusion of Cells, screening obtains secreting the hybridoma cell line of the VP2 gene monoclonal antibody of anti-ermine source CDV and parvovirus;
(3) the VP2 gene monoclonal antibody of preparation anti-ermine source CDV and parvovirus;
(4) with trisodium citrate and gold chloride reaction preparation collaurum;
(5) the anti-ermine source CDV that step (3) is made and the VP2 gene monoclonal antibody of parvovirus add respectively in the collaurum that step (4) prepares, and obtain respectively the VP2 gene monoclonal antibody of anti-ermine source CDV and parvovirus-colloid gold label thing;
(6) will resist the VP2 gene monoclonal antibody of ermine source CDV and parvovirus-colloid gold label thing to be sprayed on respectively on long collaurum pad and the short collaurum pad;
(7) preparation rabbit anti-ermine source CDV and parvovirus antibody, preparation rabbit anti-mouse antibody;
(8) antibody with the anti-ermine of rabbit source CDV and parvovirus is sprayed on two detection lines of formation on the nitrocellulose filter, and the rabbit anti-mouse antibody is sprayed on nature controlling line of formation on the nitrocellulose filter;
(9) rigid polyvinyl chloride liner plate, nitrocellulose filter, long collaurum pad, short collaurum pad, sample pad, absorption pad are bonded together the composition test strips in order, and this test strips fixedly packed in the test strips cartridge on request, obtain the described immune colloid gold test paper that can detect simultaneously fox, racoon dog, ermine canine distemper and parvovirus.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201210379154.7A CN102879573B (en) | 2012-10-09 | 2012-10-09 | Immune colloidal gold test paper capable of simultaneously detecting canine distemper and canine parvo of foxes, raccoon dogs and minks and a method for preparing immune colloidal gold test paper |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201210379154.7A CN102879573B (en) | 2012-10-09 | 2012-10-09 | Immune colloidal gold test paper capable of simultaneously detecting canine distemper and canine parvo of foxes, raccoon dogs and minks and a method for preparing immune colloidal gold test paper |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| CN102879573A true CN102879573A (en) | 2013-01-16 |
| CN102879573B CN102879573B (en) | 2015-01-21 |
Family
ID=47480972
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN201210379154.7A Active CN102879573B (en) | 2012-10-09 | 2012-10-09 | Immune colloidal gold test paper capable of simultaneously detecting canine distemper and canine parvo of foxes, raccoon dogs and minks and a method for preparing immune colloidal gold test paper |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN102879573B (en) |
Cited By (10)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN103604924A (en) * | 2013-11-22 | 2014-02-26 | 武汉中博生物股份有限公司 | Canine parvovirus colloidal gold immunochromatography test strip and preparation method |
| CN103743901A (en) * | 2013-12-19 | 2014-04-23 | 河北北方学院 | Colloidal gold test strip for detecting canine parvovirus |
| CN104407130A (en) * | 2013-11-21 | 2015-03-11 | 王海艳 | Colloidal gold test strip for detecting goatpox virus and preparation method thereof |
| CN104407144A (en) * | 2013-11-21 | 2015-03-11 | 王海艳 | Colloidal gold for goatpox virus colloidal gold detection reagent strips, colloidal-gold antibody and preparation method thereof |
| CN106226518A (en) * | 2016-07-08 | 2016-12-14 | 中国农业科学院特产研究所 | Canine distemper virus colloidal gold immunochromatographydetection detection test paper bar and preparation method thereof |
| CN107881154A (en) * | 2017-12-26 | 2018-04-06 | 中国农业科学院特产研究所 | Raccoon dog parvovirus attenuated vaccine strain and its purposes in Raccoon dog parvovirus attenuated vaccine is prepared |
| CN109613235A (en) * | 2018-12-10 | 2019-04-12 | 杭州毕肯莱博生物科技有限公司 | The colloidal gold strip and preparation method thereof of heterophile antibody HA in a kind of quick detection blood sample |
| CN109652597A (en) * | 2019-01-26 | 2019-04-19 | 沈阳农业大学 | It is a kind of for detect dog, cat, ermine parvovirus general RPA primer, RPA probe, kit and nucleic acid detection method |
| CN109975541A (en) * | 2018-12-26 | 2019-07-05 | 山东绿都生物科技有限公司 | A kind of detection card and preparation method thereof of quick detection canine distemper virus antigen |
| CN112415202A (en) * | 2020-11-06 | 2021-02-26 | 爱若维生物科技(苏州)有限公司 | Test strip for detecting feline coronavirus, preparation method thereof, kit and detection method |
Citations (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| JP2004301537A (en) * | 2003-03-28 | 2004-10-28 | Adtec Kk | Method for detecting canine distemper antigen |
| KR20050100823A (en) * | 2004-04-14 | 2005-10-20 | (주)에니젠 | Kit for detecting canine distemper virus antibody using immunochromatography and manufacturing method thereof |
| CN101074959A (en) * | 2007-06-13 | 2007-11-21 | 中国人民解放军军事医学科学院军事兽医研究所 | Gold and silver combined inspection test paper for feline and canidae animals important pestilence pathogen and its production |
| CN101078726A (en) * | 2005-09-05 | 2007-11-28 | 因韦尔尼斯医药瑞士股份有限公司 | Device for detecting immune globulin |
| WO2010036470A1 (en) * | 2008-09-02 | 2010-04-01 | The Government Of The United States Of America As Represented By The Secretary Of The Department Of Health And Human Services, Centers For Disease Control And Prevention | Method for detection of antigen-specific antibodies in biological samples |
-
2012
- 2012-10-09 CN CN201210379154.7A patent/CN102879573B/en active Active
Patent Citations (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| JP2004301537A (en) * | 2003-03-28 | 2004-10-28 | Adtec Kk | Method for detecting canine distemper antigen |
| KR20050100823A (en) * | 2004-04-14 | 2005-10-20 | (주)에니젠 | Kit for detecting canine distemper virus antibody using immunochromatography and manufacturing method thereof |
| CN101078726A (en) * | 2005-09-05 | 2007-11-28 | 因韦尔尼斯医药瑞士股份有限公司 | Device for detecting immune globulin |
| CN101074959A (en) * | 2007-06-13 | 2007-11-21 | 中国人民解放军军事医学科学院军事兽医研究所 | Gold and silver combined inspection test paper for feline and canidae animals important pestilence pathogen and its production |
| WO2010036470A1 (en) * | 2008-09-02 | 2010-04-01 | The Government Of The United States Of America As Represented By The Secretary Of The Department Of Health And Human Services, Centers For Disease Control And Prevention | Method for detection of antigen-specific antibodies in biological samples |
Cited By (12)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN104407130A (en) * | 2013-11-21 | 2015-03-11 | 王海艳 | Colloidal gold test strip for detecting goatpox virus and preparation method thereof |
| CN104407144A (en) * | 2013-11-21 | 2015-03-11 | 王海艳 | Colloidal gold for goatpox virus colloidal gold detection reagent strips, colloidal-gold antibody and preparation method thereof |
| CN103604924A (en) * | 2013-11-22 | 2014-02-26 | 武汉中博生物股份有限公司 | Canine parvovirus colloidal gold immunochromatography test strip and preparation method |
| CN103743901A (en) * | 2013-12-19 | 2014-04-23 | 河北北方学院 | Colloidal gold test strip for detecting canine parvovirus |
| CN106226518A (en) * | 2016-07-08 | 2016-12-14 | 中国农业科学院特产研究所 | Canine distemper virus colloidal gold immunochromatographydetection detection test paper bar and preparation method thereof |
| CN107881154A (en) * | 2017-12-26 | 2018-04-06 | 中国农业科学院特产研究所 | Raccoon dog parvovirus attenuated vaccine strain and its purposes in Raccoon dog parvovirus attenuated vaccine is prepared |
| CN107881154B (en) * | 2017-12-26 | 2021-03-05 | 中国农业科学院特产研究所 | Raccoon parvovirus attenuated vaccine strain and application thereof in preparation of raccoon parvovirus attenuated vaccine |
| CN109613235A (en) * | 2018-12-10 | 2019-04-12 | 杭州毕肯莱博生物科技有限公司 | The colloidal gold strip and preparation method thereof of heterophile antibody HA in a kind of quick detection blood sample |
| CN109975541A (en) * | 2018-12-26 | 2019-07-05 | 山东绿都生物科技有限公司 | A kind of detection card and preparation method thereof of quick detection canine distemper virus antigen |
| CN109975541B (en) * | 2018-12-26 | 2022-06-21 | 山东绿都生物科技有限公司 | Detection card for rapidly detecting canine distemper virus antigen and preparation method thereof |
| CN109652597A (en) * | 2019-01-26 | 2019-04-19 | 沈阳农业大学 | It is a kind of for detect dog, cat, ermine parvovirus general RPA primer, RPA probe, kit and nucleic acid detection method |
| CN112415202A (en) * | 2020-11-06 | 2021-02-26 | 爱若维生物科技(苏州)有限公司 | Test strip for detecting feline coronavirus, preparation method thereof, kit and detection method |
Also Published As
| Publication number | Publication date |
|---|---|
| CN102879573B (en) | 2015-01-21 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| CN102879573B (en) | Immune colloidal gold test paper capable of simultaneously detecting canine distemper and canine parvo of foxes, raccoon dogs and minks and a method for preparing immune colloidal gold test paper | |
| US20130309656A1 (en) | Antibody detection method and device for a saliva sample from a non-human animal | |
| CN105950563B (en) | The monoclonal antibody and application of hybridoma cell strain 7E3 and its resistant to foot and mouth disease A type virus of secretion | |
| CN103743901A (en) | Colloidal gold test strip for detecting canine parvovirus | |
| CN105695420A (en) | Mouse bone marrow hybridoma cell strains, monoclonal antibody generated by same and application | |
| CN109187967B (en) | Duplex rapid detection card for detecting and distinguishing O-type and A-type foot-and-mouth disease viruses and preparation method thereof | |
| McAllister et al. | Immunoblot assay: a rapid and sensitive method for identification of salmonid fish viruses | |
| CN106226518A (en) | Canine distemper virus colloidal gold immunochromatographydetection detection test paper bar and preparation method thereof | |
| JP4554472B2 (en) | Parvovirus antigen detection kit | |
| CN105277693A (en) | Human parainfluenza virus quantum dot immunochromatography typing detection card, preparation method and applications | |
| CN105181964B (en) | Mink Aleutian disease virus antibody colloidal gold test strip and manufacturing method thereof | |
| CN112904021A (en) | Test strip for double-line detection of antibody after vaccination of new coronavirus | |
| CN102262156B (en) | Detection test strip for coxsackie virus A16 type (CA16) IgM (Immune Globulin M) antibody | |
| CN109975541A (en) | A kind of detection card and preparation method thereof of quick detection canine distemper virus antigen | |
| CN102749446A (en) | Chlamydia pneumoniae IgM (immunoglobulin M) colloidal golden method kit and preparation method thereof | |
| Lövgren et al. | A new approach to the preparation of a Toxoplasma gondii membrane antigen for use in ELISA | |
| CN102253210B (en) | Test strip for detecting enterovirus 71 (EV71) IgM antibody | |
| Schaeffer et al. | Applications of immunofluorescence in public health virology | |
| CN105585633B (en) | The immune chromatography reagent kit of anti-human haemophilus influenzae P6 protein antibodies and the application antibody | |
| CN103983780A (en) | Colloidal gold immunochromatograohic assay test strip for detecting pigeon I-type paramyxovirus and preparation method thereof | |
| CN102980997A (en) | EB virus capsid antigen IgM antibody colloidal gold method detection reagent and preparation method thereof | |
| WO2021117862A1 (en) | Immunoassay method and immunoassay device | |
| CN105319361B (en) | Scylla paramamosain reovirus colloidal gold immunochromatographimethod reagent strip and preparation method thereof, application method | |
| CN109946457A (en) | A kind of colloidal gold strip detecting Porcine epidemic diarrhea virus | |
| And et al. | Humoral immune response to virions and dense bodies of human cytomegalovirus determined by enzyme immunofluorescence assay |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| C06 | Publication | ||
| PB01 | Publication | ||
| C10 | Entry into substantive examination | ||
| SE01 | Entry into force of request for substantive examination | ||
| C14 | Grant of patent or utility model | ||
| GR01 | Patent grant | ||
| C56 | Change in the name or address of the patentee | ||
| CP01 | Change in the name or title of a patent holder |
Address after: 066600 College of animal science and technology, Hebei Normal University of Science and Technology, Changli, Hebei, Qinhuangdao Patentee after: Hebei Science & Technology Normal College Patentee after: HEBEI XINHUA KEJI VETERINARY MEDICINE GROUP CO., LTD. Address before: 066600 College of animal science and technology, Hebei Normal University of Science and Technology, Changli, Hebei, Qinhuangdao Patentee before: Hebei Science & Technology Normal College Patentee before: Hebei Xinhua Keji Veterinary Medicine Co., Ltd. |