CN103604924A - Canine parvovirus colloidal gold immunochromatography test strip and preparation method - Google Patents
Canine parvovirus colloidal gold immunochromatography test strip and preparation method Download PDFInfo
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
- G01N33/569—Immunoassay; Biospecific binding assay; Materials therefor for microorganisms, e.g. protozoa, bacteria, viruses
- G01N33/56983—Viruses
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
- G01N33/531—Production of immunochemical test materials
- G01N33/532—Production of labelled immunochemicals
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
- G01N33/558—Immunoassay; Biospecific binding assay; Materials therefor using diffusion or migration of antigen or antibody
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
- G01N33/577—Immunoassay; Biospecific binding assay; Materials therefor involving monoclonal antibodies binding reaction mechanisms characterised by the use of monoclonal antibodies; monoclonal antibodies per se are classified with their corresponding antigens
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N2333/00—Assays involving biological materials from specific organisms or of a specific nature
- G01N2333/005—Assays involving biological materials from specific organisms or of a specific nature from viruses
- G01N2333/01—DNA viruses
- G01N2333/015—Parvoviridae, e.g. feline panleukopenia virus, human Parvovirus
Abstract
The invention discloses a canine parvovirus colloidal gold immunochromatography test strip and a preparation method. The test strip comprises a sample absorbing area, a gold label probe area, an immobilized antibody area, a water absorbing area and a bottom plate, wherein the sample absorbing area, the gold label probe area, the immobilized antibody area and the water absorbing area are sequentially paved on the bottom plate and partially overlapped each other; the gold label probe area is coated with a colloidal gold labeled anti-canine parvovirus monoclonal antibody F1; the immobilized antibody area is sequentially provided with a test line and a control line, the test line is coated with an anti-canine parvovirus monoclonal antibody B6, and the control line is coated goat anti-mouse IgG (immunoglobulin G). According to the preparation method, a glass fiber membrane, a polyester membrane coated with the anti-canine parvovirus monoclonal antibody F1, a nitrocellulose membrane coated with the test line and the control line, and water absorbing filter paper are assembled onto a polyethylene plate to obtain the canine parvovirus colloidal gold immunochromatography test strip. The test strip is fast to test, high in accuracy rate, high in specificity, and simple and convenient to carry and operate.
Description
Technical field
The present invention relates to a kind of test strip and preparation method, be specifically related to a kind of canine parvovirus colloidal gold immunochromatographydetection detection test paper bar and preparation method.
Background technology
Canine parvovirus (Canine Parvovirus, CPV) is that parvovirus belongs to member, strand small DNA virus.Virion such as is at the axisymmetric icosahedron, the rounded or hexagon of outward appearance, and without cyst membrane, virion diameter is 21nm～24nm, sedimentation coefficient is 23S～27S.In 1977, from suffering from the separated CPV of acquisition in the sick dog body of enteritis, sick dog was reduced to principal character with vomiting, hemorrhagic enteritis, leucocyte first, and the neurological susceptibility of pup is high, can cause the myocarditis of pup; In order to distinguish and called after CPV-2 with the minimum virus of dog (MCV), in the canid of many countries and regions, be in succession found subsequently, the incidence of disease is 50%～100%, mortality ratio is 0%～50%, very harmful to supporting dog industry and economic animal aquaculture, and also had influence on the existence of wild animal, therefore will set up detection method fast and effectively, and effectively treat for this disease.
The serological method for detection of CPV antibody that China has set up has hemagglutination test, hemagglutination-inhibition test, agar gel diffusion test etc., but due between CPV and Feline Panleukopenia Virus, mink enteritis virus three, serological reaction can occur to intersect, therefore these methods are often restricted in actual applications.Enzyme linked immunosorbent assay (ELISA) can detect CPV antigen, also can detect CPV antibody; PCR detection sensitivity is high, high specificity, and distinguishable wild virus infection and vaccine immunity, but these two kinds of technology all need special instrument and equipment.Utilize colloid gold label monoclonal CPV surface antibody, the colloidal gold colloidal gold detection test paper strip susceptibility of making detection CPV antigen is high, high specificity, and good stability, easy and simple to handle quick, result is easy to analyze to be judged.Wang Zhongli etc., the method of mentioning in the research > > of the article < < canine parvovirus immune colloid gold diagnostic techniques of delivering on Chinese Preventive Veterinary Medicine report is mark anti-dog parvovirus monoclonal antibody, and how anti-coated anti-dog parvovirus rabbit is.
Summary of the invention
Primary and foremost purpose of the present invention is to provide a kind of canine parvovirus colloidal gold immunochromatographydetection detection test paper bar, is fast detecting canine parvovirus, for clinical diagnosis provides reliable basis.The anti-dog parvovirus monoclonal antibody F1 of this Test paper strip adoption colloid gold label makees probe, the double antibody sandwich method of coated anti-dog parvovirus monoclonal antibody B6 and goat anti-mouse igg detects canine parvovirus, can detect for the canine parvovirus of canid and canine parvovirus susceptible animal.
Another object of the present invention is to provide the preparation method of above-mentioned canine parvovirus colloidal gold immunochromatographydetection detection test paper bar.
Object of the present invention is achieved through the following technical solutions:
A kind of canine parvovirus colloidal gold immunochromatographydetection detection test paper bar, comprise ,Jin Biao probe region, absorption of sample district, immobilization antibody district, suction zones and base plate, ,Jin Biao probe region, absorption of sample district, immobilization antibody district and suction zones are laid on successively on base plate and mutually partly overlap; Gold mark probe region is coated with the anti-dog parvovirus monoclonal antibody F1 of colloid gold label, and its labelled amount is preferably every mL colloid gold label 1～10 μ g anti-dog parvovirus monoclonal antibody F1 of 1～10 μ g/mL(); Immobilization antibody district (from gold mark probe region to suction zones direction) has detection line and control line, detection line to be coated with anti-dog parvovirus monoclonal antibody B6 successively, and package amount is preferably 0.1～10 μ g/cm; Control line is coated with goat anti-mouse igg, and package amount is preferably 0.1～10 μ g/cm.Described anti-dog parvovirus monoclonal antibody F1 and B6 are produced by anti-dog parvovirus monoclonal antibody hybridoma cell strain F1 and B6; (Liu Jie etc., the foundation of secretion anti-dog parvovirus monoclonal antibody hybridoma cell strain, China's veterinary drug magazine, 2013,47(7): 9-12), wherein, anti-dog parvovirus monoclonal antibody hybridoma cell strain F1 deposit number is CCTCC NO:C2013175, and anti-dog parvovirus monoclonal antibody hybridoma cell strain B6 deposit number is: CCTCC NO:C2013176.
The material of described base plate is preferably polyethylene board (PVC plate); The material in described absorption of sample district is preferably glass fibre membrane; The material of described gold mark probe region is preferably polyester film; The material in described immobilization antibody district is preferably nitrocellulose filter; The material of described suction zones is preferably absorbent filter.
The preparation method of above-mentioned canine parvovirus colloidal gold immunochromatographydetection detection test paper bar, comprises the steps: the anti-dog parvovirus monoclonal antibody F1 of colloid gold label to be sprayed on polyester film; On nitrocellulose filter, be coated with successively detection line (being coated with anti-dog parvovirus monoclonal antibody B6) and control line (coated goat anti-mouse igg); Glass fibre membrane, the polyester film that is coated with anti-dog parvovirus monoclonal antibody F1, the nitrocellulose filter that is coated with detection line and control line and absorbent filter are assembled into and on polyethylene board, obtain canine parvovirus colloidal gold immunochromatographydetection detection test paper bar.
Preferably, the preparation method of described canine parvovirus colloidal gold immunochromatographydetection detection test paper bar, comprises the steps:
(1) preparation of anti-dog parvovirus monoclonal antibody: select female BALB/c mouse more than body weight 20g in 8～10 week age, every lumbar injection 0.5mL norphytane, within 7 days, pneumoretroperitoneum is injected respectively anti-dog parvovirus monoclonal antibody hybridoma cell strain F1 and B6, and inoculum concentration is 2 * 10
6～4 * 10
6cell/0.5mL/ only, approximately through one week, extracts ascites after mouse web portion expands, and the centrifugal 15min of 5000rpm, gets supernatant standby; By the ammonium sulfate precipitation of 50% saturation degree 2 times for ascites, dialysis desalination obtains anti-dog parvovirus monoclonal antibody F1 and the anti-dog parvovirus monoclonal antibody B6 of purifying.
(2) method of colloid gold label anti-dog parvovirus monoclonal antibody F1: getting respectively radius is collaurum 20mL and the anti-dog parvovirus monoclonal antibody F150～200 μ g of 5～40nm, under the condition of pH8.0～9.0, by magnetic agitation, vibrate and make its combination, add PEG-20000 as stabilizing agent, and making PEG-20000 final mass concentration is 10～20%, adopt centrifuge method to remove unconjugated anti-dog parvovirus monoclonal antibody F1 and unstabilized colloid gold particle and agglutinator thereof, peony in centrifuge tube bottom is precipitated as collaurum-F1 compound (the canine parvovirus monoclonal antibody F1 of colloid gold label).
(3) collaurum-F1 compound is sprayed on polyester film: with 20mL polyglycol washing colloids gold-F1 compound, the centrifugal supernatant of removing, obtain peony precipitation, precipitation after purifying is dissolved with the borate buffer that 2mL concentration is 2mmol/L, pH6.0～8.0, with spraying equipment, be applied to 15 μ L/cm on polyester film, freeze-drying.
(4) nitrocellulose filter is coated: that detection line is coated is anti-dog parvovirus monoclonal antibody B6, and package amount is 0.1～10 μ g/cm, and what control line was coated is goat anti-mouse igg, and package amount is 0.1～10 μ g/cm, every live width 2～5mm.
(5) test strips preparation: nitrocellulose filter and the absorbent filter of the polyester film of glass fibre membrane, coated collaurum-F1 compound, coated detection line and control line are adhered on polyethylene board successively, and the strip that the test strips assembling is longitudinally cut into 4mm is canine parvovirus colloidal gold immuno-chromatography test paper strip.
The present invention has the following advantages and effect with respect to prior art tool:
During detection, with aseptic cotton carrier, pick the fecal specimens of animal to be checked, in 1mL PBS damping fluid (pH7.2～7.4), mix, until bulky grain, be sunken to pipe at the end, get supernatant and drop in this test strips absorption of sample district, sentence read result after 5～10 minutes, whether the color of contrast detection line and control line, can judge in tested animal body with canine parvovirus or infected dogs parvovirus.
(1) detect fast: only need 5～10 minutes detection time, can meet the needs of Site Detection.
(2) Detection accuracy is high, specificity good: test strips of the present invention and other dog susceptible cause of diseases do not have cross reaction, and detection sensitivity, higher than hemagglutination test, is compared with similar test strips, and coincidence rate is up to 98%.
(3) easy to carry, easy and simple to handle: the present invention need to, by Other Instruments equipment, not be applicable to veterinary hospitals at different levels, animal clinic and individual and use.
(4) test strip can be preserved at normal temperatures, and storage life can reach 1 year, and detects reproducible.
Accompanying drawing explanation
Fig. 1 is the structural representation of canine parvovirus colloidal gold immunochromatographydetection detection test paper bar; Wherein, 1 is absorption of sample district, and 2 is gold mark probe region, and 3 is immobilization antibody district, and 31 is detection line, and 32 is control line, and 4 is suction zones, and 5 is base plate.
Fig. 2 is test strips sensitivity tests result figure; Wherein, 1: negative control; 2～15: canine parvovirus positive is from 1:2
2start doubling dilution to 1:2
15.
Fig. 3 is hemagglutination test result figure; Wherein, A, B: positive reference sample from left to right 2
1～2
12doubling dilution; C: negative control.
Embodiment
Below in conjunction with embodiment and accompanying drawing, the present invention is done to further detailed description, but embodiments of the present invention are not limited to this.If do not specialize, the conventional means that in embodiment, technological means used is well known to those skilled in the art.
As shown in Figure 1, test strips surface level is followed successively by absorption of sample district 1, gold mark probe region 2, immobilization antibody district 3 and suction zones 4 and is laid on base plate 5 and mutually partly overlaps the structural representation of canine parvovirus colloidal gold immunochromatographydetection detection test paper bar from right to left; The material of absorption of sample district 1, gold mark probe region 2, immobilization antibody district 3, suction zones 4 and base plate 5 is respectively glass fibre membrane, polyester film, nitrocellulose filter, absorbent filter and polyethylene board.Gold mark probe region 2 is coated with the anti-dog parvovirus monoclonal antibody F1 of colloid gold label, and its labelled amount is 4 μ g/mL; Immobilization antibody district 3(is from gold mark probe region to suction zones direction) there are successively detection line 31 and control line 32, detection line is coated with anti-dog parvovirus monoclonal antibody B6, and package amount is 0.6 μ g/cm; Control line 32 is coated with goat anti-mouse igg, and package amount is 0.6 μ g/cm.
Above-mentioned canine parvovirus colloidal gold immunochromatographydetection detection test paper bar preparation method is as follows:
(1) preparation of anti-dog parvovirus monoclonal antibody: select female BALB/c mouse more than body weight 20g in 8～10 week age, every lumbar injection 0.5mL norphytane, within 7 days, pneumoretroperitoneum is injected respectively anti-dog parvovirus monoclonal antibody hybridoma cell strain F1 and B6, and inoculum concentration is 2 * 10
6～4 * 10
6cell/0.5mL/ only, approximately through one week, extracts ascites after mouse web portion expands, and the centrifugal 15min of 5000rpm, gets supernatant standby.By the ammonium sulfate precipitation of 50% saturation degree 2 times for ascites, dialysis desalination obtains the anti-dog parvovirus monoclonal antibody F1 of purifying and anti-dog parvovirus monoclonal antibody B6(Liu Jie etc., the foundation of secretion anti-dog parvovirus monoclonal antibody hybridoma cell strain, China's veterinary drug magazine, 2013,47(7): 9-12).Wherein, anti-dog parvovirus monoclonal antibody hybridoma cell strain F1 and B6 all on November 1st, 2013 be preserved in Chinese Typical Representative culture collection center (China. Wuhan. Wuhan University), deposit number is respectively CCTCC NO:C2013175 and CCTCC NO:C2013176.
(2) method of colloid gold label anti-dog parvovirus monoclonal antibody F1: getting respectively radius is collaurum 20mL and the anti-dog parvovirus monoclonal antibody F180 μ g of 25nm, under the condition of pH9.0, by magnetic agitation, vibrate and make its combination, add PEG-20000 as stabilizing agent, and making PEG-20000 final mass concentration is 20%, adopt centrifuge method to remove not in conjunction with F1 and unstabilized colloid gold particle and agglutinator thereof, the peony in centrifuge tube bottom is precipitated as collaurum-F1 compound.
(3) collaurum-F1 compound is sprayed on polyester film: with 20mL polyglycol washing colloids gold-F1 compound, the centrifugal supernatant of removing, obtain peony precipitation, precipitation after purifying is dissolved with the borate buffer that 2mL concentration is 2mmol/L, pH6.0, with spraying equipment, be applied to 15 μ L/cm on polyester film, freeze-drying.
(4) nitrocellulose filter is coated: that detection line is coated is anti-dog parvovirus monoclonal antibody B6, and what control line was coated is goat anti-mouse igg, every live width 2mm.
(5) test strips preparation: nitrocellulose filter and the absorbent filter of the polyester film of glass fibre membrane, coated collaurum-F1 compound, coated detection line and control line are adhered on polyethylene board successively, and the strip that the test strips assembling is longitudinally cut into 4mm is canine parvovirus colloidal gold immuno-chromatography test paper strip.
The amount that is coated with anti-dog parvovirus monoclonal antibody B6 except detection line 31 is 1.5 μ g/cm, the amount of control line 32 coated goat anti-mouse iggs is 1.5 μ g/cm, the labelled amount of the anti-dog parvovirus monoclonal antibody F1 of the colloid gold label that gold mark probe region 2 is coated is 4 μ g/mL, colloid gold label anti-dog parvovirus monoclonal antibody F1 method: getting respectively radius is 25nm colloidal gold solution 20mL and anti-dog parvovirus monoclonal antibody F180 μ g, under the condition of pH9.0, by magnetic agitation, vibrate and make its combination, add PEG-20000 as stabilizing agent, and making PEG-20000 final mass concentration is 20%, adopt centrifuge method to remove not in conjunction with anti-dog parvovirus monoclonal antibody F1 and unstabilized colloid gold particle and agglutinator thereof, peony in centrifuge tube bottom is precipitated as outside collaurum-F1 compound, all the other are identical with embodiment 1.
The amount that is coated with anti-dog parvovirus monoclonal antibody B6 except detection line 31 is 4.8 μ g/cm, the amount of control line 32 coated goat anti-mouse iggs is 4.8 μ g/cm, the labelled amount of the anti-dog parvovirus monoclonal antibody F1 of the colloid gold label that gold mark probe region 2 is coated is 10 μ g/mL, colloid gold label anti-dog parvovirus monoclonal antibody F1 method: getting respectively radius is 40nm colloidal gold solution 20mL and anti-dog parvovirus monoclonal antibody F1200 μ g, under the condition of pH9.0, by magnetic agitation, vibrate and make its combination, add PEG-20000 as stabilizing agent, and making PEG-20000 final mass concentration is 10%, adopt centrifuge method to remove not in conjunction with anti-dog parvovirus monoclonal antibody F1 and unstabilized colloid gold particle and agglutinator thereof, peony in centrifuge tube bottom is precipitated as outside collaurum-F1 compound, all the other are identical with embodiment 1.
The using method of canine parvovirus colloidal gold immunochromatographydetection detection test paper bar:
During detection, with aseptic cotton carrier, pick the fecal specimens of animal to be checked, at 1mL PBS damping fluid (pH7.2,0.01mol/L), mix, until bulky grain, be sunken to pipe at the end, get supernatant and drop in this test strips, sentence read result after 5～10 minutes, whether the color of contrast detection line and control line, can judge in tested animal body with canine parvovirus or infected dogs parvovirus.As red positive reaction appears in detection line, should treat in time or close observation; As detection line reacts without this, negative, show not have in sample canine parvovirus or canine parvovirus amount too low.Control line, no matter whether have canine parvovirus all to occur red positive reaction in sample, shows that test strips is effective, if control line is reactionless, illustrates that test strips lost efficacy.
Canine parvovirus F81 cells and supernatant is done to 2
2～2
15doubling dilution, detects by the canine parvovirus antigen test strip preparing in embodiment 1; Same sample is done to 2 simultaneously
1～2
12doubling dilution, does viral hemoagglutination test (HA) in contrast, evaluates the susceptibility of test strips.As shown in Figures 2 and 3, the minimum dilutability of the positive reference sample of ELISA test strip is 2 to result
11, and hemagglutination test detects the blood clotting valency of positive reference sample, be 2
10, two kinds of testing results show that canine parvovirus test strip doubles than hemagglutination test susceptibility, the canine parvovirus positive that hemagglutination test can't detect can be detected.
With the canine parvovirus antigen Test paper preparing in embodiment 1, detect respectively 1 part, 10 parts of Healthy Dogs fecal specimens, CDV cell toxicant sample, Inactivated rabies virus vaccine sample, hepatitis infectiosa canis virus cell toxicant sample, canine coronavirus cell toxicant sample, canine parainfluenza virus cell toxicant sample, testing result is all negative.
By canine parvovirus colloidal gold immunochromatographydetection detection test paper Tiao Yu Korea S's import canine parvovirus test strip (Bionote Inc.) of embodiment 1 preparation, clinical 60 duplicate samples are detected respectively, contrast clinical symptoms simultaneously.
Result shows (table 1), the positive coincidence rate that two test strips are treated sample product is 100%, and negative match-rate is 97.5%, and total coincidence rate is 98%, an incongruent example is further verified as the canine parvovirus positive through contrast clinical symptoms, so test strips of the present invention is better than other commodity test strips.
Two kinds of ELISA test strip sample comparing results of table 1
Above-described embodiment is preferably embodiment of the present invention; but embodiments of the present invention are not restricted to the described embodiments; other any do not deviate from change, the modification done under Spirit Essence of the present invention and principle, substitutes, combination, simplify; all should be equivalent substitute mode, within being included in protection scope of the present invention.
Claims (6)
1. a canine parvovirus colloidal gold immunochromatographydetection detection test paper bar, it is characterized in that: comprise ,Jin Biao probe region, absorption of sample district, immobilization antibody district, suction zones and base plate, ,Jin Biao probe region, absorption of sample district, immobilization antibody district and suction zones are laid on successively on base plate and mutually partly overlap; Gold mark probe region is coated with the anti-dog parvovirus monoclonal antibody F1 of colloid gold label; Immobilization antibody district has detection line and control line successively, and detection line is coated with anti-dog parvovirus monoclonal antibody B6; Control line is coated with goat anti-mouse igg; Described anti-dog parvovirus monoclonal antibody F1 and B6 are produced by anti-dog parvovirus monoclonal antibody hybridoma cell strain F1 and B6, and anti-dog parvovirus monoclonal antibody hybridoma cell strain F1 and B6 deposit number are respectively CCTCC NO:C2013175 and CCTCC NO:C2013176.
2. canine parvovirus colloidal gold immunochromatographydetection detection test paper bar according to claim 1, is characterized in that: the labelled amount of the anti-dog parvovirus monoclonal antibody F1 of described colloid gold label is 1～10 μ g/mL; The package amount of described anti-dog parvovirus monoclonal antibody B6 is 0.1～10 μ g/cm; Described goat anti-mouse igg be 0.1～10 μ g/cm.
3. canine parvovirus colloidal gold immunochromatographydetection detection test paper bar according to claim 1, is characterized in that: the material of described base plate is polyethylene board; The material in described absorption of sample district is glass fibre membrane; The material of described gold mark probe region is polyester film; The material in described immobilization antibody district is nitrocellulose filter; The material of described suction zones is absorbent filter.
4. the preparation method of the canine parvovirus colloidal gold immunochromatographydetection detection test paper bar described in claim 1-3 any one, is characterized in that comprising the steps: that the anti-dog parvovirus monoclonal antibody F1 by colloid gold label is sprayed on polyester film; On nitrocellulose filter, be coated with successively detection line and control line; Glass fibre membrane, the polyester film that is coated with anti-dog parvovirus monoclonal antibody F1, the nitrocellulose filter that is coated with detection line and control line and absorbent filter are assembled into and on polyethylene board, obtain canine parvovirus colloidal gold immunochromatographydetection detection test paper bar.
5. the preparation method of canine parvovirus colloidal gold immunochromatographydetection detection test paper bar according to claim 4, is characterized in that comprising the steps:
(1) preparation of anti-dog parvovirus monoclonal antibody: select female BALB/c mouse more than body weight 20g in 8～10 week age, every lumbar injection 0.5mL norphytane, within 7 days, pneumoretroperitoneum is injected respectively anti-dog parvovirus monoclonal antibody hybridoma cell strain F1 and B6, and inoculum concentration is 2 * 10
6～4 * 10
6cell/0.5mL/ only, through one week, extracts ascites after mouse web portion expands, and the centrifugal 15min of 5000rpm, gets supernatant standby; By the ammonium sulfate precipitation of 50% saturation degree 2 times for ascites, dialysis desalination obtains anti-dog parvovirus monoclonal antibody F1 and the anti-dog parvovirus monoclonal antibody B6 of purifying;
(2) method of colloid gold label anti-dog parvovirus monoclonal antibody F1: getting respectively radius is collaurum 20mL and anti-dog parvovirus monoclonal antibody F1 50～200 μ g of 5～40nm, under the condition of pH 8.0～9.0, by magnetic agitation, vibrate and make its combination, add PEG-20000 as stabilizing agent, and making PEG-20000 final mass concentration is 10～20%, adopt centrifuge method to remove unconjugated anti-dog parvovirus monoclonal antibody F1 and unstabilized colloid gold particle and agglutinator thereof, the peony in centrifuge tube bottom is precipitated as collaurum-F1 compound;
(3) collaurum-F1 compound is sprayed on polyester film: with 20mL polyglycol washing colloids gold-F1 compound, the centrifugal supernatant of removing, obtain peony precipitation, precipitation after purifying is dissolved with the borate buffer that 2mL concentration is 2mmol/L, pH 6.0～8.0, with spraying equipment, be applied to 15 μ L/cm on polyester film, freeze-drying;
(4) nitrocellulose filter is coated: that detection line is coated is anti-dog parvovirus monoclonal antibody B6, and package amount is 0.1～10 μ g/cm, and what control line was coated is goat anti-mouse igg, and package amount is 0.1～10 μ g/cm, every live width 2～5mm;
(5) test strips preparation: nitrocellulose filter and the absorbent filter of the polyester film of glass fibre membrane, coated collaurum-F1 compound, coated detection line and control line are adhered on polyethylene board successively, and the strip that the test strips assembling is longitudinally cut into 4mm is canine parvovirus colloidal gold immuno-chromatography test paper strip.
6. the using method of the canine parvovirus colloidal gold immunochromatographydetection detection test paper bar described in claim 1-3 any one, while it is characterized in that comprising the steps: to detect, with aseptic cotton carrier, pick the fecal specimens of animal to be checked, in PBS damping fluid, mix, until bulky grain, be sunken to pipe at the end, get supernatant and drop in this test strips absorption of sample district, sentence read result after 5～10 minutes, whether the color of contrast detection line and control line, can judge in tested animal body with canine parvovirus or infected dogs parvovirus.
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Cited By (5)
| Publication number | Priority date | Publication date | Assignee | Title |
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| CN104792984A (en) * | 2014-12-26 | 2015-07-22 | 南阳师范学院 | A canine parvovirus IgG antibody detection kit |
| 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 |
| CN107300621A (en) * | 2017-06-23 | 2017-10-27 | 石家庄洹众生物科技有限公司 | Quantitatively detect the detection reagent card and system of human complement factor H GAP-associated protein GAPs |
| CN108414753A (en) * | 2018-05-04 | 2018-08-17 | 广州敏捷生物技术有限公司 | Immunofluorescence for detecting Canine Parvovirus antigen chromatographs detection card and preparation method |
| CN111965350A (en) * | 2020-08-21 | 2020-11-20 | 苏州阿科索生物科技有限公司 | Canine parvovirus up-conversion chromatography test strip based on aptamer recognition |
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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 |
| CN107300621A (en) * | 2017-06-23 | 2017-10-27 | 石家庄洹众生物科技有限公司 | Quantitatively detect the detection reagent card and system of human complement factor H GAP-associated protein GAPs |
| CN108414753A (en) * | 2018-05-04 | 2018-08-17 | 广州敏捷生物技术有限公司 | Immunofluorescence for detecting Canine Parvovirus antigen chromatographs detection card and preparation method |
| CN111965350A (en) * | 2020-08-21 | 2020-11-20 | 苏州阿科索生物科技有限公司 | Canine parvovirus up-conversion chromatography test strip based on aptamer recognition |
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