CN113640514A - Colloidal gold test strip for detecting canine encephalitis B virus antibody and preparation method thereof - Google Patents
Colloidal gold test strip for detecting canine encephalitis B virus antibody and preparation method thereof Download PDFInfo
- Publication number
- CN113640514A CN113640514A CN202110643141.5A CN202110643141A CN113640514A CN 113640514 A CN113640514 A CN 113640514A CN 202110643141 A CN202110643141 A CN 202110643141A CN 113640514 A CN113640514 A CN 113640514A
- Authority
- CN
- China
- Prior art keywords
- canine
- colloidal gold
- test strip
- encephalitis
- virus
- 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.)
- Pending
Links
- 241000282465 Canis Species 0.000 title claims abstract description 65
- 238000012360 testing method Methods 0.000 title claims abstract description 64
- PCHJSUWPFVWCPO-UHFFFAOYSA-N gold Chemical compound [Au] PCHJSUWPFVWCPO-UHFFFAOYSA-N 0.000 title claims abstract description 52
- 206010014599 encephalitis Diseases 0.000 title claims abstract description 37
- 241000701076 Macacine alphaherpesvirus 1 Species 0.000 title claims abstract description 36
- 238000002360 preparation method Methods 0.000 title claims abstract description 20
- 238000001514 detection method Methods 0.000 claims abstract description 40
- 241000710842 Japanese encephalitis virus Species 0.000 claims abstract description 27
- 239000000427 antigen Substances 0.000 claims description 35
- 102000036639 antigens Human genes 0.000 claims description 35
- 108091007433 antigens Proteins 0.000 claims description 35
- 239000012528 membrane Substances 0.000 claims description 27
- 238000000034 method Methods 0.000 claims description 25
- 239000000020 Nitrocellulose Substances 0.000 claims description 20
- 229920001220 nitrocellulos Polymers 0.000 claims description 20
- 210000002966 serum Anatomy 0.000 claims description 18
- 238000003908 quality control method Methods 0.000 claims description 15
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 claims description 15
- 102000004169 proteins and genes Human genes 0.000 claims description 13
- 108090000623 proteins and genes Proteins 0.000 claims description 13
- 238000010521 absorption reaction Methods 0.000 claims description 12
- 238000002415 sodium dodecyl sulfate polyacrylamide gel electrophoresis Methods 0.000 claims description 12
- BPHPUYQFMNQIOC-NXRLNHOXSA-N isopropyl beta-D-thiogalactopyranoside Chemical compound CC(C)S[C@@H]1O[C@H](CO)[C@H](O)[C@H](O)[C@H]1O BPHPUYQFMNQIOC-NXRLNHOXSA-N 0.000 claims description 11
- 239000006228 supernatant Substances 0.000 claims description 10
- 239000011248 coating agent Substances 0.000 claims description 9
- 238000000576 coating method Methods 0.000 claims description 9
- 241000283707 Capra Species 0.000 claims description 8
- XSQUKJJJFZCRTK-UHFFFAOYSA-N Urea Chemical compound NC(N)=O XSQUKJJJFZCRTK-UHFFFAOYSA-N 0.000 claims description 7
- 239000004202 carbamide Substances 0.000 claims description 7
- 238000001035 drying Methods 0.000 claims description 7
- 239000003365 glass fiber Substances 0.000 claims description 7
- 241001052560 Thallis Species 0.000 claims description 6
- 238000000502 dialysis Methods 0.000 claims description 6
- 239000003480 eluent Substances 0.000 claims description 6
- 239000000758 substrate Substances 0.000 claims description 6
- 241000191940 Staphylococcus Species 0.000 claims description 5
- 238000000746 purification Methods 0.000 claims description 5
- 238000005507 spraying Methods 0.000 claims description 5
- 239000003085 diluting agent Substances 0.000 claims description 4
- 230000006698 induction Effects 0.000 claims description 4
- 239000002245 particle Substances 0.000 claims description 4
- 239000001509 sodium citrate Substances 0.000 claims description 4
- 229920002538 Polyethylene Glycol 20000 Polymers 0.000 claims description 3
- 238000002835 absorbance Methods 0.000 claims description 3
- 238000012258 culturing Methods 0.000 claims description 3
- 238000005520 cutting process Methods 0.000 claims description 3
- 239000001963 growth medium Substances 0.000 claims description 3
- 229910052751 metal Inorganic materials 0.000 claims description 3
- 239000002184 metal Substances 0.000 claims description 3
- BWHMMNNQKKPAPP-UHFFFAOYSA-L potassium carbonate Substances [K+].[K+].[O-]C([O-])=O BWHMMNNQKKPAPP-UHFFFAOYSA-L 0.000 claims description 3
- 229910000027 potassium carbonate Inorganic materials 0.000 claims description 3
- 238000001742 protein purification Methods 0.000 claims description 3
- 230000009467 reduction Effects 0.000 claims description 3
- 238000004153 renaturation Methods 0.000 claims description 3
- NLJMYIDDQXHKNR-UHFFFAOYSA-K sodium citrate Chemical compound O.O.[Na+].[Na+].[Na+].[O-]C(=O)CC(O)(CC([O-])=O)C([O-])=O NLJMYIDDQXHKNR-UHFFFAOYSA-K 0.000 claims description 3
- 239000007853 buffer solution Substances 0.000 claims description 2
- 238000005336 cracking Methods 0.000 claims description 2
- 239000012043 crude product Substances 0.000 claims description 2
- 238000007865 diluting Methods 0.000 claims description 2
- 238000001502 gel electrophoresis Methods 0.000 claims description 2
- 238000002372 labelling Methods 0.000 claims description 2
- 239000011259 mixed solution Substances 0.000 claims description 2
- 238000002156 mixing Methods 0.000 claims description 2
- 238000004806 packaging method and process Methods 0.000 claims description 2
- 208000006400 Arbovirus Encephalitis Diseases 0.000 abstract description 7
- 206010052369 Encephalitis lethargica Diseases 0.000 abstract description 7
- 201000002498 viral encephalitis Diseases 0.000 abstract description 7
- 230000007547 defect Effects 0.000 abstract description 2
- 244000052769 pathogen Species 0.000 abstract description 2
- 230000001717 pathogenic effect Effects 0.000 abstract description 2
- 241000282472 Canis lupus familiaris Species 0.000 description 24
- 241000700605 Viruses Species 0.000 description 11
- 241000282414 Homo sapiens Species 0.000 description 8
- 230000035945 sensitivity Effects 0.000 description 8
- 238000002965 ELISA Methods 0.000 description 7
- 201000010099 disease Diseases 0.000 description 5
- 208000037265 diseases, disorders, signs and symptoms Diseases 0.000 description 5
- 238000003317 immunochromatography Methods 0.000 description 4
- 208000015181 infectious disease Diseases 0.000 description 4
- 241000712083 Canine morbillivirus Species 0.000 description 3
- 238000008157 ELISA kit Methods 0.000 description 3
- 241000711798 Rabies lyssavirus Species 0.000 description 3
- 230000000694 effects Effects 0.000 description 3
- 238000005516 engineering process Methods 0.000 description 3
- 244000144980 herd Species 0.000 description 3
- 241000824799 Canis lupus dingo Species 0.000 description 2
- 241000711573 Coronaviridae Species 0.000 description 2
- 208000000655 Distemper Diseases 0.000 description 2
- 241000589902 Leptospira Species 0.000 description 2
- 241001465754 Metazoa Species 0.000 description 2
- 208000002606 Paramyxoviridae Infections Diseases 0.000 description 2
- 241000125945 Protoparvovirus Species 0.000 description 2
- 108010088160 Staphylococcal Protein A Proteins 0.000 description 2
- 238000004458 analytical method Methods 0.000 description 2
- 238000006243 chemical reaction Methods 0.000 description 2
- 239000002274 desiccant Substances 0.000 description 2
- 238000010586 diagram Methods 0.000 description 2
- 238000010790 dilution Methods 0.000 description 2
- 239000012895 dilution Substances 0.000 description 2
- 238000009826 distribution Methods 0.000 description 2
- 238000001962 electrophoresis Methods 0.000 description 2
- 239000010931 gold Substances 0.000 description 2
- 229910052737 gold Inorganic materials 0.000 description 2
- 210000000653 nervous system Anatomy 0.000 description 2
- 239000012264 purified product Substances 0.000 description 2
- 239000000243 solution Substances 0.000 description 2
- 238000003860 storage Methods 0.000 description 2
- 208000024891 symptom Diseases 0.000 description 2
- 241000701161 unidentified adenovirus Species 0.000 description 2
- LYFPBWOBIJJASK-INIZCTEOSA-N (S)-(-)-Canadine Natural products O(C)c1c(OC)ccc2c1C[N+]1[C@H](c3c(cc4OCOc4c3)CC1)C2 LYFPBWOBIJJASK-INIZCTEOSA-N 0.000 description 1
- VZTUIEROBZXUFA-INIZCTEOSA-N (S)-canadine Chemical compound C1=C2[C@@H]3CC4=CC=C(OC)C(OC)=C4CN3CCC2=CC2=C1OCO2 VZTUIEROBZXUFA-INIZCTEOSA-N 0.000 description 1
- YMHOBZXQZVXHBM-UHFFFAOYSA-N 2,5-dimethoxy-4-bromophenethylamine Chemical compound COC1=CC(CCN)=C(OC)C=C1Br YMHOBZXQZVXHBM-UHFFFAOYSA-N 0.000 description 1
- 101100298998 Caenorhabditis elegans pbs-3 gene Proteins 0.000 description 1
- 241000701101 Canine adenovirus 1 Species 0.000 description 1
- 241000701114 Canine adenovirus 2 Species 0.000 description 1
- 241000711506 Canine coronavirus Species 0.000 description 1
- 208000035473 Communicable disease Diseases 0.000 description 1
- 206010010904 Convulsion Diseases 0.000 description 1
- 241000255925 Diptera Species 0.000 description 1
- 239000006144 Dulbecco’s modified Eagle's medium Substances 0.000 description 1
- 206010024238 Leptospirosis Diseases 0.000 description 1
- 102000007056 Recombinant Fusion Proteins Human genes 0.000 description 1
- 108010008281 Recombinant Fusion Proteins Proteins 0.000 description 1
- 208000004756 Respiratory Insufficiency Diseases 0.000 description 1
- 235000011483 Ribes Nutrition 0.000 description 1
- 241000220483 Ribes Species 0.000 description 1
- 241000282887 Suidae Species 0.000 description 1
- PQECCKIOFCWGRJ-UHFFFAOYSA-N Tetrahydro-berberine Natural products C1=C2C3CC4=CC=C(OC)C(O)=C4CN3CCC2=CC2=C1OCO2 PQECCKIOFCWGRJ-UHFFFAOYSA-N 0.000 description 1
- 241000545067 Venus Species 0.000 description 1
- 208000036142 Viral infection Diseases 0.000 description 1
- 239000002250 absorbent Substances 0.000 description 1
- 230000002745 absorbent Effects 0.000 description 1
- 239000002253 acid Substances 0.000 description 1
- 229910052782 aluminium Inorganic materials 0.000 description 1
- XAGFODPZIPBFFR-UHFFFAOYSA-N aluminium Chemical compound [Al] XAGFODPZIPBFFR-UHFFFAOYSA-N 0.000 description 1
- 230000003321 amplification Effects 0.000 description 1
- 238000003556 assay Methods 0.000 description 1
- 229930016122 canadine Natural products 0.000 description 1
- 210000003169 central nervous system Anatomy 0.000 description 1
- 230000008859 change Effects 0.000 description 1
- 239000003153 chemical reaction reagent Substances 0.000 description 1
- 238000007796 conventional method Methods 0.000 description 1
- 230000036461 convulsion Effects 0.000 description 1
- NKLPQNGYXWVELD-UHFFFAOYSA-M coomassie brilliant blue Chemical compound [Na+].C1=CC(OCC)=CC=C1NC1=CC=C(C(=C2C=CC(C=C2)=[N+](CC)CC=2C=C(C=CC=2)S([O-])(=O)=O)C=2C=CC(=CC=2)N(CC)CC=2C=C(C=CC=2)S([O-])(=O)=O)C=C1 NKLPQNGYXWVELD-UHFFFAOYSA-M 0.000 description 1
- 150000004683 dihydrates Chemical class 0.000 description 1
- 239000011888 foil Substances 0.000 description 1
- 208000021760 high fever Diseases 0.000 description 1
- 238000000703 high-speed centrifugation Methods 0.000 description 1
- 238000002649 immunization Methods 0.000 description 1
- 230000003053 immunization Effects 0.000 description 1
- 238000003018 immunoassay Methods 0.000 description 1
- 238000001727 in vivo Methods 0.000 description 1
- 238000011835 investigation Methods 0.000 description 1
- OOYGSFOGFJDDHP-KMCOLRRFSA-N kanamycin A sulfate Chemical compound OS(O)(=O)=O.O[C@@H]1[C@@H](O)[C@H](O)[C@@H](CN)O[C@@H]1O[C@H]1[C@H](O)[C@@H](O[C@@H]2[C@@H]([C@@H](N)[C@H](O)[C@@H](CO)O2)O)[C@H](N)C[C@@H]1N OOYGSFOGFJDDHP-KMCOLRRFSA-N 0.000 description 1
- 229960002064 kanamycin sulfate Drugs 0.000 description 1
- 239000003550 marker Substances 0.000 description 1
- 239000002609 medium Substances 0.000 description 1
- 238000012544 monitoring process Methods 0.000 description 1
- 239000013642 negative control Substances 0.000 description 1
- 238000003199 nucleic acid amplification method Methods 0.000 description 1
- DIKWKTWMLYICAA-UHFFFAOYSA-N pachycanthine Natural products C1=C2C3C=C4C=CC(OC)=C(OC)C4=CN3CCC2=CC2=C1OCO2 DIKWKTWMLYICAA-UHFFFAOYSA-N 0.000 description 1
- 230000001575 pathological effect Effects 0.000 description 1
- 230000008569 process Effects 0.000 description 1
- 239000012474 protein marker Substances 0.000 description 1
- 230000005180 public health Effects 0.000 description 1
- 238000000197 pyrolysis Methods 0.000 description 1
- 239000000700 radioactive tracer Substances 0.000 description 1
- 230000011514 reflex Effects 0.000 description 1
- 238000011160 research Methods 0.000 description 1
- 201000004193 respiratory failure Diseases 0.000 description 1
- 238000003757 reverse transcription PCR Methods 0.000 description 1
- 210000000614 rib Anatomy 0.000 description 1
- 238000007789 sealing Methods 0.000 description 1
- 238000011896 sensitive detection Methods 0.000 description 1
- 238000000926 separation method Methods 0.000 description 1
- 230000000405 serological effect Effects 0.000 description 1
- 238000011895 specific detection Methods 0.000 description 1
- 238000013112 stability test Methods 0.000 description 1
- 238000010186 staining Methods 0.000 description 1
- 230000000638 stimulation Effects 0.000 description 1
- HRXKRNGNAMMEHJ-UHFFFAOYSA-K trisodium citrate Chemical compound [Na+].[Na+].[Na+].[O-]C(=O)CC(O)(CC([O-])=O)C([O-])=O HRXKRNGNAMMEHJ-UHFFFAOYSA-K 0.000 description 1
- 229940038773 trisodium citrate Drugs 0.000 description 1
- 230000009385 viral infection Effects 0.000 description 1
- 230000000007 visual effect Effects 0.000 description 1
- 206010048282 zoonosis Diseases 0.000 description 1
Images
Classifications
-
- 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
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
- C07K14/005—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from viruses
-
- 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
- G01N33/52—Use of compounds or compositions for colorimetric, spectrophotometric or fluorometric investigation, e.g. use of reagent paper and including single- and multilayer analytical elements
-
- 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
- G01N33/543—Immunoassay; Biospecific binding assay; Materials therefor with an insoluble carrier for immobilising immunochemicals
- G01N33/54313—Immunoassay; Biospecific binding assay; Materials therefor with an insoluble carrier for immobilising immunochemicals the carrier being characterised by its particulate form
- G01N33/54346—Nanoparticles
-
- 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
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N2770/00—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA ssRNA viruses positive-sense
- C12N2770/00011—Details
- C12N2770/24011—Flaviviridae
- C12N2770/24111—Flavivirus, e.g. yellow fever virus, dengue, JEV
- C12N2770/24122—New viral proteins or individual genes, new structural or functional aspects of known viral proteins or genes
-
- 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
- G01N2333/08—RNA viruses
- G01N2333/18—Togaviridae; Flaviviridae
- G01N2333/183—Flaviviridae, e.g. pestivirus, mucosal disease virus, bovine viral diarrhoea virus, classical swine fever virus (hog cholera virus) or border disease virus
- G01N2333/185—Flaviviruses or Group B arboviruses, e.g. yellow fever virus, japanese encephalitis, tick-borne encephalitis, dengue
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N2469/00—Immunoassays for the detection of microorganisms
- G01N2469/20—Detection of antibodies in sample from host which are directed against antigens from microorganisms
Abstract
The invention relates to a preparation method and a preparation method of a colloidal gold test strip for detecting a canine Japanese encephalitis virus antibody, and belongs to the field of pathogen detection. In order to overcome the defect that a rapid and simple detection method for detecting canine epidemic encephalitis B is lacked in the prior art, the invention provides a preparation method and a preparation method of a colloidal gold test strip for detecting a canine encephalitis B virus antibody.
Description
Technical Field
The invention relates to a preparation method and a preparation method of a colloidal gold test strip for detecting a canine Japanese encephalitis virus antibody, and belongs to the field of pathogen detection.
Background
Epidemic Encephalitis b (JE) is an infectious disease of zoonosis mainly suffering from central nervous system damage caused by epidemic Encephalitis b (JEV). The disease is spread by taking mosquitoes as a medium, has obvious seasonality and a certain geographical distribution area, mainly occurs in Asia, and the prevalence distribution range of the disease tends to be expanded continuously along with the change of human activities and global climate. The disease is spread in people and often occurs in children under 10 years old, and clinically, the disease is mainly characterized by high fever, convulsion, disturbance of consciousness, respiratory failure, pathological reflex and meningeal stimulation, and the death rate of critical cases is high or sequela exists. About 30 hundred million people live in popular areas around the world, and these areas have about 7000 million new-born annually. It is estimated that 35000-50000 cases per year, 10000 cases of which die, and about 15000 cases of which have nervous system sequelae with different degrees. Wherein, China is one of the areas with high epidemic encephalitis B of pigs, and is the country with the most number of epidemic encephalitis B, and accounts for more than 80% of the total number of diseases in the world. At present, epidemic encephalitis B becomes the leading cause of viral infection and disability of the nervous system of children in China and even Asia,
in recent years, dogs as companion animals become increasingly part of the lives of people, and are more closely related to human beings. At present, some research results in China and abroad and JEV seroepidemiological investigation on dogs in earlier period show that part of pet dogs have JEV antibodies in vivo and have higher positive rate. Comprehensive analysis of the results of serological survey of Ribes and domesticated dogs by Shimoda et al suggested that the prevalence of human JEV is closely linked to the prevalence of canine JEV infection. Further analysis considers that although the pig is the most important intermediate amplification host of the JEV, the antibody positive rate of the pig herd is high, the pig herd has a certain distance with the human residence, and part of the pig herd is also subjected to JEV immunization, so the epidemic situation of the pig JEV can not completely and accurately reflect and evaluate the risk of human infection of the JEV, relatively speaking, the domestic dog has close living relationship with the human and does not need to be vaccinated, and the epidemic situation of the domestic dog can accurately reflect and evaluate the risk of human infection of the JEV, so the dog is suitable for being used as a sentinel animal for monitoring the epidemic situation of the human JEV and evaluating the risk. Therefore, the establishment of the rapid detection technology of the canine JEV has extremely important significance for evaluating the public health safety risk of the canine JEV.
At present, common methods for detecting JEV in actual work comprise ELISA, RT-PCR, virus separation and identification and the like, the operation process is complex and is limited by the detection time and the reagent cost, and the methods all need certain conditions and equipment and are difficult to popularize in the basic level. Therefore, aiming at the popular condition of JEV in China, it is necessary to develop a quick, simple and sensitive detection method. As a new generation of immunoassay technology, Gold Immunochromatography (GICA) utilizes Colloidal Gold (Colloidal Gold) as a tracer marker, has the advantages of naked eye judgment, rapidness, accuracy, simplicity and convenience in application and the like, and is widely regarded. However, the application of GICA to the detection of canine encephalitis B antibody is not seen in the utmost. The present invention has been made in view of the above circumstances.
Disclosure of Invention
In order to overcome the defect that a rapid and simple method for detecting canine epidemic encephalitis B is absent in the prior art, the invention provides a preparation method and a preparation method of a colloidal gold test strip for detecting a canine encephalitis B virus antibody.
The invention realizes the technical effects through the following technical scheme:
a colloidal gold test strip for detecting a canine Japanese encephalitis virus antibody comprises a substrate card, wherein a sample pad, a combination pad, a nitrocellulose membrane and a water absorption pad are sequentially stuck on the substrate card from front to back, and the structure of the colloidal gold test strip is shown in figure 2, wherein the nitrocellulose membrane is stuck on the substrate card, and a detection strip formed by recombinant canine Japanese encephalitis virus ED III antigen coating and a quality control strip formed by goat anti-rabbit IgG coating are distributed on the nitrocellulose membrane; the combination pad is positioned at the front end of the nitrocellulose membrane and is prepared by spraying colloidal gold particles on a glass fiber membrane and drying; the sample pad is arranged at the front end of the combination pad and is used for sample application; the water absorption pad is positioned at the rear end of the nitrocellulose membrane and is used for absorbing redundant water in the sample.
A preparation method of a colloidal gold test strip for detecting a canine Japanese encephalitis virus antibody comprises the following steps:
1) preparation of recombinant canine encephalitis B virus ED III antigen: inoculating the recombinant engineering strain into LB culture medium, culturing at 37 deg.C to absorbance A600When the concentration is 0.6, IPTG is added thereto to induce culture, and the cells are collected and culturedPerforming SDS-PAGE to determine purity to obtain a crude product of the recombinant canine encephalitis B virus ED III antigen;
2) purification of recombinant canine encephalitis B virus ED III antigen: dissolving the collected thalli after ultrasonic pyrolysis in 8mol/L urea, centrifuging and collecting supernatant; using Ni2+Purifying the supernatant containing the recombinant canine encephalitis B virus ED III antigen by an NTA metal chelating protein purification column, eluting while taking 20 mu L of eluent sample for SDS-PAGE gel electrophoresis detection, and collecting target eluent to obtain the purified recombinant canine encephalitis B virus ED III antigen;
3) renaturation of recombinant canine encephalitis B virus ED III antigen: transferring the purified recombinant canine encephalitis B virus ED III antigen into a dialysis bag, putting the dialysis bag into dialysate at 4 ℃, and dialyzing by replacing the dialysate until the urea concentration is reduced to 0.2 mol/L; then dialyzing with PBS buffer solution for 3 times, 3 h/time; concentrating protein with PEG 20000, centrifuging at high speed, collecting supernatant, quantitatively determining concentration with NanoDrop, and storing at-80 deg.C;
4) the preparation of the recombinant canine encephalitis B virus ED III antigen immunochromatographic test strip comprises the following steps: preparing a colloidal gold bonding pad; diluting the recombinant canine encephalitis B virus ED III antigen and the goat anti-rabbit IgG to 1mg/ml and 1.2mg/ml respectively, coating the antigen and the goat anti-rabbit IgG on a nitrocellulose membrane as a detection zone and a quality control zone, and drying the antigen and the quality control zone at 37 ℃ for 2 hours; and (3) sequentially sticking the sample pad, the combination pad and the water absorption pad on the bottom lining card, cutting the sample pad, the combination pad and the water absorption pad into strips with the width of 0.4cm, and packaging the strips into shells to obtain the colloidal gold test strip for detecting the canine encephalitis B virus antibody.
In the preparation method, the recombinant engineering strain is a strain capable of producing the recombinant canine encephalitis B virus ED III antigen, and the strain capable of producing the antigen in the prior art can be used for the invention. The recombinant engineering strain can express target protein with molecular weight of 33KDa, and the purity of the purified protein is more than 95% as shown by SDS-PAGE detection.
Preferably, the final concentration of IPTG in the step 1) is 1mmol/L, and the induction culture time is 6 hours.
Preferably, the method for preparing the conjugate pad in step 4) includes the steps of: preparing 25nm colloidal gold by a sodium citrate reduction method; the prepared glueThe ratio of the bulk gold is 0.1mol/L K2CO3And adjusting the pH value to 6.6-7.0, then labeling 4 mu g of staphylococcus protein A according to 1mL of colloidal gold, spraying the staphylococcus protein A on a glass fiber membrane, and drying at 37 ℃ for 2h to prepare the bonding pad.
A method for detecting a canine Japanese encephalitis virus antibody by using a colloidal gold test strip comprises the following steps: and (3) uniformly mixing the serum sample diluent and the serum to be detected, dripping 100 mu L of the mixed solution on a sample pad, and judging the detection result according to a judgment standard for about 20 min.
In the method for detecting the canine Japanese encephalitis virus antibody by using the colloidal gold test strip, the judgment standard is as follows: if only the quality control band appears red, the result is negative; if the quality control band and the detection band are red, the result is positive; if neither strip is colored or only the detection strip is colored, the test strip is invalid and the result is invalid.
The invention also provides an application, namely the application of the colloidal gold test strip in detecting the canine Japanese encephalitis virus antibody. The embodiments 2-4 of the invention show that the method has good sensitivity, stability and specificity when being used for detecting the canine Japanese encephalitis virus antibody, and the detection method is rapid and has visual results. Example 5 has higher accuracy when used for detecting the canine Japanese encephalitis virus antibody.
The invention establishes a method for detecting a dog encephalitis B virus specific antibody by expressing the dog encephalitis B virus ED III recombinant protein and combining a colloidal gold immunochromatography technology. Compared with the prior art, the method has the following technical effects:
1) the method for detecting the canine Japanese encephalitis virus antibody by using the colloidal gold test strip is simple and rapid to operate, high in detection speed and capable of completing detection within 20 min.
2) The method for detecting the canine Japanese encephalitis virus antibody by using the colloidal gold test strip, which is established by the invention, has strong specificity and better stability and sensitivity. The detection method established by the invention is used for detecting other viruses with similar symptoms and close sources, and has no non-specific reaction. The test strip which is finished by the determination is stored for 2 weeks at 37 ℃, and the detection result is unchanged; and the method has no obvious difference with the sensitivity detection of the ELISA kit.
3) The colloidal gold immunochromatographic assay has the characteristics of rapidness, sensitivity, specificity and stability, and is suitable for field detection.
Drawings
FIG. 1 shows the induced expression and purification results of dog encephalitis B virus ED III antigen, wherein M is protein marker; 1, add IPTG to index expression; 5,6, ED III purified protein.
FIG. 2 shows the structure of the colloidal gold immunochromatographic test strip.
FIG. 3 is a schematic diagram of a standard for colloidal gold immunochromatographic test strip.
Figure 4 detection sensitivity of the canine encephalitis B virus serum colloidal gold immunochromatographic test strip.
Figure 5 specific detection of canine encephalitis B virus serum colloidal gold immunochromatographic test strip.
Detailed Description
The invention is further described below by means of specific examples, which do not limit the scope of protection of the patent in any way.
Embodiment 1 a method for preparing a colloidal gold test strip for detecting a canine Japanese encephalitis virus antibody
A preparation method of a colloidal gold test strip for detecting a canine Japanese encephalitis virus antibody comprises the following steps:
1) preparation of recombinant canine encephalitis B virus ED III antigen: inoculating the recombinant engineering strain into LB culture medium, culturing at 37 deg.C to absorbance A600When the concentration is 0.6, adding IPTG (isopropyl thiogalactoside) to the solution until the final concentration is 1mmol/L, carrying out induction culture for 6h, collecting thalli, and carrying out SDS-PAGE (sodium dodecyl sulfate-polyacrylamide gel electrophoresis) detection to obtain a recombinant canine encephalitis B virus ED III antigen;
2) purification of recombinant canine encephalitis B virus ED III antigen: dissolving the collected thalli in 8mol/L urea after ultrasonic cracking, and centrifuging to collect supernatant; using Ni2+Purifying ED III antigen by an NTA metal chelating protein purification column, collecting eluent, and carrying out SDS-PAGE (sodium dodecyl sulfate-polyacrylamide gel electrophoresis) detection on 20 mu L of eluent sample;
3) renaturation of recombinant ED iii antigen: transferring the recombinant ED III antigen into a dialysis bag, putting the dialysis bag into dialysate at 4 ℃, and slowly reducing the urea concentration in the dialysate until the urea concentration is reduced to 0.2mol/L by replacing the solution; dialyzed against PBS 3 times for 3 h/time. Thirdly, protein is concentrated by PEG 20000, supernatant is collected after high-speed centrifugation, the concentration of the supernatant is quantified by NanoDrop, and the supernatant is labeled and separately stored in a refrigerator at minus 80 ℃ for later use;
4) preparing an immunochromatography test strip:
preparing a colloidal gold bonding pad: preparing 25nm colloidal gold particles by a sodium citrate reduction method; the prepared colloidal gold is added with the concentration of 0.1mol/L K2CO3After the pH value is adjusted to 6.6-7.0, 1mL of colloidal gold is used for marking 4 mu g of staphylococcus protein A, and the colloidal gold is sprayed on a glass fiber membrane and dried for 2h at 37 ℃.
Coating: the recombinant canine Japanese encephalitis virus ED III antigen and the goat anti-rabbit IgG are respectively diluted to 1mg/ml and 1.2mg/ml, coated on a nitrocellulose membrane as a detection zone and a quality control zone, and dried at 37 ℃ for 2 h.
The immunochromatographic test strip is assembled by sequentially adhering a sample pad and a water absorption pad of a combination pad on a bottom liner card, cutting the sample pad and the water absorption pad into strips with the width of 0.4cm, encasing, and then encapsulating the strips and a drying agent in an aluminum foil bag.
Isopropyl- β -D-thiogalactopyranoside (IPTG) and Kanamycin Sulfate in this example were obtained from Beijing Jingxin Kogyo Biotech, Inc.; dihydrate and trisodium citrate (C6H5O7Na 3.2H 2O), chloroauric acid (HAuCl 4.4H2O), Staphylococcal Protein A (SPA) were purchased from Sigma; BSA was purchased from MERCK; goat anti-rabbit IgG was purchased from dingguo biotechnology; nitrocellulose membranes (NC membranes), glass fibers, absorbent paper were purchased from Millipore corporation; dog encephalitis B virus antibody ELISA kits were purchased from R & D.
Experimental results for preparation and purification of recombinant ED iii antigen: the recombinant strain is induced and expressed for 6h by 1mmol/L IPTG, SDS-PAGE electrophoresis is carried out after the expression strain is taken for treatment, and coomassie brilliant blue staining shows that an obvious visible target band is arranged at the position with molecular mass of about 40 kDa. As can be seen from FIG. 1, the purified product ED III was identified by SDSPAGE electrophoresis and showed a single band at 33kDa, which was consistent with the expected size, indicating that the purified product was ED III protein. The protein concentration was 1.58 mg/mL.
Assembling the colloidal gold immunochromatographic test strip: the assembly schematic diagram is shown in fig. 2, and the test strip is assembled according to a conventional method. The kit comprises a bottom lining card, wherein a sample pad, a combination pad, a nitrocellulose membrane and a water absorption pad are sequentially stuck on the bottom lining card from front to back, and the structure of the kit is shown in figure 2, wherein the nitrocellulose membrane is stuck on the bottom lining card, and a detection band formed by recombinant canine encephalitis B virus ED III antigen coating and a quality control band formed by goat anti-rabbit IgG coating are distributed on the nitrocellulose membrane; the combination pad is positioned at the front end of the nitrocellulose membrane and is prepared by spraying colloidal gold particles on a glass fiber membrane and drying; the sample pad is arranged at the front end of the combination pad and is used for sample application; the water absorption pad is positioned at the rear end of the nitrocellulose membrane and is used for absorbing redundant water in the sample.
The judgment standard of the colloidal gold immunochromatographic test strip is shown in fig. 3. The judgment standard is as follows: if only the quality control band appears red, the result is negative; if the quality control band and the detection band are red, the result is positive; if neither strip or only the detection strip develops color, the test strip is invalid and the result is invalid.
Example 2 test strip for colloidal gold immunochromatography test sensitivity
The prepared test paper is used for detecting 1:700, 1:1000, 1:3000, 1: 70000, 1: 10000, 1: 20000 and 1: 30000 times of diluent of the canine serum respectively, and the sensitivity of the test paper is identified. Dog Japanese encephalitis virus antibodies in ELISA kits of R & D company are diluted by 1:10, 1:100 and 1:1000, and are detected by test paper and ELISA respectively. Determining ELISA result, wherein the cut-off value (CUTOFF) is the average value of negative controls plus 0.15; the samples with the average OD value less than the critical value are negative to the Venus veneris virus antibody; and if the average OD value of the sample is larger than or equal to the critical value, the positive of the Veneza virus antibody is obtained.
The results show (fig. 4), that the prepared test strip can detect dilution to 1: 640-fold serum dilution.
Example 3 colloidal gold immunochromatographic test strip test specificity test
The test paper is used for respectively detecting dog adenovirus CAdV-1 and CAdV-2 dog serum, diluent of dog rabies virus CRV dog serum, dog distemper virus CDV dog serum, dog coronavirus CCV dog serum, parainfluenza virus CPIV dog serum, dog leptospira virus CLV dog serum and parvovirus CPV dog serum, and identifying the specificity of the test paper.
The test strip for detecting the canine Japanese encephalitis virus serum shows two red strips, the detection strip and the quality control strip are both colored, and the result is positive; the detection of the near-source virus or the viruses with similar symptoms (such as canine adenovirus 1, canine adenovirus 2, canine rabies virus, canine distemper virus, canine coronavirus, parainfluenza virus, canine leptospirosis virus and parvovirus) is negative reaction (figure 5), which indicates that the prepared test strip for the canine encephalitis B virus has strong specificity. Wherein, in FIG. 5, canadine adenoviruses 1(CAdV-1), CAdV-2, Canaine Rabies Virus (CRV), Canaine Distempers (CDV), Canaine Coronavirus (CCV), Canaine Paraflunzavirus (CPIV), Canaine Leptospira Virus (CLV), Canaine Paravorous (CPV), Dulbecco's modified Eagle's medium (control), and andMDCK cell (control).
Example 4 colloidal gold immunochromatographic test strip test stability test
And (3) sealing the test strip with a drying agent, storing at 37 ℃ and room temperature respectively, detecting positive samples every other week, and identifying the stability of the chromatographic test strip.
The specificity and sensitivity of the test strips were unchanged from those of the freshly prepared test strips after storage at 37 ℃ for 2 weeks and storage at room temperature for 4 months.
Example 5 the test strip prepared by the invention is used for measuring the canine Japanese encephalitis virus antibody
And carrying out parallel detection on 368 parts of healthy dog serum by using the prepared test strip and a common ELISA method, namely a dog encephalitis B virus antibody detection kit, counting results, and calculating the coincidence rate of the two detection methods.
The percent of agreement (%) is (true positive + true negative)/(true positive + true negative + false positive + false negative) × 100%.
368 parts of canine serum were detected by prepared test strips and ELISA, respectively. The results show that: the test paper strip detects that 20 samples are positive, the specificity is 94.57%, and no positive sample is detected by ELISA; 348 samples are detected to be negative by the test strip, and 12 samples are detected to be negative by ELISA; the coincidence rate of the detection results of the two methods is 96.74% (356/369).
Claims (7)
1. A colloidal gold test strip for detecting a canine Japanese encephalitis virus antibody comprises a substrate card, wherein a sample pad, a combination pad, a nitrocellulose membrane and a water absorption pad are sequentially stuck on the substrate card from front to back, wherein the nitrocellulose membrane is stuck on the substrate card, and a detection band formed by recombinant canine Japanese encephalitis virus ED III antigen coating and a quality control band formed by goat anti-rabbit IgG coating are distributed on the nitrocellulose membrane; the combination pad is positioned at the front end of the nitrocellulose membrane and is prepared by spraying colloidal gold particles on a glass fiber membrane and drying; the sample pad is arranged at the front end of the combination pad and is used for sample application; the water absorption pad is positioned at the rear end of the nitrocellulose membrane and is used for absorbing redundant water in the sample.
2. A preparation method of a colloidal gold test strip for detecting a canine Japanese encephalitis virus antibody comprises the following steps:
1) preparation of recombinant canine encephalitis B virus ED III antigen: inoculating the recombinant engineering strain into LB culture medium, culturing at 37 deg.C to absorbance A600When the concentration is 0.6, adding IPTG (isopropyl-beta-thiogalactoside) for induction culture, collecting thalli, and determining the purity by SDS-PAGE (sodium dodecyl sulfate-polyacrylamide gel electrophoresis) to obtain a crude product of the recombinant canine encephalitis B virus ED III antigen;
2) purification of recombinant canine encephalitis B virus ED III antigen: carrying out ultrasonic cracking on the collected thalli, dissolving the thalli in 8mol/L urea, and centrifuging to collect supernatant; using Ni2+Purifying the supernatant containing the recombinant canine encephalitis B virus ED III antigen by an NTA metal chelating protein purification column, eluting while taking 20 mu L of eluent sample for SDS-PAGE gel electrophoresis detection, and collecting target eluent to obtain the purified recombinant canine encephalitis B virus ED III antigen;
3) renaturation of recombinant canine encephalitis B virus ED III antigen: transferring the purified recombinant canine encephalitis B virus ED III antigen into a dialysis bag, putting the dialysis bag into dialysate at 4 ℃, and dialyzing by replacing the dialysate until the urea concentration is reduced to 0.2 mol/L; then dialyzing with PBS buffer solution for 3 times, 3 h/time; concentrating protein with PEG 20000, centrifuging at high speed, collecting supernatant, quantitatively determining concentration with NanoDrop, and storing at-80 deg.C;
4) the preparation of the recombinant canine encephalitis B virus ED III antigen immunochromatographic test strip comprises the following steps: preparing a colloidal gold bonding pad; diluting the recombinant canine Japanese encephalitis virus ED III antigen and the goat anti-rabbit IgG to 1mg/ml and 1.2mg/ml respectively, coating the antigens on a nitrocellulose membrane as a detection zone and a quality control zone, and drying the nitrocellulose membrane at 37 ℃ for 2 hours; and (3) sequentially sticking the sample pad, the combination pad and the water absorption pad on the bottom lining card, cutting the sample pad, the combination pad and the water absorption pad into strips with the width of 0.4cm, and packaging the strips into shells to obtain the colloidal gold test strip for detecting the canine encephalitis B virus antibody.
3. The method for preparing a colloidal gold test strip for detecting canine Japanese encephalitis virus antibody according to claim 1, wherein the final concentration of IPTG in step 1) is 1mmol/L, and the induction culture time is 6 hours.
4. The method for preparing a colloidal gold test strip for detecting canine Japanese encephalitis virus antibody according to claim 1, wherein the method for preparing the conjugate pad in step 4) comprises the following steps: preparing 25nm colloidal gold by a sodium citrate reduction method; the prepared colloidal gold is added with the concentration of 0.1mol/L K2CO3And adjusting the pH value to 6.6-7.0, then labeling 4 mu g of staphylococcus protein A according to 1mL of colloidal gold, spraying the staphylococcus protein A on a glass fiber membrane, and drying at 37 ℃ for 2h to prepare the bonding pad.
5. A method for detecting a dog Japanese encephalitis virus antibody by using a colloidal gold test strip is characterized by comprising the following steps: and (3) uniformly mixing the serum sample diluent and the serum to be detected, dripping 100 mu L of the mixed solution on a sample pad, and judging the detection result according to a judgment standard for about 20 min.
6. A method for detecting a dog Japanese encephalitis virus antibody by using a colloidal gold test strip is characterized in that the judgment standard is as follows: only the quality control band appears red, and the result is negative; if the quality control band and the detection band are red, the result is positive; if the quality control band and the detection band do not develop color or only the detection band develops color, the test strip is invalid, and the result is invalid.
7. The use of the colloidal gold test strip of claim 1 for detecting canine Japanese encephalitis virus antibody.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202110643141.5A CN113640514A (en) | 2021-06-09 | 2021-06-09 | Colloidal gold test strip for detecting canine encephalitis B virus antibody and preparation method thereof |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202110643141.5A CN113640514A (en) | 2021-06-09 | 2021-06-09 | Colloidal gold test strip for detecting canine encephalitis B virus antibody and preparation method thereof |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| CN113640514A true CN113640514A (en) | 2021-11-12 |
Family
ID=78415901
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN202110643141.5A Pending CN113640514A (en) | 2021-06-09 | 2021-06-09 | Colloidal gold test strip for detecting canine encephalitis B virus antibody and preparation method thereof |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN113640514A (en) |
Citations (8)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101363866A (en) * | 2008-05-26 | 2009-02-11 | 北京庄笛浩禾生物医学科技有限公司 | Test paper strip for detecting encephalitis virus IgM antibody colloidal gold, method for making same and applications |
| CN101363864A (en) * | 2008-05-26 | 2009-02-11 | 北京庄笛浩禾生物医学科技有限公司 | Test paper strip for detecting encephalitis virus specificity IgG antibody, method for making same and applications |
| CN102393454A (en) * | 2011-08-24 | 2012-03-28 | 中国检验检疫科学研究院 | Colloidal gold test strip for detecting dengue virus antibody and preparation method and application thereof |
| CN103116022A (en) * | 2013-01-23 | 2013-05-22 | 广西壮族自治区兽医研究所 | Test strip for quickly detecting swine Japanese encephalitis antibody and preparation method of test strip |
| CN104497109A (en) * | 2014-12-05 | 2015-04-08 | 任瑞文 | Recombinant antigen protein and kit for detecting epidemic encephalitis B virus antibody |
| CN105116147A (en) * | 2015-08-25 | 2015-12-02 | 四川农业大学 | Colloidal gold immunochromatographic test strip for detecting DHVA-1 (Duck Hepatitis A Virus Type I) antibody and preparation method thereof |
| CN106153926A (en) * | 2015-04-08 | 2016-11-23 | 北京中检安泰诊断科技有限公司 | Encephalitis b virus IgG antibody detection kit and preparation method thereof |
| CN110702925A (en) * | 2019-11-11 | 2020-01-17 | 南京农业大学 | Preparation and detection method of fluorescence labeling protein chip for simultaneously detecting CSFV, PPV, JEV and PRRSV antibodies |
-
2021
- 2021-06-09 CN CN202110643141.5A patent/CN113640514A/en active Pending
Patent Citations (8)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101363866A (en) * | 2008-05-26 | 2009-02-11 | 北京庄笛浩禾生物医学科技有限公司 | Test paper strip for detecting encephalitis virus IgM antibody colloidal gold, method for making same and applications |
| CN101363864A (en) * | 2008-05-26 | 2009-02-11 | 北京庄笛浩禾生物医学科技有限公司 | Test paper strip for detecting encephalitis virus specificity IgG antibody, method for making same and applications |
| CN102393454A (en) * | 2011-08-24 | 2012-03-28 | 中国检验检疫科学研究院 | Colloidal gold test strip for detecting dengue virus antibody and preparation method and application thereof |
| CN103116022A (en) * | 2013-01-23 | 2013-05-22 | 广西壮族自治区兽医研究所 | Test strip for quickly detecting swine Japanese encephalitis antibody and preparation method of test strip |
| CN104497109A (en) * | 2014-12-05 | 2015-04-08 | 任瑞文 | Recombinant antigen protein and kit for detecting epidemic encephalitis B virus antibody |
| CN106153926A (en) * | 2015-04-08 | 2016-11-23 | 北京中检安泰诊断科技有限公司 | Encephalitis b virus IgG antibody detection kit and preparation method thereof |
| CN105116147A (en) * | 2015-08-25 | 2015-12-02 | 四川农业大学 | Colloidal gold immunochromatographic test strip for detecting DHVA-1 (Duck Hepatitis A Virus Type I) antibody and preparation method thereof |
| CN110702925A (en) * | 2019-11-11 | 2020-01-17 | 南京农业大学 | Preparation and detection method of fluorescence labeling protein chip for simultaneously detecting CSFV, PPV, JEV and PRRSV antibodies |
Non-Patent Citations (3)
| Title |
|---|
| 王振东等: "委内瑞拉马脑炎病毒重组抗原的制备纯化及其胶体金免疫层析检测方法的建立", 《中国生物工程杂志》, vol. 31, no. 07, 15 July 2011 (2011-07-15), pages 104 - 108 * |
| 钟登科等: "上海市犬流行性乙型脑炎血清流行病学调查与分析", 《中国动物传染病学报》, vol. 24, no. 05, pages 7 * |
| 陈沙彬等: "乙型脑炎病毒E结构域蛋白表达、纯化与胶体金的制备", 《实用预防医学》, vol. 22, no. 12, pages 1445 - 1448 * |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| CN111220803B (en) | Novel coronavirus antibody detection reagent, preparation method thereof and novel coronavirus antibody detection card | |
| CN111856003A (en) | Novel coronavirus (2019-nCoV) IgM and IgG antibody detection test strip, kit and method | |
| CN102520169A (en) | ELISA (Enzyme-Linked Immuno Sorbent Assay) detection kit of animal rabies neutralizing antibody and application thereof | |
| EP4113121A1 (en) | Antigen for 2019 novel coronavirus and detection use thereof | |
| CN101701959A (en) | Fluorescent test strip for rapidly testing and immunizing Newcastle disease virus and application | |
| CN109856407B (en) | Canine distemper virus antibody fluorescence detection test strip and preparation method and application thereof | |
| CN202141725U (en) | Magnetic bead immunochromatographic kit for qualitative/ quantitative detection of surface antigen of hepatitis B virus | |
| CN113640514A (en) | Colloidal gold test strip for detecting canine encephalitis B virus antibody and preparation method thereof | |
| CN113341138A (en) | Combined detection card for simultaneously detecting ASFV specific IgM and IgG antibodies and preparation method and application thereof | |
| CN112904021A (en) | Test strip for double-line detection of antibody after vaccination of new coronavirus | |
| CN210347666U (en) | African swine fever antibody colloidal gold detection card | |
| CN101833003A (en) | Vesicular stomatitis virus antibody colloidal goldtest paper tape and preparation method thereof | |
| CN101706499A (en) | FLAG fusion tag colloidal gold test strip and preparation method thereof | |
| CN102033128B (en) | Edwardsiella tarda rapid detection test paper as well as rapid detection method and application | |
| CN202599960U (en) | Colloidal gold test strip for rapid detection of O-type foot-and-mouth disease virus, classical swine fever virus and porcine reproductive and respiratory syndrome virus | |
| CN202433383U (en) | Joint inspection test paper for influenza A virus antigen and influenza B virus antigen | |
| KR101792860B1 (en) | Monoclonal antibody specific to nucleoprotein of influenza A virus and rapid fluorescence-linked immunochromatographic diagnostic kit with enhanced sensitivity using the same | |
| CN113322268A (en) | African swine fever virus p72 recombinant protein and colloidal gold immunochromatographic test paper constructed by same | |
| CN101852802A (en) | Test paper for detecting newcastle disease and infectious bronchitis viruses in one step | |
| CN113671178A (en) | African swine fever virus antibody detection test paper established based on capsid protein p72 and preparation method thereof | |
| CN202854145U (en) | Joint detection card for measles, parotitis and rubella IgG (Intravenous Gamma Globulin) antibodies | |
| CN102305861B (en) | Reagent strip for carrying out joint detection on Toxoplasma gondii IgM (immunoglobulin M) and IgG (immunoglobulin G) antibodies and preparation method thereof | |
| CN109856397A (en) | A kind of colloidal-gold detecting-card and preparation method thereof of quick detection Rabbit pest virus | |
| CN105116145B (en) | A kind of method of monoclonal antibody gold mark testing inspection lily mottle virus | |
| CN103146714A (en) | Mycobacterium tuberculosis CFP10 antigen protein serial recombinant expression method and application in tuberculosis detection thereof |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| PB01 | Publication | ||
| PB01 | Publication | ||
| SE01 | Entry into force of request for substantive examination | ||
| SE01 | Entry into force of request for substantive examination |