CN110967480A - Preparation and application of pig epidemic diarrhea virus IgA antibody ELISA kit - Google Patents
Preparation and application of pig epidemic diarrhea virus IgA antibody ELISA kit Download PDFInfo
- Publication number
- CN110967480A CN110967480A CN201811141426.3A CN201811141426A CN110967480A CN 110967480 A CN110967480 A CN 110967480A CN 201811141426 A CN201811141426 A CN 201811141426A CN 110967480 A CN110967480 A CN 110967480A
- Authority
- CN
- China
- Prior art keywords
- antibody
- kit
- strain
- epidemic diarrhea
- diarrhea 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
- 206010012735 Diarrhoea Diseases 0.000 title description 12
- 241000700605 Viruses Species 0.000 title description 12
- 238000008157 ELISA kit Methods 0.000 title description 11
- 238000002360 preparation method Methods 0.000 title description 8
- 241001135549 Porcine epidemic diarrhea virus Species 0.000 claims abstract description 82
- 239000000427 antigen Substances 0.000 claims abstract description 56
- 210000002966 serum Anatomy 0.000 claims abstract description 54
- 238000001514 detection method Methods 0.000 claims abstract description 50
- 102000036639 antigens Human genes 0.000 claims abstract description 41
- 108091007433 antigens Proteins 0.000 claims abstract description 41
- 235000013336 milk Nutrition 0.000 claims abstract description 37
- 210000004080 milk Anatomy 0.000 claims abstract description 37
- 239000008267 milk Substances 0.000 claims abstract description 37
- 102000004190 Enzymes Human genes 0.000 claims abstract description 19
- 108090000790 Enzymes Proteins 0.000 claims abstract description 19
- 210000003296 saliva Anatomy 0.000 claims abstract description 14
- 238000006243 chemical reaction Methods 0.000 claims abstract description 12
- 239000013642 negative control Substances 0.000 claims abstract description 10
- 239000013641 positive control Substances 0.000 claims abstract description 10
- 239000003153 chemical reaction reagent Substances 0.000 claims abstract description 8
- 239000000243 solution Substances 0.000 claims description 34
- 239000011248 coating agent Substances 0.000 claims description 28
- 238000000576 coating method Methods 0.000 claims description 28
- 238000005406 washing Methods 0.000 claims description 27
- 230000002779 inactivation Effects 0.000 claims description 20
- WSFSSNUMVMOOMR-UHFFFAOYSA-N Formaldehyde Chemical compound O=C WSFSSNUMVMOOMR-UHFFFAOYSA-N 0.000 claims description 18
- 238000000034 method Methods 0.000 claims description 18
- 239000003085 diluting agent Substances 0.000 claims description 13
- VEZXCJBBBCKRPI-UHFFFAOYSA-N beta-propiolactone Chemical compound O=C1CCO1 VEZXCJBBBCKRPI-UHFFFAOYSA-N 0.000 claims description 11
- KRKNYBCHXYNGOX-UHFFFAOYSA-N citric acid Chemical compound OC(=O)CC(O)(C(O)=O)CC(O)=O KRKNYBCHXYNGOX-UHFFFAOYSA-N 0.000 claims description 9
- 239000007788 liquid Substances 0.000 claims description 9
- 108091003079 Bovine Serum Albumin Proteins 0.000 claims description 8
- 239000008363 phosphate buffer Substances 0.000 claims description 8
- 108010001336 Horseradish Peroxidase Proteins 0.000 claims description 7
- 239000012091 fetal bovine serum Substances 0.000 claims description 6
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 claims description 6
- 241000283074 Equus asinus Species 0.000 claims description 5
- 241001494479 Pecora Species 0.000 claims description 5
- 238000005119 centrifugation Methods 0.000 claims description 5
- 239000008055 phosphate buffer solution Substances 0.000 claims description 5
- 229960000380 propiolactone Drugs 0.000 claims description 5
- 235000020183 skimmed milk Nutrition 0.000 claims description 5
- 239000012089 stop solution Substances 0.000 claims description 5
- BNIILDVGGAEEIG-UHFFFAOYSA-L disodium hydrogen phosphate Chemical compound [Na+].[Na+].OP([O-])([O-])=O BNIILDVGGAEEIG-UHFFFAOYSA-L 0.000 claims description 4
- LFQSCWFLJHTTHZ-UHFFFAOYSA-N Ethanol Chemical compound CCO LFQSCWFLJHTTHZ-UHFFFAOYSA-N 0.000 claims description 3
- AQLJVWUFPCUVLO-UHFFFAOYSA-N urea hydrogen peroxide Chemical compound OO.NC(N)=O AQLJVWUFPCUVLO-UHFFFAOYSA-N 0.000 claims description 3
- 229940078916 carbamide peroxide Drugs 0.000 claims description 2
- 239000003795 chemical substances by application Substances 0.000 claims 4
- WBODDOZXDKQEFS-UHFFFAOYSA-N 1,2,3,4-tetramethyl-5-phenylbenzene Chemical group CC1=C(C)C(C)=CC(C=2C=CC=CC=2)=C1C WBODDOZXDKQEFS-UHFFFAOYSA-N 0.000 claims 1
- 230000003068 static effect Effects 0.000 claims 1
- 238000002965 ELISA Methods 0.000 abstract description 15
- 230000035945 sensitivity Effects 0.000 abstract description 14
- 210000000436 anus Anatomy 0.000 abstract description 5
- 239000000523 sample Substances 0.000 description 39
- 241000282898 Sus scrofa Species 0.000 description 23
- 238000011156 evaluation Methods 0.000 description 12
- 238000004321 preservation Methods 0.000 description 12
- 238000012360 testing method Methods 0.000 description 10
- 241000283707 Capra Species 0.000 description 8
- 210000003022 colostrum Anatomy 0.000 description 8
- 235000021277 colostrum Nutrition 0.000 description 8
- 241000282887 Suidae Species 0.000 description 7
- 238000007789 sealing Methods 0.000 description 7
- 229960005486 vaccine Drugs 0.000 description 7
- 239000000872 buffer Substances 0.000 description 6
- 230000001900 immune effect Effects 0.000 description 6
- 101710139375 Corneodesmosin Proteins 0.000 description 5
- 230000003053 immunization Effects 0.000 description 5
- 102000002260 Alkaline Phosphatase Human genes 0.000 description 4
- 108020004774 Alkaline Phosphatase Proteins 0.000 description 4
- NBIIXXVUZAFLBC-UHFFFAOYSA-N Phosphoric acid Chemical compound OP(O)(O)=O NBIIXXVUZAFLBC-UHFFFAOYSA-N 0.000 description 4
- WCUXLLCKKVVCTQ-UHFFFAOYSA-M Potassium chloride Chemical compound [Cl-].[K+] WCUXLLCKKVVCTQ-UHFFFAOYSA-M 0.000 description 4
- FAPWRFPIFSIZLT-UHFFFAOYSA-M Sodium chloride Chemical compound [Na+].[Cl-] FAPWRFPIFSIZLT-UHFFFAOYSA-M 0.000 description 4
- 102000005936 beta-Galactosidase Human genes 0.000 description 4
- 108010005774 beta-Galactosidase Proteins 0.000 description 4
- 201000010099 disease Diseases 0.000 description 4
- 208000037265 diseases, disorders, signs and symptoms Diseases 0.000 description 4
- 238000005516 engineering process Methods 0.000 description 4
- 238000002649 immunization Methods 0.000 description 4
- CSCPPACGZOOCGX-UHFFFAOYSA-N Acetone Chemical compound CC(C)=O CSCPPACGZOOCGX-UHFFFAOYSA-N 0.000 description 3
- 102100031673 Corneodesmosin Human genes 0.000 description 3
- DGAQECJNVWCQMB-PUAWFVPOSA-M Ilexoside XXIX Chemical compound C[C@@H]1CC[C@@]2(CC[C@@]3(C(=CC[C@H]4[C@]3(CC[C@@H]5[C@@]4(CC[C@@H](C5(C)C)OS(=O)(=O)[O-])C)C)[C@@H]2[C@]1(C)O)C)C(=O)O[C@H]6[C@@H]([C@H]([C@@H]([C@H](O6)CO)O)O)O.[Na+] DGAQECJNVWCQMB-PUAWFVPOSA-M 0.000 description 3
- 238000003556 assay Methods 0.000 description 3
- 230000007547 defect Effects 0.000 description 3
- 238000007865 diluting Methods 0.000 description 3
- 238000011841 epidemiological investigation Methods 0.000 description 3
- 210000003608 fece Anatomy 0.000 description 3
- 238000000338 in vitro Methods 0.000 description 3
- 208000015181 infectious disease Diseases 0.000 description 3
- 238000002372 labelling Methods 0.000 description 3
- 239000000203 mixture Substances 0.000 description 3
- 239000002773 nucleotide Substances 0.000 description 3
- 125000003729 nucleotide group Chemical group 0.000 description 3
- 230000001717 pathogenic effect Effects 0.000 description 3
- 150000003013 phosphoric acid derivatives Chemical class 0.000 description 3
- 150000003839 salts Chemical class 0.000 description 3
- 230000009385 viral infection Effects 0.000 description 3
- GEYOCULIXLDCMW-UHFFFAOYSA-N 1,2-phenylenediamine Chemical group NC1=CC=CC=C1N GEYOCULIXLDCMW-UHFFFAOYSA-N 0.000 description 2
- VOZKAJLKRJDJLL-UHFFFAOYSA-N 2,4-diaminotoluene Chemical compound CC1=CC=C(N)C=C1N VOZKAJLKRJDJLL-UHFFFAOYSA-N 0.000 description 2
- YRNWIFYIFSBPAU-UHFFFAOYSA-N 4-[4-(dimethylamino)phenyl]-n,n-dimethylaniline Chemical compound C1=CC(N(C)C)=CC=C1C1=CC=C(N(C)C)C=C1 YRNWIFYIFSBPAU-UHFFFAOYSA-N 0.000 description 2
- 241001533384 Circovirus Species 0.000 description 2
- 208000005156 Dehydration Diseases 0.000 description 2
- WZUVPPKBWHMQCE-UHFFFAOYSA-N Haematoxylin Chemical compound C12=CC(O)=C(O)C=C2CC2(O)C1C1=CC=C(O)C(O)=C1OC2 WZUVPPKBWHMQCE-UHFFFAOYSA-N 0.000 description 2
- 229910019142 PO4 Inorganic materials 0.000 description 2
- 229940096437 Protein S Drugs 0.000 description 2
- 101710198474 Spike protein Proteins 0.000 description 2
- 206010047700 Vomiting Diseases 0.000 description 2
- 229910000147 aluminium phosphate Inorganic materials 0.000 description 2
- 210000004369 blood Anatomy 0.000 description 2
- 239000008280 blood Substances 0.000 description 2
- 210000004027 cell Anatomy 0.000 description 2
- 230000018044 dehydration Effects 0.000 description 2
- 238000006297 dehydration reaction Methods 0.000 description 2
- 238000011161 development Methods 0.000 description 2
- 238000003745 diagnosis Methods 0.000 description 2
- 238000001035 drying Methods 0.000 description 2
- 238000002474 experimental method Methods 0.000 description 2
- 239000012894 fetal calf serum Substances 0.000 description 2
- 238000001914 filtration Methods 0.000 description 2
- 238000009472 formulation Methods 0.000 description 2
- 210000004408 hybridoma Anatomy 0.000 description 2
- 230000036039 immunity Effects 0.000 description 2
- 238000012544 monitoring process Methods 0.000 description 2
- 229910000402 monopotassium phosphate Inorganic materials 0.000 description 2
- 244000052769 pathogen Species 0.000 description 2
- 235000021317 phosphate Nutrition 0.000 description 2
- -1 poly hexamethylene guanidine hydrochloride Polymers 0.000 description 2
- 239000001103 potassium chloride Substances 0.000 description 2
- 235000011164 potassium chloride Nutrition 0.000 description 2
- 239000008213 purified water Substances 0.000 description 2
- 238000012216 screening Methods 0.000 description 2
- 239000011734 sodium Substances 0.000 description 2
- 239000011780 sodium chloride Substances 0.000 description 2
- LWIHDJKSTIGBAC-UHFFFAOYSA-K tripotassium phosphate Chemical compound [K+].[K+].[K+].[O-]P([O-])([O-])=O LWIHDJKSTIGBAC-UHFFFAOYSA-K 0.000 description 2
- 210000002845 virion Anatomy 0.000 description 2
- 230000008673 vomiting Effects 0.000 description 2
- BVKZGUZCCUSVTD-UHFFFAOYSA-L Carbonate Chemical compound [O-]C([O-])=O BVKZGUZCCUSVTD-UHFFFAOYSA-L 0.000 description 1
- 108010001857 Cell Surface Receptors Proteins 0.000 description 1
- 241001461743 Deltacoronavirus Species 0.000 description 1
- QXNVGIXVLWOKEQ-UHFFFAOYSA-N Disodium Chemical compound [Na][Na] QXNVGIXVLWOKEQ-UHFFFAOYSA-N 0.000 description 1
- 241000709661 Enterovirus Species 0.000 description 1
- 102000016359 Fibronectins Human genes 0.000 description 1
- 108010067306 Fibronectins Proteins 0.000 description 1
- 208000005577 Gastroenteritis Diseases 0.000 description 1
- 239000007836 KH2PO4 Substances 0.000 description 1
- 241000710778 Pestivirus Species 0.000 description 1
- 229920001213 Polysorbate 20 Polymers 0.000 description 1
- 241001673669 Porcine circovirus 2 Species 0.000 description 1
- 241001135989 Porcine reproductive and respiratory syndrome virus Species 0.000 description 1
- 241000520007 Porcine rubulavirus Species 0.000 description 1
- 241000125945 Protoparvovirus Species 0.000 description 1
- 241000702670 Rotavirus Species 0.000 description 1
- 101710172711 Structural protein Proteins 0.000 description 1
- 241000701093 Suid alphaherpesvirus 1 Species 0.000 description 1
- 241000711484 Transmissible gastroenteritis virus Species 0.000 description 1
- 238000002835 absorbance Methods 0.000 description 1
- 239000002253 acid Substances 0.000 description 1
- 230000004075 alteration Effects 0.000 description 1
- 230000002924 anti-infective effect Effects 0.000 description 1
- 230000004596 appetite loss Effects 0.000 description 1
- 238000009739 binding Methods 0.000 description 1
- 239000012620 biological material Substances 0.000 description 1
- 210000001185 bone marrow Anatomy 0.000 description 1
- 238000009395 breeding Methods 0.000 description 1
- 230000001488 breeding effect Effects 0.000 description 1
- 239000007853 buffer solution Substances 0.000 description 1
- 238000003759 clinical diagnosis Methods 0.000 description 1
- 238000007796 conventional method Methods 0.000 description 1
- LXWYCLOUQZZDBD-LIYNQYRNSA-N csfv Chemical compound C([C@H](NC(=O)[C@H](CC(C)C)NC(=O)[C@H](CC(C)C)NC(=O)[C@H](CCC(O)=O)NC(=O)[C@H](CC(C)C)NC(=O)[C@@H](N)[C@@H](C)CC)C(=O)N[C@@H](CCCCN)C(=O)N[C@@H](CC=1C=CC=CC=1)C(=O)N[C@@H](CCCNC(N)=N)C(=O)N[C@@H](CC(O)=O)C(=O)N[C@@H](CC(N)=O)C(=O)N[C@@H]([C@@H](C)CC)C(=O)N[C@@H](CCCCN)C(=O)N[C@@H](CO)C(=O)N[C@@H](CO)C(=O)N[C@@H](C(C)C)C(=O)N[C@@H](CCCNC(N)=N)C(=O)N[C@@H](CCC(O)=O)C(O)=O)C1=CC=C(O)C=C1 LXWYCLOUQZZDBD-LIYNQYRNSA-N 0.000 description 1
- 210000000805 cytoplasm Anatomy 0.000 description 1
- 206010061428 decreased appetite Diseases 0.000 description 1
- 230000007123 defense Effects 0.000 description 1
- 239000012470 diluted sample Substances 0.000 description 1
- ZPWVASYFFYYZEW-UHFFFAOYSA-L dipotassium hydrogen phosphate Chemical compound [K+].[K+].OP([O-])([O-])=O ZPWVASYFFYYZEW-UHFFFAOYSA-L 0.000 description 1
- 229910000397 disodium phosphate Inorganic materials 0.000 description 1
- 238000010494 dissociation reaction Methods 0.000 description 1
- 230000005593 dissociations Effects 0.000 description 1
- 230000000694 effects Effects 0.000 description 1
- 206010014599 encephalitis Diseases 0.000 description 1
- 210000001035 gastrointestinal tract Anatomy 0.000 description 1
- 244000144980 herd Species 0.000 description 1
- 150000004677 hydrates Chemical class 0.000 description 1
- 150000002466 imines Chemical class 0.000 description 1
- 230000028993 immune response Effects 0.000 description 1
- 210000000987 immune system Anatomy 0.000 description 1
- 238000003018 immunoassay Methods 0.000 description 1
- 238000011534 incubation Methods 0.000 description 1
- 229910052500 inorganic mineral Inorganic materials 0.000 description 1
- 230000000968 intestinal effect Effects 0.000 description 1
- 208000011140 intestinal infectious disease Diseases 0.000 description 1
- 210000004347 intestinal mucosa Anatomy 0.000 description 1
- 230000006651 lactation Effects 0.000 description 1
- 208000019017 loss of appetite Diseases 0.000 description 1
- 235000021266 loss of appetite Nutrition 0.000 description 1
- 238000004519 manufacturing process Methods 0.000 description 1
- 238000000691 measurement method Methods 0.000 description 1
- 230000034217 membrane fusion Effects 0.000 description 1
- 102000006240 membrane receptors Human genes 0.000 description 1
- 239000011707 mineral Substances 0.000 description 1
- 235000010755 mineral Nutrition 0.000 description 1
- 238000002156 mixing Methods 0.000 description 1
- 238000012986 modification Methods 0.000 description 1
- 230000004048 modification Effects 0.000 description 1
- 235000019796 monopotassium phosphate Nutrition 0.000 description 1
- 229910000403 monosodium phosphate Inorganic materials 0.000 description 1
- 235000019799 monosodium phosphate Nutrition 0.000 description 1
- 230000003472 neutralizing effect Effects 0.000 description 1
- 231100000915 pathological change Toxicity 0.000 description 1
- 230000036285 pathological change Effects 0.000 description 1
- PJNZPQUBCPKICU-UHFFFAOYSA-N phosphoric acid;potassium Chemical compound [K].OP(O)(O)=O PJNZPQUBCPKICU-UHFFFAOYSA-N 0.000 description 1
- 239000000256 polyoxyethylene sorbitan monolaurate Substances 0.000 description 1
- 235000010486 polyoxyethylene sorbitan monolaurate Nutrition 0.000 description 1
- 230000002516 postimmunization Effects 0.000 description 1
- 229910000160 potassium phosphate Inorganic materials 0.000 description 1
- 235000011009 potassium phosphates Nutrition 0.000 description 1
- 159000000001 potassium salts Chemical class 0.000 description 1
- 108090000623 proteins and genes Proteins 0.000 description 1
- 238000000746 purification Methods 0.000 description 1
- 238000011160 research Methods 0.000 description 1
- 230000000241 respiratory effect Effects 0.000 description 1
- 230000003248 secreting effect Effects 0.000 description 1
- 230000000405 serological effect Effects 0.000 description 1
- 229910052708 sodium Inorganic materials 0.000 description 1
- 235000015424 sodium Nutrition 0.000 description 1
- 239000001488 sodium phosphate Substances 0.000 description 1
- 229910000162 sodium phosphate Inorganic materials 0.000 description 1
- AKHNMLFCWUSKQB-UHFFFAOYSA-L sodium thiosulfate Chemical compound [Na+].[Na+].[O-]S([O-])(=O)=S AKHNMLFCWUSKQB-UHFFFAOYSA-L 0.000 description 1
- 235000019345 sodium thiosulphate Nutrition 0.000 description 1
- 238000003860 storage Methods 0.000 description 1
- 238000006467 substitution reaction Methods 0.000 description 1
- 239000000758 substrate Substances 0.000 description 1
- 208000012153 swine disease Diseases 0.000 description 1
- 208000011580 syndromic disease Diseases 0.000 description 1
- 238000010998 test method Methods 0.000 description 1
- 231100000419 toxicity Toxicity 0.000 description 1
- 230000001988 toxicity Effects 0.000 description 1
- 210000003501 vero cell Anatomy 0.000 description 1
- 230000003612 virological effect Effects 0.000 description 1
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
- G01N33/531—Production of immunochemical test materials
- G01N33/532—Production of labelled immunochemicals
- G01N33/535—Production of labelled immunochemicals with enzyme label or co-enzymes, co-factors, enzyme inhibitors or enzyme substrates
-
- 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
- G01N33/536—Immunoassay; Biospecific binding assay; Materials therefor with immune complex formed in liquid phase
-
- 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
Landscapes
- Health & Medical Sciences (AREA)
- Life Sciences & Earth Sciences (AREA)
- Immunology (AREA)
- Engineering & Computer Science (AREA)
- Molecular Biology (AREA)
- Biomedical Technology (AREA)
- Chemical & Material Sciences (AREA)
- Hematology (AREA)
- Urology & Nephrology (AREA)
- Biotechnology (AREA)
- Biochemistry (AREA)
- Cell Biology (AREA)
- Food Science & Technology (AREA)
- Medicinal Chemistry (AREA)
- Physics & Mathematics (AREA)
- Analytical Chemistry (AREA)
- Microbiology (AREA)
- General Health & Medical Sciences (AREA)
- General Physics & Mathematics (AREA)
- Pathology (AREA)
- Virology (AREA)
- Tropical Medicine & Parasitology (AREA)
- Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)
Abstract
The invention provides a porcine epidemic diarrhea virus IgA antibody ELISA detection kit, wherein the kit comprises a support medium coated with a porcine epidemic diarrhea virus antibody-antigen complex, the porcine epidemic diarrhea virus antibody-antigen complex is a porcine epidemic diarrhea virus IgG monoclonal antibody-inactivated antigen complex, an enzyme-labeled anti-porcine IgA secondary antibody, and a detection reagent, a positive control and a negative control for detecting the reaction of the porcine IgA and the porcine epidemic diarrhea virus antibody-antigen complex antigen antibody. The kit has high sensitivity, and can accurately detect target samples in multiple milk, serum, saliva, anus swab and excrement samples.
Description
Technical Field
The invention relates to preparation and application of a porcine epidemic diarrhea virus IgA antibody ELISA kit, and belongs to the field of immunoassay.
Background
Porcine Epidemic Diarrheia (PED) is a highly-contagious, viral intestinal infectious disease in pigs caused by Porcine Epidemic Diarrhea Virus (PEDV), characterized by watery diarrhea, vomiting, dehydration, decreased appetite, and high mortality in suckling piglets. The disease is susceptible to pigs of various ages, the incidence rate of suckling piglets, skeleton pigs and fattening pigs reaches 100 percent, particularly the suckling piglets are the most serious, and the fatality rate reaches 100 percent.
In practice, serological testing remains a common method for clinical diagnosis and immunological evaluation. Currently, the PEDV antibody detection technology mainly focuses on detection of the level of PEDV IgG antibody in blood, and clinical detection finds that PED is frequently generated in swinery despite the existence of higher level of PEDV IgG antibody in serum after vaccine immunization. Therefore, traditional detection of the level of PEDV IgG antibodies in serum does not truly reflect the immunoprotection status of PEDV vaccines. The immune response caused by PEDV is mainly intestinal mucosal immunity, the mucosal immune system mainly plays a role by generating secretory IgA (sIgA) and IgM, and for enteroviruses, IgA is the type of antibody which is generated most by intestinal mucosa after pathogen infection, is also an important defense line for preventing pathogen from invading the intestinal tract, and plays an important role in body anti-infection immunity. Thus, the level of PEDV IgA antibody in the pig is more responsive to the condition of infection of the organism by PEDV or the immunoprotection efficacy of the vaccine.
At present, the IgA detection still has certain difficulty, and standard PEDV IgA detection technology and products are not available at home and abroad. At present, only the PEDV IgA antibody ELISA detection kit produced by Han Guaijie exists in domestic commercial products, but the kit can only detect the milk of the sow, and particularly has the problems that the milk is difficult to collect by the sow during lactation and the quality of the milk needs to be checked. Because samples such as blood and the like cannot be tested by using the kit, the immune effect of the piglet group can be indirectly and fuzzily evaluated only, and the immune effect of each piglet cannot be directly evaluated; clinical PEDV infection could not be evaluated and epidemiological monitoring could not be performed.
Disclosure of Invention
In order to overcome the defects of the prior art, the invention provides the pig epidemic diarrhea virus IgA antibody ELISA detection kit capable of detecting various targets, and particularly, the kit has high sensitivity and good specificity.
The invention relates to a porcine epidemic diarrhea virus IgA antibody ELISA detection kit, wherein the kit comprises a support medium coated with a porcine epidemic diarrhea virus antibody-antigen complex, the porcine epidemic diarrhea virus antibody-antigen complex is a porcine epidemic diarrhea virus IgG monoclonal antibody-inactivated antigen complex, an enzyme-labeled anti-porcine IgA secondary antibody, and a detection reagent, a positive control and a negative control for detecting the reaction of porcine IgA and the porcine epidemic diarrhea virus antibody-antigen complex antigen antibody.
In one embodiment of the invention, the porcine epidemic diarrhea virus IgG monoclonal antibody is 8A3A10, PEDV-McAB1 or PEDV-McAB 2.
The porcine epidemic diarrhea virus monoclonal antibody 8A3A10 is secreted and expressed by a mouse bone marrow hybridoma cell 8A3A10, wherein the preservation number of the hybridoma cell 8A3A10 strain is CCTCC No: C2014197, the preservation unit is China center for type culture Collection, the preservation address is Wuhan university in Wuhan City of China, the preservation date is 11 months and 3 days in 2014, and the Chinese patent CN104694480A is referred to.
The porcine epidemic diarrhea virus monoclonal antibodies PEDV-McAB1 and PEDV-McAB2 are disclosed in Chinese patent CN 105461805A.
In one embodiment of the present invention, the inactivated antigen is an inactivated antigen of CV777 strain, HN1301 strain, HN1302 strain, HN1303 strain, Iowa18984 strain, AJ1102 strain, ZJ08 strain, LW/L strain, SD10 strain, or a culture thereof; the inactivated antigen is prepared by centrifugation followed by inactivation.
The strain CV777 is commercially available.
As a preferred embodiment of the invention, the centrifugation condition is 5000-15000 r/min for 5-30 min, the inactivation step is formaldehyde inactivation or β -propiolactone inactivation, the formaldehyde concentration is 0.05-0.5% (v/v), the mixture is kept still at 25-37 ℃ or shaken at 50-200 r/min for 24-48 h, the β -propiolactone (β -PL) BPL is inactivated, the BPL concentration is 0.01-0.05%, and the inactivation time is 8-16 h.
In one embodiment of the present invention, the amount of the porcine epidemic diarrhea virus IgG monoclonal antibody 8A3a10 coated is 30-1000 ng/well; preferably, the coating amount of the porcine epidemic diarrhea virus IgG monoclonal antibody 8A3A10 is 50-500 ng/hole; more preferably, the coating amount of the porcine epidemic diarrhea virus IgG monoclonal antibody 8A3A10 is 50-300 ng/hole; the addition amount of the HN1301 strain inactivated antigen is 10-160 mu g/hole; preferably, the addition amount of the HN1301 strain inactivated antigen is 20-120 mu g/hole; more preferably, the addition amount of the HN1301 strain inactivated antigen is 20-80 mu g/hole.
In one embodiment of the invention, the enzyme-labeled anti-pig IgA secondary antibody is horseradish peroxidase, alkaline phosphatase, or β -D-galactosidase-labeled anti-pig IgA secondary antibody, preferably horseradish peroxidase-labeled anti-pig IgA secondary antibody.
In a preferred embodiment of the present invention, the enzyme-labeled secondary anti-porcine IgA antibody is an enzyme-labeled secondary goat-porcine IgA antibody, the detection reagents for detecting the reaction between the porcine IgA and the porcine epidemic diarrhea virus antibody-antigen complex antigen-antibody are a developer a solution containing 14.7g of disodium hydrogen phosphate, 9.3g of citric acid and 0.3g of urea peroxide in 1L of water, and a developer B solution containing 0.2g of Tetramethyldiphenyldiamine (TMB) and 100ml of absolute ethanol in 1L of water.
As an embodiment of the invention, the kit further comprises a stop solution, wherein the stop solution is 2M H2SO4And (3) solution.
In one embodiment of the present invention, the kit reaction system is a phosphate buffer containing BSA, OVA, skim milk, donkey serum, sheep serum, or fetal bovine serum.
In a preferred embodiment of the present invention, the reaction system is a PBS solution containing 15% to 25% fetal bovine serum.
As an embodiment of the invention, the kit further comprises a sample diluent and a washing solution, wherein the sample diluent is a phosphate buffer solution containing BSA, OVA, skim milk, donkey serum, sheep serum or fetal calf serum; the washing solution is phosphate buffer solution.
As an embodiment of the present invention, the positive control is serum, milk, saliva, anal swab, feces, which are positive for IgA antibodies identified by the IPMA method; the negative control is serum, milk, saliva, anal swab and excrement which are negative by the IgA antibody identified by the IPMA method.
In one embodiment of the invention, the support medium is a microtiter plate.
As an embodiment of the invention, the kit detects a sample selected from the group consisting of milk, serum, saliva, anal swab, and a stool sample.
The invention relates to a preparation method of the kit, which comprises the following steps:
coating porcine epidemic diarrhea virus antibodies with a coating solution, and coating for 16-24 hours at 2-8 ℃ or 1-3 hours at 37 ℃; washing with a washing solution, adding a sealing solution, sealing for 16-24 hours at 2-8 ℃ or sealing for 1-3 hours at 37 ℃; washing with a washing solution, adding a porcine epidemic diarrhea virus antigen, and capturing for 0.5-2 hours at 37 ℃; washing with a washing solution, and drying to obtain a support medium coated with the porcine epidemic diarrhea virus antibody-antigen complex;
preparing a detection reagent for detecting the antigen-antibody reaction of the porcine epidemic diarrhea virus;
and (3) assembling the support medium coated with the porcine epidemic diarrhea virus antibody-antigen complex prepared in the steps (1) to (2), a detection reagent for detecting the porcine epidemic diarrhea virus antigen-antibody reaction, and the enzyme-labeled goat anti-porcine IgA antibody, the positive control and the negative control into a kit.
In one embodiment of the present invention, the coating solution in step (1) is a carbonate buffer solution with a pH value of 0.05mol/L to 0.1mol/L and a pH value of 9.0 to 9.6; the confining liquid is phosphate buffer containing 1-3% BSA, 1-3% OVA, or 3-5% skimmed milk, or 10-30% donkey serum, or 10-30% sheep serum, or 10-30% fetal calf serum; the washing solution is phosphate buffer solution.
As an embodiment of the invention, the antibody in the step (1) is porcine epidemic diarrhea virus monoclonal antibody 8A3A10, PEDV-McAB1 or PEDV-McAB 2.
In one embodiment of the present invention, the antibody in step (1) is coated in an amount of 30 to 1000 ng/well, preferably 50 to 500 ng/well, and more preferably 50 to 300 ng/well.
In one embodiment of the present invention, the capture amount of the antigen in the step (1) is 10 to 160. mu.g/well, preferably 20 to 120. mu.g/well, and more preferably 20 to 80. mu.g/well.
As an embodiment of the invention, the enzyme used for labeling the goat anti-pig IgA antibody in the step (2) is horseradish peroxidase, or alkaline phosphatase, or β -D-galactosidase.
The invention also relates to a method for detecting the pig epidemic diarrhea virus IgA antibody in a sample by using the kit, wherein the method comprises the following steps:
step 1) diluting a detection sample by using a sample diluent, and adding the diluted detection sample, a positive control and a negative control into a detection hole coated with a porcine epidemic diarrhea virus antibody-antigen complex;
and 2) adding the enzyme-labeled goat anti-pig IgA antibody into the detection hole, and carrying out binding reaction with the pig epidemic diarrhea virus IgA antibody in the detection sample.
As a preferred embodiment of the present invention, the antibody-antigen complex in step 1) is attached to a support medium, preferably a microtiter plate, and the enzyme used in the labeling of the goat anti-pig IgA antibody in step 2) is horseradish peroxidase, alkaline phosphatase, or β -D-galactosidase.
As an embodiment of the present invention, the sample in step 1) includes milk, serum, saliva, anal swab, and feces.
In an embodiment of the present invention, in the step 1), the milk and the serum are diluted by 10 times with a sample diluent, 1 branch of the anus swab is dissolved in 1ml of the sample diluent, and 0.1g of the feces is dissolved in 1ml of the sample diluent.
The invention relates to application of the kit, which comprises the steps of piglet immune effect evaluation, wild virus infection evaluation, in-vitro sample epidemiological investigation and the like.
The kit can be applied to the detection of pig epidemic diarrhea virus IgA antibody with non-diagnosis purpose, not only can be applied to the evaluation of sow milk, but also can be applied to the accurate detection of a plurality of targets such as serum, saliva, anus swab, excrement and the like, so that the immune effect evaluation of each pig, the clinical evaluation after wild virus infection of the sow, the epidemiological investigation of in vitro samples and the like are realized, and the defects that the existing product can only detect the sow milk, detect the serum and other samples and has low sensitivity are overcome.
Detailed Description
The term "porcine epidemic diarrhea virus variant strain" is also called highly pathogenic porcine epidemic diarrhea virus or porcine epidemic diarrhea virus epidemic strain, which is epidemic from 2010, and the clinical manifestation after the onset is that the toxicity of the strain is obviously enhanced, so that pigs of any age such as sows, suckling piglets, shelf pigs and fattening pigs can be infected, particularly the suckling piglets are the most serious, and the disease death rate reaches 100%; watery diarrhea, vomiting, dehydration, loss of appetite, and high mortality in suckling piglets are the main features, but are not limited thereto. The disease still occurs after the existing vaccine is immunized. Variants include, but are not limited to, PEDV whose S protein has a nucleotide sequence substantially identical to the gene sequence of the S protein of PEDV SC1402 strain (sequence accession number KP 162057.1); preferably, the variant porcine epidemic diarrhea virus is PEDV having a nucleotide sequence with substantially the same S protein gene sequence. By substantially identical is meant that the nucleotide sequence of PEDV preferably comprises a sequence that is 85% to 100% identical to the sequence of the PEDV SC1402 strain S protein gene. Including but not limited to HN1301 strain, HN1302 strain, HN1303 strain, Iowa18984 strain, AJ1102 strain, ZJ08 strain, LW/L strain, SD10 strain and its culture. Wherein, HN1301 strains (with the preservation number of CCTCC NO. V201341) and HN1302 strains (with the preservation number of CCTCC NO. V201342) are preserved in China center for type culture Collection, the preservation addresses are Wuhan university in Wuhan City, and the preservation dates are all 2013, 9 and 16 days, and are disclosed in China patent CN 104513827A; HN1303 strain with preservation number of CCTCC NO. V201514, preservation unit of China center for type culture Collection, preservation address of Wuhan university in Wuhan City, and preservation date of 2015, 3 months and 4 days, is disclosed in Chinese patent CN 106148287A. Iowa18984 is disclosed in Hoang, H.A., Killian, M.L., Madson, D.M., Arruda, et al, full-Length Genome Sequence of a plane-ClonedVirulate molecular approximate Virus Isolate (USA/Iowa/18984/2013) from a Midwest U.S. Swine Herd.genome Announc 1.2013.AJ1102 is disclosed in Bi, J.Zeng, S.S., Xiao, S.Complete Genome Sequence of clinical idiomatic differential Virus along AJ.isolated a sucklung of salt of mineral acid in JV.J.6386, CN.10932. CN.11. J.Pat.3. J.3. J.J.P.S.J.S.J.P.J.P.J.P.P.J.P.P.J.P.P.J.P.P.P.J.P.P.P.J.P.P.P.P.. The LW/L strain is disclosed in Chinese patent CN 103041385A. Strain SD10 is disclosed in chinese patent CN 103820399A.
The term "culture" refers to a subculture of a variant of porcine epidemic diarrhea virus.
The term "porcine epidemic diarrhea virus S protein" is one of four structural proteins in porcine epidemic diarrhea virus, which is a spike protein (spike protein), a fibronectin located on the surface of virions, and plays an important biological role in invading host cells by membrane fusion after the virions bind to cell surface receptors and in mediating the production of neutralizing antibodies in infected hosts.
The term "enzyme" includes horseradish peroxidase, alkaline phosphatase, β -D-galactosidase, wherein the substrate used by horseradish peroxidase is o-phenylenediamine (OPD), Tetramethylbenzidine (TMB), preferably Tetramethylbenzidine (TMB).
The term "phosphate buffer" refers to a solution containing phosphoric acid or a salt thereof and adjusted to a desired pH, and is one of the most widely used buffers in biochemical studies. Typically, phosphate buffers are prepared from phosphoric acid or phosphates (including but not limited to sodium and potassium salts). Some phosphates are known in the art, such as sodium and potassium dihydrogen phosphate, disodium and dipotassium hydrogen phosphate, sodium and potassium phosphate. Phosphate salts are known to exist as hydrates of salts. The pH of the buffer is broad, e.g., in the range of about 4 to about 10, preferably in the range of about 5 to about 9, more preferably in the range of about 6 to about 8, and most preferably about 7.4, due to secondary dissociation of the buffer. Further preferably, the phosphate buffer is a phosphate buffer containing sodium chloride and potassium chloride.
The present invention will be further described with reference to specific embodiments, and the advantages and features of the present invention will become more apparent as the description proceeds. These examples are only illustrative and do not limit the scope of the present invention in any way. It will be understood by those skilled in the art that various changes in form and details may be made therein without departing from the spirit and scope of the invention, and that such changes and modifications may be made without departing from the spirit and scope of the invention.
The washing solution used in the examples of the present invention is a phosphate buffer such as PBS buffer (pH7.4, 0.01mol/L), and the formulation of 1L volume of PBS buffer is exemplified by: NaCl 8.0g, KCl 0.2g, Na2HPO4·12H2O 2.9g、KH2PO40.24g, but not limited to this formulation; unless otherwise specified, the samples were diluted with PBS buffer.
The chemical reagents used in the invention are all analytically pure and purchased from the national pharmaceutical group.
The experimental methods are conventional methods unless specified otherwise; the biomaterial is commercially available unless otherwise specified.
Example 1 preparation and identification of porcine epidemic diarrhea Virus antigen
1.1 selection of strains for antigens
Selecting a representative strain from 2 types of strains of classical strain and variant strain of porcine epidemic diarrhea virus for culture propagation, such as: the classical strain is CV777 strain, and the variant strains are HN1301 strain, HN1302 strain, HN1303 strain and their cultures.
1.2 methods of antigen preparation
The culture prepared in the example 1.1 is firstly centrifuged (centrifuged at 5000-15000 r/min for 5-30 min) and then inactivated, wherein the inactivation method comprises a formaldehyde method (M1 for short), the concentration range of formaldehyde is 0.05-0.5% (v/v), standing at 25-37 ℃ or shaking table is 50-200 r/min for 24-48 h, binary imine BEI inactivation (M2 for short), BEA is firstly cyclized for 2 h, the virus inactivation is preferably carried out within the concentration range of 3 mM-20 mM, shaking table is 50-200 r/min at 25-37 ℃ for 12-24 h, sodium thiosulfate is required for 1-3 h after inactivation, β -propiolactone (β -PL) BPL is inactivated (M3 for short), the concentration range of BPL is 0.01-0.05%, the inactivation time is 8-16 h, poly hexamethylene guanidine hydrochloride (PHMG) is simply called M4, the concentration range is 1-5%, the inactivation time is 12-24 h, the antigen is diluted to 20 g, the titer of PHMG is detected according to the method of an IPM 1P 591 sample, and the positive titer of the test method is detected according to the example 1.1.
TABLE 1 evaluation of coating antigens prepared from classical and variant PEDV strains
The above results show that: the detection of multiple strains revealed that the detection results of inactivated antigens prepared by M1 and M3 were optimal for both the classical strain (CV777 strain) and the variant strain (HN1301, HN1302, and HN1303 strain), and therefore, the inactivated antigens were prepared by these two methods and used in subsequent experiments.
The classical porcine epidemic diarrhea virus strain CV777 is used as a coating antigen for detecting the current clinical positive serum titer is lower, is not suitable for the current clinical detection and is discarded; the protein content difference of a plurality of variant strains or culture products thereof after being treated by centrifugation and inactivation methods is not obvious, and the positive serum titer of the present clinical detection as the coating antigen is also equivalent.
Example 2 selection of coating antibody for porcine epidemic diarrhea Virus IgA antibody ELISA kit
Monoclonal antibodies 8A3A10, PEDV-McAB1 and PEDV-McAB2 were used as coating antibodies, respectively, and the antibodies were prepared in an amount of 100 ng/well by the method of example 3.1, and 40 parts of positive milk (containing 20 parts of colostrum), 20 parts of positive serum, 40 parts of negative milk (containing 20 parts of colostrum) and 20 parts of negative serum (all of which were confirmed to be positive for IgA antibody by IPMA assay) were assayed by the assay method, and the coincidence rate of the samples with the IPMA method was calculated, and 1 part of clinical positive milk sample P1(IPMA titer 1: 256) was assayed, as shown in Table 2.
TABLE 2 evaluation results of porcine epidemic diarrhea virus antibody for kit coating
Shows that: the 3 monoclonal antibodies can be used for preparing a PEDV IgA antibody ELISA kit. The detection result of the kit 1 has good detection sensitivity and sample coincidence rate, and the kit 1 is selected for subsequent tests in order to facilitate more accurate subsequent detection.
The IPMA detection method comprises the following steps: the Vero cells are paved into a 96-hole cell plate, PEDV virus liquid is inoculated, the pathological changes reach 30% -50%, the antigen plate is fixed by 80% cold acetone, and the antigen plate is frozen at minus 20 ℃ for standby. And adding the diluted sample to be detected into an antigen plate, setting a positive control and a negative control at the same time, incubating for 1 hour at 37 ℃, and washing. Adding the diluted goat anti-pig IgA enzyme-labeled secondary antibody into an antigen plate, incubating for 1 hour at 37 ℃, and washing. And adding the diluted AEC color developing solution into the hole, and washing after color development at room temperature in a dark place. The diluted hematoxylin solution was added to the wells and stained at room temperature. Observing under a microscope, and judging the result, wherein the judgment standard is as follows: the control was true, the nucleus was blue, and the cytoplasm was positive if it appeared reddish brown, and negative if it did not appear.
Example 3 preparation and detection methods of porcine epidemic diarrhea Virus IgA antibody ELISA kit
3.1 kit preparation
Coating a plate: diluting the porcine epidemic diarrhea virus antibody by using a coating solution, adding the diluted porcine epidemic diarrhea virus antibody into the coating solution according to a volume of 100 mu l/hole, coating the porcine epidemic diarrhea virus antibody for 16-24 hours at a temperature of 2-8 ℃ or coating the porcine epidemic diarrhea virus antibody for 1-3 hours at a temperature of 37 ℃; washing with a washing solution, adding a sealing solution, sealing for 16-24 hours at the temperature of 2-8 ℃ or sealing for 1-3 hours at the temperature of 37 ℃; washing with a washing solution, adding a porcine epidemic diarrhea virus antigen, and capturing for 0.5-2 hours at 37 ℃; and (5) washing, drying and sealing the plate.
Sample diluent: PBS solution containing 15% -25% fetal bovine serum.
Enzyme-labeled goat anti-porcine IgA antibody: diluting the enzyme-labeled goat anti-pig IgA antibody with a sample diluent to 1: 8000-1: 30000, and subpackaging.
Washing liquid: PBS solution containing 0.5% -1% Tween 20.
Color development liquid: dissolving 14.7g of disodium hydrogen phosphate, 9.3g of citric acid and 0.3g of carbamide peroxide in purified water, metering to 1L, filtering with a volume of 0.22 mu m, and performing aseptic subpackage to obtain the developer A solution. Taking 0.2g of tetramethylbiphenyl diamine (TMB) and 100ml of absolute ethyl alcohol, dissolving in purified water to a constant volume of 1L, filtering with a diameter of 0.22 mu m, and performing sterile subpackage to obtain a developer B solution.
Stopping liquid: 2M H2SO4And (3) solution.
The components are assembled into a kit.
3.2 detection method establishment
The operation steps are as follows:
(1) the milk, serum and saliva samples are diluted by 10 times by using sample diluent, 1 anus swab is dissolved in 1ml of sample diluent, and 0.1g of excrement is dissolved in 1ml of sample diluent; 100 mul of each sample is added into a reaction hole, and a positive control and a negative control are arranged at the same time, and the incubation is carried out for 45-90 minutes at 37 ℃.
(2) Washing with a washing solution, adding 100 mu l of enzyme-labeled goat anti-pig IgA antibody into each hole, and incubating for 30-45 minutes at 37 ℃.
(3) Washing with washing solution, adding 50 μ l of developer A and B into each well, mixing, and developing at 37 deg.C or room temperature in dark for 15 min.
(4) Washing with washing solution, adding 50 μ l of stop solution into each well, and measuring absorbance OD at 450nm of each well with enzyme labeling instrument within 10 min450nmThe judgment is made based on the following result judgment criteria.
And (4) result judgment standard: the cut-off value A is 0.2 × (positive control mean-negative control mean), when the OD of the sample is450nmGreater than or equal to A is positive and OD450nm< A was negative.
3.3 determination of the amount of antibody against porcine epidemic diarrhea Virus for coating
A kit was prepared in the same manner as in example 3.1, using monoclonal antibody 8A3A10 as an antibody for coating according to the result of example 2.1 in the amount of coating shown in Table 3, and 40 parts of positive milk (containing 20 parts of colostrum), 20 parts of positive serum, 40 parts of negative milk (containing 20 parts of colostrum), and 20 parts of negative serum (all confirmed to be positive for IgA antibody by IPMA assay) were measured by the measurement method, and the coincidence rate of the sample with the IPMA method was calculated while 1 part of the clinically positive milk sample P1(IPMA titer 1: 256) was measured, and the results are shown in Table 3.
TABLE 3 screening results of the amount of porcine epidemic diarrhea virus antibody coating for kit coating
Shows that: the monoclonal antibody coating amount is 30-1000 ng/hole, the detection requirement can be met, and the coincidence rate of the milk and serum samples reaches 60-100%; further, when the coating amount is 50-500 ng/hole, the ELISA titer of the sample P1 is the highest, and the coincidence rate of the milk and serum samples reaches 85% -100%; particularly, when the coating amount is 50-300 ng/hole, the detection sensitivity and the sample conformity rate are highest.
3.4 determination of the amount of porcine epidemic diarrhea Virus antigen used for coating
The kit was prepared according to example 3.1 with the inactivated culture of the epidemic diarrhea HN1301 strain M3 in example 1.2 as the kit antigen, the kit was prepared according to example 3.1 with the antigen capturing amount in Table 4, 40 parts of positive milk (containing 20 parts of colostrum), 20 parts of positive serum, 40 parts of negative milk (containing 20 parts of colostrum), and 20 parts of negative serum (all identified by IPMA to confirm whether IgA antibody is positive) were detected by the detection method, and the coincidence rate of the sample with the IPMA method was calculated, and 1 part of the clinically positive milk sample P1(IPMA titer 1: 256) was detected, and the results are shown in Table 4.
TABLE 4 screening results of Capture amount of porcine epidemic diarrhea Virus for Capture by kit
The above results show that: the ELISA titer of the kit for detecting P1, which is prepared by using the kit with the antigen capture amount of 10-160 mu g/hole, is 1: 256-1: 2048, and the positive and negative coincidence rate of the detected milk and serum samples is 75-100%. Furthermore, when the antigen capture amount is 20-120 mug/hole, the positive and negative coincidence rate of the detected milk and serum samples is higher and reaches 85% -100%. Particularly, when the antigen capture amount is 20-80 mug/hole, the ELISA titer for detecting P1 is high (1: 1024-1: 2048), and the positive and negative coincidence rate for detecting milk and serum samples is highest (90-100%).
Example 4 application of porcine epidemic diarrhea Virus IgA antibody ELISA kit
4.1 sensitivity
The prepared kit was used to detect ELISA titers in milk and serum samples, and the results compared to IPMA titers are shown in Table 5. The result of the negative sample ELISA is negative, and the positive sample ELISA titers are all greater than the IPMA titer, which indicates that the kit has good detection sensitivity.
TABLE 5 kit sensitivity test results
4.2 specificity
The prepared kit is used for detecting pig transmissible gastroenteritis virus TGEV positive serum, pig Deltacoronavirus PDcov positive serum, pig rotavirus PoRV positive serum, pig pseudorabies virus PRV positive serum, pig parvovirus PPV positive serum, pig circovirus type 2 PCV2 positive serum, pig circovirus type 3 PCV3 positive serum, pig breeding and respiratory syndrome virus PRRSV positive serum, pig pestivirus CSFV positive serum and pig encephalitis B JEV positive serum, and the result is that: all the results are negative, which indicates that the kit has no cross reaction with other virus IgA positive serums.
4.3 repeatability
3 batches of kits were prepared to repeatedly detect the positive milk sample P2 and the positive serum sample P3 10 times respectively, and the results were: are all made ofPositive, and within and between batches of the kit OD450nmThe coefficient of variation of the values is less than 15%, and the method has good detection repeatability.
4.4 shelf life
And 3 batches of the kit are prepared and stored at 2-8 ℃, and the samples are taken in 3, 6, 9, 12 and 15 months respectively. The sensitivity, specificity and reproducibility of the detection were determined with reference to examples 4.1, 4.2 and 4.3. And (3) detection results: at different storage times, the ELISA test results of the negative samples are negative, the ELISA titers of the positive samples are all greater than the IPMA titer, the ELISA test results of a series of swine disease other virus positive sera in example 4.2 are all negative, and the OD in the kit batch and between batches is450nmThe coefficient of variation of the values is less than 15%, and the results show that the kit still has good sensitivity, specificity and repeatability when stored at 2-8 ℃ for 15 months. This is longer than the 12-month shelf life of commercial kits.
4.5 clinical applications
The prepared kit is used for detecting clinical samples of various targets, including 100 parts of milk (40 parts of colostrum), 100 parts of serum, 50 parts of saliva, 50 parts of anus swab and 50 parts of excrement sample (all identified by IPMA), and the Korean and fast PEDV IgA antibody ELISA kit (short for Anjie kit, the specification of which shows that only milk samples can be detected) is used for detection and comparison, so that the coincidence rate of the samples and the IPMA is calculated. The results are shown in Table 6. The results show that: the ELISA test result of the clinical sample has good coincidence rate with the IPMA test result, is superior to the test result of the agile kit, and particularly can detect various targets accurately with high sensitivity, while the agile kit can not be used for detecting saliva samples, anal swab samples and excrement samples.
TABLE 6 test results of clinical samples of the kit
4.5 post-immunization clinical detection of different strains
Vaccines prepared by using CV777 strain, HN1301 strain, HN1302 strain and ZJ08 strain (porcine transmissible gastroenteritis and porcine epidemic diarrhea bigeminal live vaccines (HB08 strain and ZJ08 strain) of rapes used for immunodetection replace the ZJ08 strain) are used for immunizing sows and piglets with PEDV antigens and specific antibodies being negative. 20 parts of sow milk, 20 parts of piglet serum, 20 parts of saliva, 20 parts of anal swab and 20 parts of excrement sample are collected for IPMA and ELISA detection. The results are shown in Table 7.
TABLE 7 clinical sample test results after immunization of vaccines with different strains
The above results show that: the ELISA kit prepared by the invention can detect PEDV specific IgA antibodies in different target samples after the immunization of a classical strain and an epidemic strain, has good sensitivity, is far superior to the detection effect of an agile kit, and has very large broad spectrum of sample sources. The Korean agile kit specification requires the use of colostrum for detecting samples, so that the accuracy of the detection result can be ensured.
According to the research of Dingzhenjiang et al (establishment and clinical evaluation of pig epidemic diarrhea IgA antibody detection method in sow milk, national agriculture technology field Lisheng pig industry technology system: the fifth national large-scale pig farm major epidemic disease monitoring and purification workshop Levender, page 105-. Therefore, the ELISA kit can accurately detect a plurality of detection target samples with low IgA content, shows high sensitivity, has higher adaptability of clinical application, and is more flexible and convenient. The ELISA kit can accurately detect IgA titer generated after the immunization of not only classical strains (CV777 strains), but also variant strains (HN1301 strains, HN1302 strains and ZJ08 strains), and has wider clinical application.
In conclusion, the kit can be applied to the detection of the pig epidemic diarrhea virus IgA antibody for non-diagnosis purposes, can be applied to the evaluation of sow milk, and can also be applied to the accurate detection of a plurality of targets such as serum, anal swab, excrement, saliva and the like, so that the immune effect evaluation of each pig, the clinical evaluation of the sow after wild virus infection, the epidemiological investigation of in-vitro samples and the like are realized, and the defects that the existing products can only detect the sow milk, detect serum and other samples and have low sensitivity are overcome.
Although the present invention has been described with reference to the preferred embodiments, it should be understood that various changes, substitutions and alterations can be made herein without departing from the spirit and scope of the invention as defined by the appended claims.
Claims (9)
1. The kit comprises a supporting medium coated with a porcine epidemic diarrhea virus antibody-antigen complex, wherein the porcine epidemic diarrhea virus antibody-antigen complex is a porcine epidemic diarrhea virus IgG monoclonal antibody-inactivated antigen complex, an enzyme-labeled anti-porcine IgA secondary antibody, and a detection reagent, a positive control and a negative control for detecting the reaction of the porcine IgA and the porcine epidemic diarrhea virus antibody-antigen complex antigen antibody; preferably, the porcine epidemic diarrhea virus IgG monoclonal antibody is 8A3A10, PEDV-McAB1 or PEDV-McAB 2.
2. The kit according to claim 1, wherein the inactivated antigen is an inactivated antigen of CV777 strain, HN1301 strain, HN1302 strain, HN1303 strain, Iowa18984 strain, AJ1102 strain, ZJ08 strain, LW/L strain, SD10 strain or a culture thereof;
the inactivated antigen is prepared by centrifugation and then inactivation, preferably, the centrifugation condition is 5000-15000 r/min for 5-30 minutes, the inactivation step is formaldehyde inactivation or β -propiolactone inactivation, the concentration range of formaldehyde is 0.05-0.5% (v/v), the inactivation step is static at 25-37 ℃ or shaking table is 50-200 r/min, the inactivation time is 24-48 hours, the inactivation step is β -propiolactone (β -PL) BPL, the concentration range of BPL is 0.01-0.05%, and the inactivation time is 8-16 hours.
3. The kit according to claim 2, wherein the porcine epidemic diarrhea virus IgG monoclonal antibody 8A3A10 is coated in an amount of 30-1000 ng/well; preferably, the coating amount of the porcine epidemic diarrhea virus IgG monoclonal antibody 8A3A10 is 50-500 ng/hole; more preferably, the coating amount of the porcine epidemic diarrhea virus IgG monoclonal antibody 8A3A10 is 50-300 ng/hole;
the addition amount of the HN1301 strain inactivated antigen is 10-160 mu g/hole; preferably, the addition amount of the HN1301 strain inactivated antigen is 20-120 mu g/hole; more preferably, the addition amount of the HN1301 strain inactivated antigen is 20-80 mu g/hole.
4. The kit of claim 1, wherein the enzyme-labeled anti-porcine IgA secondary antibody is a horseradish peroxidase-labeled anti-porcine IgA secondary antibody; preferably, the enzyme-labeled anti-pig IgA secondary antibody is an enzyme-labeled sheep-pig IgA secondary antibody, the detection reagents for detecting the reaction of pig IgA and the antibody-antigen complex antigen-antibody of the porcine epidemic diarrhea virus are a color-developing agent A liquid and a color-developing agent B liquid, the color-developing agent A liquid contains 14.7g of disodium hydrogen phosphate, 9.3g of citric acid and 0.3g of carbamide peroxide in 1L of water, and the color-developing agent B liquid contains 0.2g of tetramethyl biphenyl diamine (TMB) and 100ml of absolute ethyl alcohol in 1L of water;
the kit also comprises a stop solution, wherein the stop solution is 2M H2SO4And (3) solution.
5. The kit of claim 1, wherein the reaction system of the kit is phosphate buffer containing BSA, or OVA, or skim milk, or donkey serum, or sheep serum, or fetal bovine serum; preferably, the reaction system is PBS solution containing 15% -25% fetal bovine serum.
6. The kit of claim 1, wherein the kit further comprises a sample diluent and a washing solution, wherein the sample diluent is a phosphate buffer solution containing BSA, OVA, skim milk, donkey serum, sheep serum or fetal bovine serum; the washing solution is phosphate buffer solution.
7. The kit of claim 1, wherein the positive control is serum, milk, saliva, anal swab, stool that identifies IgA antibodies as positive by IPMA methods; the negative control is serum, milk, saliva, anal swab and excrement which are negative by the IgA antibody identified by the IPMA method.
8. The kit of claim 1, wherein the support medium is a microtiter plate.
9. The kit of claim 1, wherein the kit detects a sample selected from the group consisting of milk, serum, saliva, anal swab, and a stool sample.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201811141426.3A CN110967480A (en) | 2018-09-28 | 2018-09-28 | Preparation and application of pig epidemic diarrhea virus IgA antibody ELISA kit |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201811141426.3A CN110967480A (en) | 2018-09-28 | 2018-09-28 | Preparation and application of pig epidemic diarrhea virus IgA antibody ELISA kit |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| CN110967480A true CN110967480A (en) | 2020-04-07 |
Family
ID=70026947
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN201811141426.3A Pending CN110967480A (en) | 2018-09-28 | 2018-09-28 | Preparation and application of pig epidemic diarrhea virus IgA antibody ELISA kit |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN110967480A (en) |
Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN111474346A (en) * | 2020-04-15 | 2020-07-31 | 浙江理工大学绍兴生物医药研究院有限公司 | Pig epidemic diarrhea virus IgA and IgG antibody detection kit and preparation method and application thereof |
| CN111830257A (en) * | 2020-07-17 | 2020-10-27 | 南京农业大学 | Swine lawsonia intracellularis IPMA antigen detection method and application thereof |
| CN115184344A (en) * | 2022-01-30 | 2022-10-14 | 北京市疾病预防控制中心 | Novel coronavirus IgG antibody detection method suitable for novel biological sample oral fluid and application thereof |
Citations (12)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| KR20010004480A (en) * | 1999-06-29 | 2001-01-15 | 이재진 | A method for diagnosis of Porcine epidemic diarrhea virus and a kit for diagnosis |
| KR20090121673A (en) * | 2008-05-22 | 2009-11-26 | 주식회사 바이오노트 | Method for predicting protection against pedv of pig by measuring iga titer and a kit for the same |
| CN103777010A (en) * | 2014-01-27 | 2014-05-07 | 深圳市宝舜泰生物医药股份有限公司 | ELISA (Enzyme-Linked Immuno Sorbent Assay) detection kit for porcine epidemic diarrhea viruses and preparation method thereof |
| CN104694480A (en) * | 2015-03-25 | 2015-06-10 | 洛阳普莱柯万泰生物技术有限公司 | Hybridoma cell strain and secretion monoclonal antibody and application thereof |
| CA2943816A1 (en) * | 2014-04-03 | 2015-10-08 | Boehringer Ingelheim Vetmedica, Inc. | Porcine epidemic diarrhea virus vaccine |
| CN105461805A (en) * | 2015-12-17 | 2016-04-06 | 洛阳普莱柯万泰生物技术有限公司 | Monoclonal antibody for resisting porcine epidemic diarrhea viruses and application thereof |
| CN105527437A (en) * | 2015-12-17 | 2016-04-27 | 洛阳普莱柯万泰生物技术有限公司 | Detection kit and application thereof |
| CN106188249A (en) * | 2016-07-13 | 2016-12-07 | 北京农学院 | For detecting the antigen of PEDV variant antibody and method and test kit |
| CN106990253A (en) * | 2017-05-24 | 2017-07-28 | 河南科技学院 | A kind of ELISA method based on restructuring S1 Protein Detection Porcine epidemic diarrhea virus IgA antibodies |
| CN107167611A (en) * | 2017-05-24 | 2017-09-15 | 广东海大畜牧兽医研究院有限公司 | A kind of Porcine epidemic diarrhea virus antibody sIgA ELISA detection method |
| CN107219366A (en) * | 2017-06-09 | 2017-09-29 | 江苏省农业科学院 | A kind of sandwich ELISA lcits for detecting pig colostrum moderate resistance PEDV Specific IgA antibodies |
| CN107312088A (en) * | 2017-05-24 | 2017-11-03 | 中国农业科学院哈尔滨兽医研究所 | Porcine epidemic diarrhea virus specificity SIgA ELISA detection kits and its application |
-
2018
- 2018-09-28 CN CN201811141426.3A patent/CN110967480A/en active Pending
Patent Citations (12)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| KR20010004480A (en) * | 1999-06-29 | 2001-01-15 | 이재진 | A method for diagnosis of Porcine epidemic diarrhea virus and a kit for diagnosis |
| KR20090121673A (en) * | 2008-05-22 | 2009-11-26 | 주식회사 바이오노트 | Method for predicting protection against pedv of pig by measuring iga titer and a kit for the same |
| CN103777010A (en) * | 2014-01-27 | 2014-05-07 | 深圳市宝舜泰生物医药股份有限公司 | ELISA (Enzyme-Linked Immuno Sorbent Assay) detection kit for porcine epidemic diarrhea viruses and preparation method thereof |
| CA2943816A1 (en) * | 2014-04-03 | 2015-10-08 | Boehringer Ingelheim Vetmedica, Inc. | Porcine epidemic diarrhea virus vaccine |
| CN104694480A (en) * | 2015-03-25 | 2015-06-10 | 洛阳普莱柯万泰生物技术有限公司 | Hybridoma cell strain and secretion monoclonal antibody and application thereof |
| CN105461805A (en) * | 2015-12-17 | 2016-04-06 | 洛阳普莱柯万泰生物技术有限公司 | Monoclonal antibody for resisting porcine epidemic diarrhea viruses and application thereof |
| CN105527437A (en) * | 2015-12-17 | 2016-04-27 | 洛阳普莱柯万泰生物技术有限公司 | Detection kit and application thereof |
| CN106188249A (en) * | 2016-07-13 | 2016-12-07 | 北京农学院 | For detecting the antigen of PEDV variant antibody and method and test kit |
| CN106990253A (en) * | 2017-05-24 | 2017-07-28 | 河南科技学院 | A kind of ELISA method based on restructuring S1 Protein Detection Porcine epidemic diarrhea virus IgA antibodies |
| CN107167611A (en) * | 2017-05-24 | 2017-09-15 | 广东海大畜牧兽医研究院有限公司 | A kind of Porcine epidemic diarrhea virus antibody sIgA ELISA detection method |
| CN107312088A (en) * | 2017-05-24 | 2017-11-03 | 中国农业科学院哈尔滨兽医研究所 | Porcine epidemic diarrhea virus specificity SIgA ELISA detection kits and its application |
| CN107219366A (en) * | 2017-06-09 | 2017-09-29 | 江苏省农业科学院 | A kind of sandwich ELISA lcits for detecting pig colostrum moderate resistance PEDV Specific IgA antibodies |
Non-Patent Citations (1)
| Title |
|---|
| 孔园园等: "猪流行性腹泻IgA抗体间接ELISA检测方法的建立", 《猪业科学》 * |
Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN111474346A (en) * | 2020-04-15 | 2020-07-31 | 浙江理工大学绍兴生物医药研究院有限公司 | Pig epidemic diarrhea virus IgA and IgG antibody detection kit and preparation method and application thereof |
| CN111830257A (en) * | 2020-07-17 | 2020-10-27 | 南京农业大学 | Swine lawsonia intracellularis IPMA antigen detection method and application thereof |
| CN111830257B (en) * | 2020-07-17 | 2023-10-24 | 南京农业大学 | Pig-derived lawsonia intracellularis IPMA antigen detection method and application thereof |
| CN115184344A (en) * | 2022-01-30 | 2022-10-14 | 北京市疾病预防控制中心 | Novel coronavirus IgG antibody detection method suitable for novel biological sample oral fluid and application thereof |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| Choi et al. | Rapid competitive enzyme-linked immunosorbent assay for detection of antibodies to peste des petits ruminants virus | |
| EP3084441B1 (en) | Improved diagnostic test for csfv antibodies | |
| Oh et al. | Comparison of an enzyme-linked immunosorbent assay with serum neutralization test for serodiagnosis of porcine epidemic diarrhea virus infection. | |
| CN110873792B (en) | African swine fever virus antibody detection kit | |
| CN110967480A (en) | Preparation and application of pig epidemic diarrhea virus IgA antibody ELISA kit | |
| CN105837686B (en) | Monoclonal antibody and application thereof | |
| CN111929433A (en) | African swine fever virus antibody ELISA detection kit and preparation method thereof | |
| CN111474346B (en) | Porcine epidemic diarrhea virus IgA and IgG antibody detection kit, and preparation method and application thereof | |
| CN111925436A (en) | Monoclonal antibody of African swine fever virus P30 protein and application thereof | |
| CN108956987B (en) | Application of fiber2 protein and recombinant protein thereof in detecting duck type 2 adenovirus antibody in duck serum | |
| CN107312088B (en) | Porcine epidemic diarrhea virus specificity SIgA ELISA detection kit and application thereof | |
| KR101667122B1 (en) | Improved vaccine diagnostics | |
| CN112760294A (en) | Canine type I adenovirus monoclonal antibody/polyclonal antibody, double-antibody sandwich ELISA kit and application | |
| CN108918869B (en) | Application of fiber2 protein and recombinant protein thereof in detecting serum type 4 avian adenovirus antibody | |
| CN109799351A (en) | Porcine circovirus 2 type double-antibody sandwich elisa kit and its application | |
| CN109521199B (en) | Colloidal gold detection kit for detecting avian leukosis virus and application thereof | |
| CN109280644B (en) | Anti-human IgG monoclonal antibody, hybridoma cell strain and application thereof | |
| CN109425738B (en) | Porcine circovirus type 3 antibody detection kit and application thereof | |
| CN110607282B (en) | Bovine parvovirus monoclonal antibody and application thereof in detecting bovine parvovirus infection | |
| Alkahalaf et al. | Comparison of enzyme-linked immunosorbent assays and virus neutralization test for detection of antibodies to avian pneumovirus | |
| RU2526131C2 (en) | Vero cell lysis proteins, method for production thereof and kit for determining host cell proteins for vero cells containing lysis proteins | |
| KR101101386B1 (en) | Immunogenic peptide specific for a north American type PRRSV and use thereof | |
| Cai et al. | Detection of asymptomatic antigenemia in pigs infected by porcine reproductive and respiratory syndrome virus (PRRSV) by a novel capture immunoassay with monoclonal antibodies against the nucleocapsid protein of PRRSV | |
| CN112444626B (en) | African swine fever virus antibody ELISA detection kit and preparation method thereof | |
| EP4033247A1 (en) | Multi-species immunoassays for detecting antibodies anti-sars-cov-2 using protein a for detection of captured antibodies |
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 | ||
| RJ01 | Rejection of invention patent application after publication | ||
| RJ01 | Rejection of invention patent application after publication |
Application publication date: 20200407 |