CN112379105A - 一种prrsv的ipma中和抗体检测方法 - Google Patents
一种prrsv的ipma中和抗体检测方法 Download PDFInfo
- Publication number
- CN112379105A CN112379105A CN202011240812.5A CN202011240812A CN112379105A CN 112379105 A CN112379105 A CN 112379105A CN 202011240812 A CN202011240812 A CN 202011240812A CN 112379105 A CN112379105 A CN 112379105A
- Authority
- CN
- China
- Prior art keywords
- prrsv
- antibody
- ipma
- detecting
- serum
- 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
- 241001135989 Porcine reproductive and respiratory syndrome virus Species 0.000 title claims abstract description 65
- 230000003472 neutralizing effect Effects 0.000 title claims abstract description 20
- 238000000034 method Methods 0.000 title claims abstract description 18
- 239000000427 antigen Substances 0.000 title abstract description 8
- 102000036639 antigens Human genes 0.000 title abstract description 8
- 108091007433 antigens Proteins 0.000 title abstract description 8
- 229920000831 ionic polymer Polymers 0.000 title abstract description 4
- 210000002966 serum Anatomy 0.000 claims abstract description 30
- 238000001514 detection method Methods 0.000 claims abstract description 14
- 241000700605 Viruses Species 0.000 claims abstract description 13
- 238000006243 chemical reaction Methods 0.000 claims description 13
- 238000012258 culturing Methods 0.000 claims description 13
- 238000011161 development Methods 0.000 claims description 10
- 230000036647 reaction Effects 0.000 claims description 8
- 238000005406 washing Methods 0.000 claims description 8
- 241000283707 Capra Species 0.000 claims description 7
- 239000006285 cell suspension Substances 0.000 claims description 5
- 230000001717 pathogenic effect Effects 0.000 claims description 5
- 108091003079 Bovine Serum Albumin Proteins 0.000 claims description 4
- 239000006144 Dulbecco’s modified Eagle's medium Substances 0.000 claims description 4
- LFQSCWFLJHTTHZ-UHFFFAOYSA-N Ethanol Chemical compound CCO LFQSCWFLJHTTHZ-UHFFFAOYSA-N 0.000 claims description 4
- 210000004369 blood Anatomy 0.000 claims description 4
- 239000008280 blood Substances 0.000 claims description 4
- 238000005119 centrifugation Methods 0.000 claims description 4
- 230000029087 digestion Effects 0.000 claims description 4
- 239000012530 fluid Substances 0.000 claims description 4
- 238000000227 grinding Methods 0.000 claims description 4
- 230000003287 optical effect Effects 0.000 claims description 4
- 101100028789 Arabidopsis thaliana PBS1 gene Proteins 0.000 claims description 3
- 239000012894 fetal calf serum Substances 0.000 claims description 3
- 230000008014 freezing Effects 0.000 claims description 3
- 238000007710 freezing Methods 0.000 claims description 3
- 239000001963 growth medium Substances 0.000 claims description 3
- 238000010257 thawing Methods 0.000 claims description 3
- 238000010008 shearing Methods 0.000 claims description 2
- 229960005486 vaccine Drugs 0.000 abstract description 6
- 208000005342 Porcine Reproductive and Respiratory Syndrome Diseases 0.000 abstract description 5
- 238000011156 evaluation Methods 0.000 abstract description 4
- 230000003993 interaction Effects 0.000 abstract description 4
- 239000007788 liquid Substances 0.000 abstract description 3
- 238000012360 testing method Methods 0.000 abstract description 3
- 230000001900 immune effect Effects 0.000 abstract description 2
- 230000036039 immunity Effects 0.000 abstract description 2
- 238000004519 manufacturing process Methods 0.000 abstract description 2
- 238000006386 neutralization reaction Methods 0.000 abstract description 2
- 230000002265 prevention Effects 0.000 abstract description 2
- 230000035945 sensitivity Effects 0.000 abstract description 2
- 241000282887 Suidae Species 0.000 abstract 1
- 239000003795 chemical substances by application Substances 0.000 abstract 1
- 210000004027 cell Anatomy 0.000 description 38
- 238000002965 ELISA Methods 0.000 description 8
- 210000001519 tissue Anatomy 0.000 description 8
- 238000010790 dilution Methods 0.000 description 7
- 239000012895 dilution Substances 0.000 description 7
- 239000000243 solution Substances 0.000 description 7
- 208000015181 infectious disease Diseases 0.000 description 6
- 230000001575 pathological effect Effects 0.000 description 6
- 238000012408 PCR amplification Methods 0.000 description 4
- 101710172711 Structural protein Proteins 0.000 description 3
- 238000003556 assay Methods 0.000 description 3
- 238000004364 calculation method Methods 0.000 description 3
- 239000002299 complementary DNA Substances 0.000 description 3
- 238000007865 diluting Methods 0.000 description 3
- 210000004185 liver Anatomy 0.000 description 3
- 210000004072 lung Anatomy 0.000 description 3
- 210000000952 spleen Anatomy 0.000 description 3
- 239000005723 virus inoculator Substances 0.000 description 3
- 238000008157 ELISA kit Methods 0.000 description 2
- 101710141454 Nucleoprotein Proteins 0.000 description 2
- 230000005875 antibody response Effects 0.000 description 2
- 230000005540 biological transmission Effects 0.000 description 2
- 239000011248 coating agent Substances 0.000 description 2
- 238000000576 coating method Methods 0.000 description 2
- 201000010099 disease Diseases 0.000 description 2
- 208000037265 diseases, disorders, signs and symptoms Diseases 0.000 description 2
- 238000011081 inoculation Methods 0.000 description 2
- 210000002751 lymph Anatomy 0.000 description 2
- 238000012423 maintenance Methods 0.000 description 2
- 238000005259 measurement Methods 0.000 description 2
- 239000002609 medium Substances 0.000 description 2
- 238000002360 preparation method Methods 0.000 description 2
- 108090000623 proteins and genes Proteins 0.000 description 2
- 230000000405 serological effect Effects 0.000 description 2
- 241000282552 Chlorocebus aethiops Species 0.000 description 1
- 208000035473 Communicable disease Diseases 0.000 description 1
- 101710091045 Envelope protein Proteins 0.000 description 1
- 102000004190 Enzymes Human genes 0.000 description 1
- 108090000790 Enzymes Proteins 0.000 description 1
- 241000588724 Escherichia coli Species 0.000 description 1
- 101800000120 Host translation inhibitor nsp1 Proteins 0.000 description 1
- 101800000512 Non-structural protein 1 Proteins 0.000 description 1
- 101800000511 Non-structural protein 2 Proteins 0.000 description 1
- 101800000510 Non-structural protein 7 Proteins 0.000 description 1
- 229930182555 Penicillin Natural products 0.000 description 1
- JGSARLDLIJGVTE-MBNYWOFBSA-N Penicillin G Chemical compound N([C@H]1[C@H]2SC([C@@H](N2C1=O)C(O)=O)(C)C)C(=O)CC1=CC=CC=C1 JGSARLDLIJGVTE-MBNYWOFBSA-N 0.000 description 1
- 101710188315 Protein X Proteins 0.000 description 1
- 238000010802 RNA extraction kit Methods 0.000 description 1
- 102100021696 Syncytin-1 Human genes 0.000 description 1
- 238000000246 agarose gel electrophoresis Methods 0.000 description 1
- 238000000137 annealing Methods 0.000 description 1
- 238000011278 co-treatment Methods 0.000 description 1
- 238000004040 coloring Methods 0.000 description 1
- 210000000805 cytoplasm Anatomy 0.000 description 1
- 231100000433 cytotoxic Toxicity 0.000 description 1
- 230000001472 cytotoxic effect Effects 0.000 description 1
- 238000004925 denaturation Methods 0.000 description 1
- 230000036425 denaturation Effects 0.000 description 1
- 238000013461 design Methods 0.000 description 1
- 238000010586 diagram Methods 0.000 description 1
- 239000012154 double-distilled water Substances 0.000 description 1
- 244000309457 enveloped RNA virus Species 0.000 description 1
- 239000012091 fetal bovine serum Substances 0.000 description 1
- 238000010166 immunofluorescence Methods 0.000 description 1
- 230000002163 immunogen Effects 0.000 description 1
- 238000002955 isolation Methods 0.000 description 1
- 210000003292 kidney cell Anatomy 0.000 description 1
- 210000001165 lymph node Anatomy 0.000 description 1
- 239000003550 marker Substances 0.000 description 1
- 239000000463 material Substances 0.000 description 1
- 238000012986 modification Methods 0.000 description 1
- 230000004048 modification Effects 0.000 description 1
- -1 nsps Proteins 0.000 description 1
- 231100000915 pathological change Toxicity 0.000 description 1
- 230000036285 pathological change Effects 0.000 description 1
- 229940049954 penicillin Drugs 0.000 description 1
- 230000002093 peripheral effect Effects 0.000 description 1
- 238000012257 pre-denaturation Methods 0.000 description 1
- 230000001681 protective effect Effects 0.000 description 1
- 102000004169 proteins and genes Human genes 0.000 description 1
- 238000004445 quantitative analysis Methods 0.000 description 1
- 230000000241 respiratory effect Effects 0.000 description 1
- 239000002356 single layer Substances 0.000 description 1
- 238000003892 spreading Methods 0.000 description 1
- 230000007480 spreading Effects 0.000 description 1
- 238000002255 vaccination Methods 0.000 description 1
- 230000003612 virological effect Effects 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
- G01N33/68—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids
- G01N33/6854—Immunoglobulins
-
- 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
-
- 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
-
- 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
- G01N33/58—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving labelled substances
- G01N33/582—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving labelled substances with fluorescent label
-
- 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
-
- 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
Landscapes
- Health & Medical Sciences (AREA)
- Life Sciences & Earth Sciences (AREA)
- Engineering & Computer Science (AREA)
- Immunology (AREA)
- Molecular Biology (AREA)
- Biomedical Technology (AREA)
- Chemical & Material Sciences (AREA)
- Hematology (AREA)
- Urology & Nephrology (AREA)
- Food Science & Technology (AREA)
- Biochemistry (AREA)
- Cell Biology (AREA)
- Biotechnology (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)
- Proteomics, Peptides & Aminoacids (AREA)
- Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)
Abstract
本发明公开了一种PRRSV的IPMA中和抗体检测方法,属于分子生物学检测技术领域。本发明公开的一种PRRSV的IPMA中和抗体检测方法,包括将等体积的PRRSV病毒液和临床血清接种到Marc145细胞上,固定、加一抗、加二抗、显色。本发明方法为感染PRRSV猪的预防和疫苗评价、免疫评价提供一定技术支持;且血清中和试验可以用来检测PRRSV抗体水平的高低以及对PRRS疫苗免疫效果的评定,有望应用于实际疫苗的生产中。本发明方法利用全病毒和抗体相互作用后感染靶细胞,真实的反应了病毒与中和抗体相互作用的情况;具有特异性强、敏感性高、操作简单的特点。
Description
技术领域
本发明涉及生物技术领域,更具体的说是涉及一种PRRSV的IPMA中和抗体检测方法。
背景技术
猪繁殖与呼吸综合征(Porcine reproductive and respiratory syndrome,PRRS)是由猪繁殖与呼吸综合征病毒(PRRSV)引起的一种高接触性呼吸道传染疾病,传播速度快,传播途径广,发病率高,死亡率高,严重危害养猪业的发展。
PRRSV是一种正链包膜RNA病毒。PRRSV感染的抗体反应非常复杂,并且仍不是很清楚。然而,已经建立了多种方法来检测PRRSV特异性抗体作为PRRSV感染的血清学标志物,例如酶联免疫吸附测定(ELISA)以及基于免疫荧光和免疫层析带的测定。PRRSV特异性中和抗体(NAb)通常在接种后(dpi)28天后出现,但在感染后第一周内产生的非保护性抗体可能对PRRSV感染的早期检测更为有用。这些早期抗体包括对PRRSV N蛋白或nsps等结构蛋白具有特异性的非中和抗体,N蛋白和某些非结构蛋白(nsp1,nsp2和nsp7)已被证明具有高度免疫原性。
目前检测PRRSV特异性抗体的大多数商用ELISA试剂盒(例如IDEXX Herd ChekPRRS ELISA)都采用抗N抗体作为PRRSV感染或改良活病毒(MLV)免疫状态的血清学标志物。尽管诸如ELISA之类的商业测试对于确定血清样品中PRRSV特异性抗体的存在非常敏感,但是ELISA不适合用于抗体水平的定量分析。该缺点是由于以下事实:从ELISA获得的OD值通常在窄范围(0.1至2)内变化。此外,大多数ELISA试剂盒使用原核表达的单个重组PRRSV结构抗原(通常为PRRSV-N蛋白)作为包被抗原。因此,此类系统无法评估针对其他PRRSV包膜蛋白或非结构蛋白(nsps)的PRRSV特异性抗体反应。在此应注意,由于表达和纯化多种ELISA板包被抗原需要付出巨大的努力,因此可能不会开发使用多种PRRSV抗原的系统。此外,由于ELISA包膜抗原在大肠杆菌中表达的一般性质,经常报告假阳性或假阴性结果。基于此,迫切需要开发一种用于准确检测PRRSV特异性抗体的改进方法。
因此,提供一种PRRSV的IPMA中和抗体检测方法是本领域技术人员亟需解决的问题。
发明内容
有鉴于此,本发明提供了一种PRRSV的IPMA中和抗体检测方法,用于准确检测和定量PRRSV特异性抗体。
为了实现上述目的,本发明采用如下技术方案:
一种PRRSV的IPMA中和抗体检测方法,具体步骤如下:
(1)铺板:把Marc145细胞消化离心后制备成含1.0×105cells/ml的细胞悬液,每孔100μl铺到96孔板上,置于37℃5%CO2培养箱中培养12h,待细胞完全贴壁,将等体积的PRRSV病毒液和临床血清37℃孵育1h,接种到Marc145细胞上,加入胎牛血清含量为2%的DMEM培养基进行培养,置于CO2培养箱中继续培养48h,观察细胞生长情况,待细胞长满后,取出96孔板;同时设立正常接毒细胞对照孔;
(2)固定:把取出的96孔板用PBS冲洗2-3次,用预冷的无水乙醇于-20℃固定30min,细胞反应板制备成功;
(3)加一抗:把细胞反应板用PBS洗过2-3次,加入100μl用0.01M PBS1:400稀释的PRRSV标准阳性血清,置于37℃中作用1.0h;
(4)加二抗:加入100μl用0.01M PBS(pH7.4)稀释1000倍的羊抗猪IgG-HRP二抗,37℃作用1h;
(5)显色:DAB显色5min,终止反应,置光学显微镜下观察显色结果。
进一步,所述PRRSV病毒液的制备方法如下:将PRRSV的病料组织用PBS冲洗,剪碎后按照1g组织样品加入1mL PBS,反复冻融三次,制成病料研磨液。
进一步,所述临床血清的制备方法如下:将采集的血液进行离心,收集血清,用于后期临床血清检测。
经由上述的技术方案可知,与现有技术相比,本发明公开提供了一种PRRSV的IPMA中和抗体检测方法,为感染PRRSV猪的预防和疫苗评价、免疫评价提供一定技术支持;且血清中和试验可以用来检测PRRSV抗体水平的高低以及对PRRS疫苗免疫效果的评定,有望应用于实际疫苗的生产中。本发明方法利用全病毒和抗体相互作用后感染靶细胞,真实的反应了病毒与中和抗体相互作用的情况;具有特异性强、敏感性高、操作简单的特点。
附图说明
为了更清楚地说明本发明实施例或现有技术中的技术方案,下面将对实施例或现有技术描述中所需要使用的附图作简单地介绍,显而易见地,下面描述中的附图仅仅是本发明的实施例,对于本领域普通技术人员来讲,在不付出创造性劳动的前提下,还可以根据提供的附图获得其他的附图。
图1附图为本发明各病料组织的PCR扩增结果;
其中,M:DL 2000 Marker;1:淋巴结;2:肝脏;3:脾脏;4:肺脏;5:空白对照(模板为ddH2O);
图2附图为本发明IPMA检测PRRSV中和抗体的显色结果;
其中,A为中和抗体试验,B为正常接毒细胞对照。
具体实施方式
下面将结合本发明实施例中的附图,对本发明实施例中的技术方案进行清楚、完整地描述,显然,所描述的实施例仅仅是本发明一部分实施例,而不是全部的实施例。基于本发明中的实施例,本领域普通技术人员在没有做出创造性劳动前提下所获得的所有其他实施例,都属于本发明保护的范围。
血液样品和病料组织采集自周边猪场,将采集的血液进行离心,收集血清,用于后期临床血清检测;病料组织进行研磨,检测PRRSV并分离病毒;细胞株为Marc145细胞(非洲绿猴肾细胞),各种病毒抗血清由实验室保存。
实施例1 PRRSV分离及鉴定
将疑似PRRSV的病料组织(淋巴、肝脏、脾脏、肺脏)用PBS冲洗,剪碎后按照1g组织样品加入1mL PBS,反复冻融三次,制成病料研磨液(作为PRRSV病毒液使用),使用RNA提取试剂盒,从上述病料中提取PRRSV的RNA,然后把RNA反转录为cDNA。
利用DNAMAN软件设计PRRSV目的基因引物,具体引物序列如下:
PRRSV-F:5’-GGCCAGCCAGTCAATCAG-3’;SEQ ID NO.1;
PRRSV-R:5’-GGCAAACTAAACTCCACAGTG-3’;SEQ ID NO.2。
利用设计好的引物,分别以上述提取的PRRSV的淋巴、肝脏、脾脏、肺脏cDNA为模板进行PCR扩增。PCR扩增体系为25μL:模板1μL,PRRSV-F0.5μL,PRRSV-R 0.5μL,Ex Taq酶12.5μL,双蒸水10.5μL。反应条件为:98℃预变性5min;95℃变性30s,55℃退火30s,72℃延伸50s,30个循环;72℃延伸10min,结束后4℃保存。PCR产物进行琼脂糖凝胶电泳,结果见图1。结果显示,以病料提取的cDNA为模板进行PCR扩增,出现一条特异性目的条带,长度约为537bp,与预期大小一致;病料中含有PRRSV。
实施例2建立稳定的IPMA中和抗体检测方法
把Marc145细胞消化离心后制备成含1.0×105ceLs/mL的细胞悬液,每孔100μL铺到96孔板上,置于37℃5%CO2培养箱中培养12h,待细胞完全贴壁,将等体积的PRRSV病毒(50μL)和临床血清37℃孵育1h,然后接种到96孔板内的Marc145细胞上,培养48h待细胞长满后取出96孔板,用预冷的无水乙醇固定。加入PRRSV标准阳性血清,37℃孵育1h,然后加入羊抗猪IgG-HRP,37℃孵育1h,DAB或AEC显色,置普通光学显微镜下观察,产生红棕色的不溶性产物的为阴性,反之为阳性。将鉴定为阳性的血清进行倍比稀释,重复上述操作,可获得抗体效价。
(1)铺板:把Marc145细胞消化离心后制备成含1.0×105cells/ml的细胞悬液,每孔100μl铺到96孔板上,置于37℃5%CO2培养箱中培养12h,待细胞完全贴壁,将等体积的PRRSV病毒液和临床血清37℃孵育1h,接种到Marc145细胞上,加入胎牛血清含量为2%的DMEM培养基进行培养,置于CO2培养箱中继续培养48h,观察细胞生长情况,待细胞长满后,取出96孔板;同时设立正常接毒细胞对照孔;
(2)固定:把取出的96孔板用PBS冲洗2-3次,然后用预冷的无水乙醇于-20℃固定30min,也可以长时间保存备用,即细胞反应板制备成功;
(3)加一抗:把96孔板用PBS洗过2-3次,然后加入100μl用0.01M PBS1:400稀释的PRRSV标准阳性血清(IDEXX),置于37℃中作用1.0h;
(4)加二抗:加入100μl用0.01M PBS(pH7.4)稀释1000倍的羊抗猪IgG-HRP二抗,37℃作用1h;
(5)显色:DAB显色5min,达到预期结果,终止反应,置光学显微镜下观察显色结果,见图2。
IPMA结果判定方法:PRRSV未被血清中和而感染的细胞核或胞浆呈红棕色着染,即为阴性(如图2B),而被中和未感染的不能着染,即为阳性(如图2A)。
实施例3 IPMA反应条件
1)PRRSV的TCID50测定
(1)将Marc145细胞悬液铺到96孔板上,每孔100μL,使细胞量达到2-3×105个/mL,培养12h,直至细胞完全贴壁;
(2)在青霉素瓶或离心管中将PRRSV病毒液作连续10倍的稀释,从10-1-10-10;
(3)将稀释好的病毒接种到细胞长成单层的96孔板上,每一稀释度接种一纵排共8孔,每孔接种100μL;
(4)剩下两纵排不接毒,设正常细胞对照(每孔100μL维持液,维持液为含2%胎牛血清的DMEM培养基);
(5)培养48h待细胞长满后取出固定,置于-20℃备用;
(6)利用IPMA方法进行检测,观察记录发生病变(CPE)的孔数,结果见表1;
(7)TCID50的计算,按Reed-Muench两氏法计算。
表1 TCID50测定结果统计
按Reed-Muench两氏法计算TCID50,计算方法如下:
距离比=(高于50%病变率的百分数-50%)/(高于50%病变率的百分数-低于50%病变率的百分数)=(55.5-50)/(55.5-8.3)=0.1
lg TCID50=距离比×稀释度对数之间的差+高于50%病变率的稀释度的对数=0.1×(-1)+(-4)=-4.1。
根据上述数据可得TCID50=10-4.1/0.1ml=10-5.1/ml
含义:将该病毒稀释104.1接种100μl可使50%的细胞发生病变。
2)病毒接种量和固定时间
分别用1000个TCID50、100个TCID50、10个TCID50、1个TCID50、0.1个TCID50的病毒接种Marc145细胞,并设置未接毒对照。利用IPMA检测方法分别以阳性和阴性血清作为一抗进行检测,观察结果确定最佳病毒接种量。将PRRSV(最佳病毒接种量)接种到长至60%-70%左右的Marc145细胞瓶内,待细胞长满后将其分别铺到4个96孔板上,在37℃5%CO2培养箱培养12、24、36、48h后,逐一取出固定。然后利用IPMA检测方法分别以阳性和阴性血清作为一抗进行检测,观察结果确定固定时间。
结果发现,以100个TCID50的病毒接种Marc145细胞培养48h后固定制备IPMA细胞反应板最佳。
3)血清稀释浓度、羊抗猪IgG-HRP工作浓度
将感染PRRSV的猪血清分别按1:50、1:100、1:200、1:400等进行连续倍比稀释,然后分别作为一抗加入制备好的细胞反应板内,利用IPMA抗体检测方法分别进行检测,观察结果确定最佳血清稀释浓度。
将制备好的细胞反应板取出,加入1:400倍稀释的阳性和阴性血清,然后分别加入1:100、1:200、1:400、1:800、1:1000倍稀释的羊抗猪IgG-HRP,观察实验结果确定最佳二抗稀释浓度。
结果发现,待检血清以1:50开始,可根据抗体强弱情况在此基础上进行连续2倍稀释检测抗体效价,羊抗猪IgG-HRP工作浓度1:1000最佳,DAB显色5min。
对所公开的实施例的上述说明,使本领域专业技术人员能够实现或使用本发明。对这些实施例的多种修改对本领域的专业技术人员来说将是显而易见的,本文中所定义的一般原理可以在不脱离本发明的精神或范围的情况下,在其它实施例中实现。因此,本发明将不会被限制于本文所示的这些实施例,而是要符合与本文所公开的原理和新颖特点相一致的最宽的范围。
序列表
<110> 新乡学院
<120> 一种PRRSV的IPMA中和抗体检测方法
<160> 2
<170> SIPOSequenceListing 1.0
<210> 1
<211> 18
<212> DNA
<213> Artificial Sequence
<400> 1
ggccagccag tcaatcag 18
<210> 2
<211> 21
<212> DNA
<213> Artificial Sequence
<400> 2
ggcaaactaa actccacagt g 21
Claims (3)
1.一种PRRSV的IPMA中和抗体检测方法,其特征在于,具体步骤如下:
(1)铺板:把Marc145细胞消化离心后制备成含1.0×105cells/ml的细胞悬液,每孔100μl铺到96孔板上,置于37℃5%CO2培养箱中培养12h,待细胞完全贴壁,将等体积的PRRSV病毒液和临床血清37℃孵育1h,接种到Marc145细胞上,加入胎牛血清含量为2%的DMEM培养基进行培养,置于CO2培养箱中继续培养48h,观察细胞生长情况,待细胞长满后,取出96孔板;同时设立正常接毒细胞对照孔;
(2)固定:把取出的96孔板用PBS冲洗2-3次,用预冷的无水乙醇于-20℃固定30min,细胞反应板制备成功;
(3)加一抗:把细胞反应板用PBS洗过2-3次,加入100μl用0.01M PBS 1:400稀释的PRRSV标准阳性血清,置于37℃中作用1.0h;
(4)加二抗:加入100μl用0.01M PBS(pH7.4)稀释1000倍的羊抗猪IgG-HRP二抗,37℃作用1h;
(5)显色:DAB显色5min,终止反应,置光学显微镜下观察显色结果。
2.根据权利要求1所述的一种PRRSV的IPMA中和抗体检测方法,其特征在于,所述PRRSV病毒液的制备方法如下:将PRRSV的病料组织用PBS冲洗,剪碎后按照1g组织样品加入1mLPBS,反复冻融三次,制成病料研磨液。
3.根据权利要求1所述的一种PRRSV的IPMA中和抗体检测方法,其特征在于,所述临床血清的制备方法如下:将采集的血液进行离心,收集血清,用于后期临床血清检测。
Priority Applications (1)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
CN202011240812.5A CN112379105A (zh) | 2020-11-09 | 2020-11-09 | 一种prrsv的ipma中和抗体检测方法 |
Applications Claiming Priority (1)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
CN202011240812.5A CN112379105A (zh) | 2020-11-09 | 2020-11-09 | 一种prrsv的ipma中和抗体检测方法 |
Publications (1)
Publication Number | Publication Date |
---|---|
CN112379105A true CN112379105A (zh) | 2021-02-19 |
Family
ID=74578177
Family Applications (1)
Application Number | Title | Priority Date | Filing Date |
---|---|---|---|
CN202011240812.5A Pending CN112379105A (zh) | 2020-11-09 | 2020-11-09 | 一种prrsv的ipma中和抗体检测方法 |
Country Status (1)
Country | Link |
---|---|
CN (1) | CN112379105A (zh) |
Cited By (2)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
CN113552336A (zh) * | 2021-07-22 | 2021-10-26 | 金宇保灵生物药品有限公司 | 一种基于ifa的brv血清中和抗体效价水平的检测方法 |
CN117517653A (zh) * | 2023-09-22 | 2024-02-06 | 深圳市第三人民医院(深圳市肝病研究所) | 非诊断目的的Mpox病毒抗体中和活性FRNT检测方法 |
Citations (2)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
CA2700078A1 (en) * | 2009-04-15 | 2010-10-15 | Variation Biotechnologies Inc. | Method and kit for detection of hepatitis a virus neutralizing antibodies |
CN104062443A (zh) * | 2014-07-14 | 2014-09-24 | 江苏省疾病预防控制中心 | 一种病毒中和抗体定量检测试剂盒及其应用 |
-
2020
- 2020-11-09 CN CN202011240812.5A patent/CN112379105A/zh active Pending
Patent Citations (2)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
CA2700078A1 (en) * | 2009-04-15 | 2010-10-15 | Variation Biotechnologies Inc. | Method and kit for detection of hepatitis a virus neutralizing antibodies |
CN104062443A (zh) * | 2014-07-14 | 2014-09-24 | 江苏省疾病预防控制中心 | 一种病毒中和抗体定量检测试剂盒及其应用 |
Non-Patent Citations (2)
Title |
---|
高小静: ""PRRSV的IPMA和IFA临床抗体检测方法的建立及应用"", 《万方学位论文》 * |
黄立平等: "猪圆环病毒1型抗体IPMA检测方法的建立及应用", 《中国兽医科学》 * |
Cited By (2)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
CN113552336A (zh) * | 2021-07-22 | 2021-10-26 | 金宇保灵生物药品有限公司 | 一种基于ifa的brv血清中和抗体效价水平的检测方法 |
CN117517653A (zh) * | 2023-09-22 | 2024-02-06 | 深圳市第三人民医院(深圳市肝病研究所) | 非诊断目的的Mpox病毒抗体中和活性FRNT检测方法 |
Similar Documents
Publication | Publication Date | Title |
---|---|---|
Sandvik | Laboratory diagnostic investigations for bovine viral diarrhoea virus infections in cattle | |
Benetka et al. | Prevalence of feline coronavirus types I and II in cats with histopathologically verified feline infectious peritonitis | |
Sandvik | Selection and use of laboratory diagnostic assays in BVD control programmes | |
Hasoksuz et al. | Detection of respiratory and enteric shedding of bovine coronaviruses in cattle in Northwestern Turkey | |
AK et al. | Serological and epidemiological investigation of bluetongue, maedi-visna and caprine arthritis-encephalitis viruses in small ruminant in Kirikkale District in Turkey | |
Rémond et al. | Diagnosis and screening of foot-and-mouth disease | |
Fulton et al. | Multiple diagnostic tests to identify cattle with Bovine viral diarrhea virus and duration of positive test results in persistently infected cattle | |
CN112379105A (zh) | 一种prrsv的ipma中和抗体检测方法 | |
CN104789697A (zh) | 一种同时检测csfv、prrsv、prv、pcv2的四色荧光定量pcr方法及其试剂盒 | |
Pratelli et al. | Diagnosis of canine coronavirus infection using nested-PCR | |
Koopmans et al. | Enzyme-linked immunosorbent assay reactivity of torovirus-like particles in fecal specimens from humans with diarrhea | |
CN112362880A (zh) | 一种prrsv的ifa中和抗体检测方法 | |
CN104673936A (zh) | 猪传染性胃肠炎病毒N基因RT-qPCR检测方法及其引物和TaqMan探针 | |
CN108330214A (zh) | 快速检测牛白血病前病毒的rpa引物及试剂和试剂盒 | |
EP1533370A1 (en) | Novel atypical pneumonia-causing virus | |
Gill et al. | Comparative prevalence and molecular characterization of group A rotavirus in cow calves of Punjab, India | |
CN112362868A (zh) | 一种prrsv的ipma抗体检测方法 | |
CN108676922A (zh) | 用于检测猪流行性腹泻病毒野毒株的引物和探针及TaqMan实时荧光定量PCR方法 | |
Villarreal | Diagnosis of infectious bronchitis: an overview of concepts and tools | |
CN114703179A (zh) | 检测PDCoV的RT-RAA-LFD引物对、探针、试纸条、试剂盒及其应用 | |
CN112379104A (zh) | 一种prrsv的ifa抗体检测方法 | |
Atwa et al. | An epidemiological survey of bovine viral diarrhea infection in calves in Egypt with identification of high prevalence of persistent infected animals | |
US9702016B2 (en) | Diagnostic test for virus | |
Cardoso et al. | Validation of an immunohistochemistry assay to detect turkey coronavirus: a rapid and simple screening tool for limited resource settings | |
Priya et al. | Standardization and application of immunofluorescence and immunoperoxidase tests for detection of bluetongue virus antigen |
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 |
Application publication date: 20210219 |
|
RJ01 | Rejection of invention patent application after publication |