Preparation of monoclonal antibody against N protein of Porcine epidemic diarrhea virus and establishment of indirect immuno-fluorescence assay
Journal of South China Agricultural University
; 41(5):27-35, 2020.
Article
in Chinese
| CAB Abstracts | ID: covidwho-2040361
ABSTRACT
Objective:
To prepare monoclonal antibodies against porcine epidemic diarrhea virus (PEDV) N protein, and develop an indirect immuno-fluorescence assay method used for detecting PEDV.Method:
The expressed recombinantly PEDV N protein was used as an immunogen and 8-week-old female BALB/c mice were immunized. Then their spleen cells with high antibody titer were isolated and fused with SP2/0 cells. The hybridoma cell lines secreting monoclonal antibodies against PEDV N protein were screened. In Vero cells infected with PEDV, monoclonal antibody of anti-PEDV N protein was used as the primary antibody and FITC-goat-anti-mouse IgG was used as the secondary antibody to develop indirect immuno-fluorescence assay method used for detecting PEDV.Result:
The prepared hybridoma cell lines could stably secrete anti-PEDV N protein antibodies, ELISA antibody titer in cell supernatant was above 13 200, and in mouse ascites above 11 000 000. While monoclonal antibodies were applied in established indirect immuno-fluorescence assay, the optimal conditions were that cells were fixed with 80% () acetone at -20 degrees C for 30 min;The primary antibody was diluted 1 000 times by PBS buffer solution and incubated at 4 degrees C overnight;The secondary antibody was diluted 100 times by PBS buffer solution and incubated at 37 degrees C for 1 h. Transmissible gastroenteritis virus (TGEV), classical swine fever virus (CSFV), porcine reproductive and respiratory syndrome virus (PRRSV), porcine reproductive virus (PRV), porcine enteric a corone virus (PEAV), porcine rotavirus (PoRV) and PEDV were detected by established indirect immuno-fluorescence assay method, only PEDV showed positive, all the else viruses showed negative.
Meat Producing Animals [LL120]; Prion, Viral, Bacterial and Fungal Pathogens of Animals [LL821]; Diagnosis of Animal Diseases [LL886]; Techniques and Methodology [ZZ900]; antibody testing; cell lines; diagnosis; diagnostic techniques; ELISA; IgG; immunodiagnosis; immunofluorescence; monoclonal antibodies; porcine epidemic diarrhoea; viral proteins; pigs; Porcine epidemic diarrhea virus; Sus scrofa; Sus; Suidae; Suiformes; Artiodactyla; mammals; vertebrates; Chordata; animals; eukaryotes; Alphacoronavirus; Coronavirinae; Coronaviridae; Nidovirales; positive-sense ssRNA Viruses; ssRNA Viruses; RNA Viruses; viruses; antibody detection; antibody tests; enzyme linked immunosorbent assay; serological diagnosis; fluorescent antibody technique; IFAT; swine; hogs; Porcine epidemic diarrhoea virus
Full text:
Available
Collection:
Databases of international organizations
Database:
CAB Abstracts
Language:
Chinese
Journal:
Journal of South China Agricultural University
Year:
2020
Document Type:
Article
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