Author: Lee, Hyojin; Kim, Eun-Ju; Song, Jae-Young; Choi, Jeong Soo; Lee, Ji Youn; Cho, In-Soo; Shin, Yeun-Kyung
Title: Development and evaluation of a competitive enzyme-linked immunosorbent assay using a monoclonal antibody for diagnosis of severe fever with thrombocytopenia syndrome virus in bovine sera Document date: 2016_9_20
ID: 0pe8vgin_12
Snippet: IFA was performed as previously described, with slight modification [23] . Briefly, Vero E6 cells grown to 80% confluency in 150 cm 2 were infected with 2 mL of 1 × 10 3 TCID 50 /mL SFTSV. After 6 days, the infected cells (∼3,000 cells per well) were spotted onto the microscope slides with reaction wells (Paul Marienfeld, Germany). The cells were immediately fixed with a methanol : acetone (1 : 1) solution for 30 min, after which non-specific .....
Document: IFA was performed as previously described, with slight modification [23] . Briefly, Vero E6 cells grown to 80% confluency in 150 cm 2 were infected with 2 mL of 1 × 10 3 TCID 50 /mL SFTSV. After 6 days, the infected cells (∼3,000 cells per well) were spotted onto the microscope slides with reaction wells (Paul Marienfeld, Germany). The cells were immediately fixed with a methanol : acetone (1 : 1) solution for 30 min, after which non-specific signals were blocked with 5% horse serum in a total volume of 20 ïL per well. The cells were incubated with mouse mAbs, ï¡-NP specific polyclonal rabbit sera or field bovine serum samples for 1 h at room temperature, followed by detection using fluorescein isothiocyanate (FITC)-labeled secondary antibodies. The FITC-conjugated anti-mouse, rabbit, and bovine antibodies (KPL, USA) were diluted to a concentration of 2.5 ïg/mL prior to use. The cells were observed using a Nikon TE-2000U fluorescence microscope (Nikon, Japan). Each cell lysate was separated on an 8-16% gradient SDS-PAGE gel and transferred to membranes for Western blot analysis. The ï¡-GAPDH antibody was used as a loading control. (B) Rabbit anti-NP and mouse anti-SFTSV polyclonal antibodies were diluted 1 : 1,000 for immunoblotting and 1:200 for IFA. Three mAbs were diluted 1 : 1,000 for immunoblotting and 1 : 50 for IFA, and the secondary antibodies for rabbit and mouse IgG were diluted 1 : 1,000 for immunoblotting and 1 : 200 for IFA. DAPI staining (4',6-diamidino-2-phenylindole) was used to stain the cell nuclei.
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