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TitleDevelopment and evaluation of diagnostic and live-imagery tools for the study of vector-borne viral
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LanguageEnglish
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Page 80

CHAPTER 2 – Yellow fever virus NS1 Detection - Methods

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cells, specific antibodies in the diluted serum sample will attach to the antigens coupled to

the solid surface. In a second step, the attached antibodies are stained with fluorescein-

labeled anti-guinea pig (or anti-rabbit) antibodies and visualized with the fluorescence

microscope (Fig. 22).





Figure 22: Schematic description of the antigen/antibody interactions in an IFA YFV slide for

detection of NS1 antigen with the guinea-pig polyclonal antisera



To detect antibodies against YFV NS1, the EUROIMMUN slide was incubated for 45

minutes at room temperature (RT) with the polyclonal anti-NS1 antisera and primary

antibodies diluted at 1:25, 1:50, 1:100 and 1:200 in 1x PBS with 0,1% Tween (PBST).

MAB6330 (a mouse anti-YFV antibody [216]) was tested at a 1:100 dilution as a positive

control. After washing with TBST, the EUROIMMUN slide was then incubated for 45 minutes

at room temperature in the dark with FITC labeled anti-guinea pig (or anti-mouse for the

positive control) secondary antibodies diluted at 1:200 in 1x PBST in order to detect primary

antibodies. After washing with TBST, the slide was covered with mounting medium

composed of glycerol only, for a standard observation or glycerol containing DAPI, a

fluorescent dye staining the DNA content and nuclei, for simultaneous observation of the cell

nucleus. The slide was then mounted with a cover slip and observed with a fluorescence

microscope with the appropriate color filters to analyze the immunofluorescence of the

secondary antibodies (FITC - green channel) and cell nucleus (DAPI - blue channel).

“Flavivirus Profile 2” IFA slides from EUROIMMUN were used for cross-reactivity

testing. Flavivirus profile slides from EUROIMMUN are designed for flavivirus serological

diagnosis and allow reliable differential diagnosis in infections with similar or unclear

symptoms (Fig. 23).

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