virus: DENV-2 rt-pcr_denv: P rt-pcr_zikv: ND collection date: 12/3/2019 onset of symptoms: 60 age: 30 sex: M igg-denv: ND igm-denv: ND igg-zikv: ND igm-zikv: ND prnt50: ND city: Ribeirao Preto country: Brazil
Extracted molecule
protein
Extraction protocol
Blood samples were collected via venipuncture, and the resulting sera were separated and stored at -80°C.
Label
NA
Label protocol
The secondary antibody was the Goat anti-Human IgG Fc Cross-Adsorbed antibody conjugated to the DyLigt 650 (Invitrogen, USA).
Hybridization protocol
The peptide microarray slide was incubated for 15 min at room temperature in standard buffer (PBS, pH 7.4, 0.05% Tween 20), then, in blocking buffer (PBS, pH 7.4, 0.05% Tween 20, 1% BSA) with shaking (140 rpm) for 60 min at room temperature and in staining buffer (standard buffer with 10% blocking buffer) with shaking (140 rpm) for 15 min at room for temperature for equilibration. The sub-arrays were incubated with individual or a pool of up to four (equal volumes) serum samples (1:80 dilution in staining buffer, pH 7.4) overnight at 4°C. The arrays were washed 3x1 min at 140 rpm with the standard buffer, followed by incubation with the secondary antibody (Goat anti-Human IgG Fc Creoss-Adsorbed Secondary Antibody - DyLigt 650, Invitrogen, USA, diluted 1:5000 in a staining buffer) for 30 min with shaking (140 rpm) at room temperature, in the dark. The arrays were washed 3x1 min in the standard buffer with shaking (140 rpm). Then, the slide was dipped two times into the dipping buffer (1 mM Tris, pH 7.4) and dried carefully in a stream of air.
Scan protocol
The array images were obtained with the Axon GenePix 4000B scanner (Molecular Devices, USA) or InnoScan 710 (Innopsys, France), using a 635-nm laser and a 10-μm resolution.
Description
DZ_07
Data processing
Each spot median fluorescence intensity, with local background subtraction, was computed with the GenePix Pro 7 Molecular Devices, USA, or Mapix, Innopsys, France, software. Fluorescence intensity values <1 were converted to 0. Then, fluorescence intensity values were normalized against the negative controls (HA peptides). This normalization was done by dividing the fluorescence intensity value of each spot by the mean fluorescence intensity of the negative control spots. Normalized fluorescence intensity values <1 were converted to 1. The normalized data were log2-transformed to reduce variability. The final fluorescence intensity value for each peptide was the mean of the fluorescence intensity value of the duplicated peptide spots.
