Rida rf absorbens

When necessary (i.e. for samples without previous determination of B19V-specific IgM and/or IgG), anti-B19V antibodies were determined using enzyme-linked immunosorbent assays (Ridascreen, R-biopharm), following the manufacturer’s instructions. For IgM assay serum, samples were treated with RIDA RF-Absorbens (R-biopharm) for the precipitation of IgG antibodies.

Proteins from purified EV-A71 virions and protein lysates were mixed with Laemmli buffer and separated by 10% SDS-PAGE. The proteins were transferred onto nitrocellulose membranes, which were blocked with 5% skimmed milk in 0.05% Tween-20 phosphate buffered saline (0.05% PBST) for 1 hour at room temperature. The pooled human serum samples were pre-treated additionally with RIDA RF-Absorbens (R-Biopharm AG, Germany) or DTT (Invitrogen, USA) for IgM- and IgG-specific antibody detection, respectively. The membrane was then incubated with 1:5000 diluted anti-GFP-HRP (Miltenyi Biotec, Germany), 1:300 diluted pooled human serum, 1:100 diluted Light Diagnostics EV-A71 monoclonal antibody 3323 (mAb 3323; Millipore, USA) or 1:1000 diluted EV-A71-specific mAb 979 (Millipore, USA) for 1 hour at room temperature, followed by secondary antibody incubation with the 1:5000 diluted HRP-conjugated polyclonal rabbit anti-human IgM (KPL, USA), 1:3000 diluted Amersham ECL human IgG, HRP-linked whole Ab from sheep (GE Healthcare, USA) or 1:5000 diluted HRP-conjugated goat anti-mouse (Gene Tex, USA) antibody. The immunoblot was developed with Clarity Western ECL Substrate (Bio-Rad, USA) and detected by chemiluminescence. The sizes of the protein bands were determined using the Precision Plus Protein WesternC Standard (Bio-Rad, USA).

The proteins were resolved with 12% SDS-PAGE under non-reducing or reducing conditions and electro-transferred onto a nitrocellulose membrane (GE Healthcare, Germany). The membrane was blocked with 10% skimmed milk in 0.05% PBS-Tween 20 (PBST). For IgM detection, the pooled sera were treated with RIDA RF-Absorbens (R-Biopharm, Germany) in 1% bovine serum albumin (BSA)-PBS prior to blotting. The immunoreactivity of recombinant CHIKV proteins and virus antigen were evaluated at 1:100 and 1:400 dilutions. The bound antigen-antibody complex was detected by goat anti-human IgM-HRP (KPL, USA, cat. no. 474–1003) at 1:5000 dilution in 1% BSA-0.05% PBST. The membrane was visualized by chemiluminescence (Bio-Rad, USA) and images were acquired by BioSpectrum AC imaging system (UVP, USA). Mouse anti-His tagged antibodies (Merck Millipore, USA, cat. no. 05–949) were included as the control.