Liquichip ni nta beads

Sera from patients were analyzed before treatment at baseline, after treatment, and at the time of relapse, if applicable. To detect and quantify anti-DSG3 Ab IgG subclasses, we developed an ALBIA-DSG3, which consisted of coupling human recombinant DSG3 protein to fluorescent beads (LiquiChip Ni-NTA Beads; Qiagen, Hilden, Germany) according to the manufacturer’s protocol. To determine the isotype of serum anti-DSG3 Abs, DSG3-coated beads were incubated with sera diluted at 1:150, then incubated with anti-IgG1 (1:125), anti-IgG2 (1:125), anti-IgG3 (1:200), or anti-IgG4 (1:200) biotinylated secondary antibody (SouthernBiotech, Birmingham, AL, USA), and finally with streptavidin–R-phycoerythrin (Qiagen). The mean fluorescence intensity (MFI) was determined on a Bio-Plex apparatus using Manager software version 4.0 (Bio-Rad, Hercules, CA, USA). Negative control with no serum and positive control [anti-DSG3 Calibrator of ELISA kit (EUROIMMUN)] were included in every assay. The anti-DSG3 Ab serum levels were determined with the formula (MFI^serum/MFI^Calibrator) × 100, in which the calibrator was the anti-DSG3-positive control previously mentioned that was used on every 96-well plate and set arbitrarily to 100 arbitrary units (AU). For each isotype, we considered a positivity threshold corresponding to + 3 standard deviations relative to the mean value obtained from the sera of 36 HD.

Macaque plasma reactivities against 9 recombinant T. cruzi proteins and parasite lysate were tested in a multiplex assay previously described for testing human and dog serum samples [20 (link),22 (link)]. Briefly, macaque plasma was diluted 1:500 and incubated with a pool of the recombinant T. cruzi proteins attached to addressable Liquichip Ni-NTA beads (Qiagen, CA) and T. cruzi amastigote/trypomastigote lysate coupled to Carboxy-Ni-NTA beads (Qiagen Inc). Antibody binding was detected with goat anti-human IgG conjugated to phycoerythrin and quantified on a Bio-Plex Suspension Array System (Bio-Rad, Hercules, CA, USA). Weighted median fluorescence intensity (MFI) for samples in duplicate was calculated and the ratio of the specific MFI for each antigen versus a negative control (green fluorescent protein) was estimated for each antigen.

Canine plasma samples were tested with a recombinant T. cruzi protein multiplex assay previously described in detail for testing T. cruzi reactivity of human sera samples [20 (link)]. Briefly, sera or plasma are diluted 1:500 and incubated with a pool of 11 recombinant proteins attached to addressable Liquichip Ni-NTA beads (Qiagen Inc, Valencia, CA, USA) and T. cruzi amastigote lysate coupled to Carboxy-Ni-NTA beads (Qiagen Inc). Following washing, antibody binding was detected with goat anti-dog IgG conjugated to phycoerythrin (Santa Cruz Biotechnology Inc, Santa Cruz, CA, USA) and quantified on a Bio-Plex Suspension Array System (Bio-Rad, Hercules, CA, USA). Serum samples were assayed in duplicate and weighted mean fluorescence intensity (MFI) was calculated. The ratio of the specific MFI for each antigen versus a negative control (green fluorescent protein) protein was calculated for each antigen and sera or plasma in the assay.