Canine Antibody Profiling for Chagas Disease

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.

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Hartley A.N., Cooley G., Gwyn S., Orozco M.M, & Tarleton R.L. (2014). Frequency of IFNγ-producing T cells correlates with seroreactivity and activated T cells during canine Trypanosoma cruzi infection. Veterinary Research, 45(1), 6.

Publication 2014

Liquichip Ni-NTA beads Carboxy-Ni-NTA beads Bio-Plex Suspension Array System

Anti igg Antibody Antigen Assay Fluorescence Goat Green fluorescent protein Human Phycoerythrin Plasma Protein Protein a Protein t Recombinant proteins Sera Sera reactivity T cruzi

Corresponding Organization :

Other organizations : University of Georgia, University of Buenos Aires, Fundación Ciencias Exactas y Naturales

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Protocol cited in 2 other protocols

Variable analysis

independent variables

Recombinant T. cruzi protein multiplex assay

dependent variables

Antibody binding as quantified by mean fluorescence intensity (MFI)

control variables

Sample dilution (1:500)
Incubation of sera or plasma with recombinant proteins and T. cruzi amastigote lysate
Washing
Detection of antibody binding with goat anti-dog IgG conjugated to phycoerythrin
Quantification on a Bio-Plex Suspension Array System

controls

Negative control: Green fluorescent protein

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