Immunoblotting experiments using the recombinant proteins and a canine serological panel.

<p>For immunoblotting experiments, the rLiHyp1 (36.6 kDa), rLiHyp6 (21.4 kDa), and rHRF (19.4 kDa) proteins (10 μg, each) were submitted to a 12% SDS-PAGE and blotted onto a nitrocellulose membrane, which were blocked with a PBS-T containing 5% BSA solution, and incubated with a pool of sera of asymptomatic and symptomatic VL dogs, or with a pool containing sera of non-infected dogs (1:200 and 1:100 diluted in PBS-T, respectively). Peroxidase conjugated anti-dog IgG (1:10,000) was used as a second antibody. The reactivities against the rLiHyp1, rLiHyp6 and rHRF proteins are shown. A low range protein ladder (Invitrogen^TM, Life Technologies, USA) was used (Mr). The individual reactions of the rLiHyp1, rLiHyp6, and rHRF proteins with the pools of sera from non-infected or <i>L</i>. <i>infantum</i>-infected dogs (NI and CVL, respectively) are shown. Immunoblottings were derived from three independent experiments, and one representative preparation is showed in this study.