Validation of an enzyme-linked immunosorbent assay (ELISA) for the measurement of canine S100A12.

BACKGROUND Canine S100 calcium-binding protein A12 (cS100A12) shows promise as biomarker of inflammation in dogs. A previously developed cS100A12-radioimmunoassay (RIA) requires radioactive tracers and is not sensitive enough for fecal cS100A12 concentrations in 79% of tested healthy dogs. An ELISA assay may be more sensitive than RIA and does not require radioactive tracers. OBJECTIVE The purpose of the study was to establish a sandwich ELISA for serum and fecal cS100A12, and to establish reference intervals (RI) for normal healthy canine serum and feces. METHODS Polyclonal rabbit anti-cS100A12 antibodies were generated and tested by Western blotting and immunohistochemistry. A sandwich ELISA was developed and validated, including accuracy and precision, and agreement with cS100A12-RIA. The RI, stability, and biologic variation in fecal cS100A12, and the effect of corticosteroids on serum cS100A12 were evaluated. RESULTS Lower detection limits were 5 μg/L (serum) and 1 ng/g (fecal), respectively. Intra- and inter-assay coefficients of variation were ≤ 4.4% and ≤ 10.9%, respectively. Observed-to-expected ratios for linearity and spiking recovery were 98.2 ± 9.8% (mean ± SD) and 93.0 ± 6.1%, respectively. There was a significant bias between the ELISA and the RIA. The RI was 49-320 μg/L for serum and 2-484 ng/g for fecal cS100A12. Fecal cS100A12 was stable for 7 days at 23, 4, -20, and -80°C; biologic variation was negligible but variation within one fecal sample was significant. Corticosteroid treatment had no clinically significant effect on serum cS100A12 concentrations. CONCLUSIONS The cS100A12-ELISA is a precise and accurate assay for serum and fecal cS100A12 in dogs.

The Fatty Acid Biosynthesis Enzyme FabI Plays a Key Role in the Development of Liver-Stage Malarial Parasites

The fatty acid synthesis type II pathway has received considerable interest as a candidate therapeutic target in Plasmodium falciparum asexual blood-stage infections. This apicoplast-resident pathway, distinct from the mammalian type I process, includes FabI. Here, we report synthetic chemistry and transfection studies concluding that Plasmodium FabI is not the target of the antimalarial activity of triclosan, an inhibitor of bacterial FabI. Disruption of fabI in P. falciparum or the rodent parasite P. berghei does not impede blood-stage growth. In contrast, mosquito-derived, FabI-deficient P. berghei sporozoites are markedly less infective for mice and typically fail to complete liver-stage development in vitro. This defect is characterized by an inability to form intrahepatic merosomes that normally initiate blood-stage infections. These data illuminate key differences between liver- and blood-stage parasites in their requirements for host versus de novo synthesized fatty acids, and create new prospects for stage-specific antimalarial interventions.