RESUMEN
Precision livestock farming utilizing advanced diagnostic tools, including biosensors, can play a key role in the management of livestock operations to improve the productivity, health, and well-being of animals. Detection of ketosis, a metabolic disease that occurs in early lactation dairy cows due to a negative energy balance, is one potential on-farm use of biosensors. Beta-hydroxybutyrate (ßHB) is an excellent biomarker for monitoring ketosis in dairy cows because ßHB is one of the main ketones produced during this metabolic state. In this report, we developed a low-cost, Keto-sensor (graphene-based sensor) for the detection of ßHB concentrations in less than a minute. On this device, graphene nanosheets were layered onto a screen-printed electrode (SPE), and then, a stabilized enzyme (beta-hydroxybutyrate dehydrogenase, NAD+, and glycerol) was used to functionalize the graphene surface enabled by EDC-NHS conjugation chemistry. The Keto-sensor offers an analytical sensitivity of 10 nm and a limit of detection (LoD) of 0.24 nm within a detection range of 0.01 µm-3.00 mm. Spike testing indicates that the Keto-sensor can detect ßHB in serum samples from bovines with subclinical ketosis. The Keto-sensor developed in this study shows promising results for early detection of subclinical ketosis on farms.
RESUMEN
Research on C-X-C motif chemokine ligand 10 (CXCL10) has been widely reported for humans and select animal species, yet immune reagents are limited for pig chemokines. Our goal is to provide veterinary immunologists and the biomedical community with new commercial immune reagents and standardized assays. Recombinant porcine CXCL10 (rPoCXCL10) protein was produced by yeast expression and used to generate a panel of α CXCL10 monoclonal antibodies (mAbs). All mAbs were assessed for cross-inhibition and reactivity to orthologous yeast expressed CXCL10 proteins. Characterization of a panel of nine α PoCXCL10 mAbs identified six distinct antigenic determinants. A sensitive quantitative sandwich ELISA was developed with anti-PoCXCL10-1.6 and -1.9 mAb; reactivity was verified with both rPoCXCL10 and native PoCXCL10, detected in supernatants of peripheral blood mononuclear cells stimulated with rPoIFNγ or PMA/Ionomycin. Immunostaining of in vitro rPoIFNγ stimulated pig spleen and blood cells verified CXCL10 + cells as CD3-CD4-CD172+, with occasional CD3-CD4 + CD172 + subsets. Comparison studies determined that α PoCXCL10-1.4 mAb was the ideal mAb clone for intracellular staining, whereas with α PoCXCL10-1.1 and -1.2 mAbs were best for immunohistochemistry analyses. These techniques and tools will be useful for evaluating swine immune development, responses to infectious diseases and vaccines, as well as for improving utility of pigs as an important biomedical model.