Assay development for essential enzyme activity in the tegument of live SchistosomesMadhu Sundaraneedi1 , Luke Becker2 , Giovanni Abbenante3 , Alex Loukas2 , Grant Collins1 , Mark Pearson2, 1School of Physical, Environmental and Mathematical Sciences, University of New South Wales, Australia , 2Australian Institute for Tropical Health and Medicine, James Cook University, Australia , 3BMG LABTECH Australia, 12/2017
Schistosomiasis is a parasitic disease that affects over 200 million people in tropical, developing nations, causing severe morbidity and over 300,000 deaths annually. Schistosomiasis is treated with a single drug and no vaccine is available.
We selected three Schistosoma surface-associated enzymes that are indispensable to parasitic survival: alkaline phosphatase; phosphodiesterase SmNPP-5 and an acetylcholinesterase. The activity of these molecules on the surface of live and intact larval and adult Schistosoma can be assayed in real-time of cultured parasites, providing a tool to assess the efficacy of drugs or vaccines targeting these enzymes. The colorimetric assay was read on a FLUOstar® Omega microplate reader.
The versatile instrument supports development of assays and drugs. Apart from its absorbance spectrometer, the FLUOstar Omega is equipped with fluorescence and luminescence detection capabilities allowing fast and reliable endpoint and kinetic measurements in all detection modes.
Fluorescence Polarization based assay for rapid, precise, high-throughput measurement of IgG & Fc containing derivativesDr. Carolanne Doherty, Valitacell, NIBRT, Fosters Avenue, Blackrock, Dublin, Ireland, 11/2017
The accurate, rapid and high-throughput measurement of IgG is essential in the development and manufacture of most therapeutic antibodies. Monoclonal antibodies are becoming increasingly dominant in biopharmaceuticals, where a vast number of samples must be screened for the development of each potential therapeutic.
Here we report the development of a novel, rapid, and simple fluorescence polarization based assay for high-throughput titer measurement of IgG and Fc-containing derivatives. It uses fluorescently labeled protein G that binds IgG. Upon binding, the depolarization of emitted light decreases, this can be detected to report on the presence of IgG. The CLARIOstar®, PHERAstar® and POLARstar® exhibit excellent assay quality when used with the ValitaTITER assay, which enables a high-throughput, simple, precise method for quantification of IgG. Moreover, the assay can be performed using sample straight cell culture supernatant, which means there are no complex sample preparation steps.
Calcium retention capacity assay evaluates inhibition of mitochondrial permeability transition poreM. Awais , D. Latawiec , R. Sutton, Liverpool Pancreatitis Research Group, Institute of Translational Medicine, University of Liverpool, UK, 11/2017
Mitochondrial dysfunction is central to the pathogenesis of acute pancreatitis, ischemia-reperfusion injury of the heart, brain and kidney, muscular dystrophies and neurodegeneration. Mitochondrial dysfunction is the result of a sudden increase in permeability of the inner mitochondrial membrane (IMM), via persistent opening of a multi-protein channel known as the mitochondrial permeability transition pore (MPTP). This is followed by uncontrolled proton flow across the IMM and unregulated flux of water, ions and small solutes into and out of the mitochondrial matrix. This results in rupture of the outer mitochondrial membrane (OMM) and eventually cell death by necrosis. Therefore, MPTP is an attractive target for cell death prevention in a host of disease states.
The calcium retention capacity assay challenges isolated mitochondria with spikes of calcium ions. Upon opening of the MPTP, Ca2+ leaks into the assay buffer and increases fluorescence of the membrane-impermeable CalciumGreen™ dye. The Omega multi-mode plate reader has proven excellent robustness for performing the multiple injections as well as reliable fluorescent detection of the assay.
Detection of plant-synthesized nanoparticles and their antibacterial capacitySalem W. and Schild S., University of Graz , Institute of Molecular Biosciences , BioTechMed-Graz , Austria, 03/2017
Metallic nanoparticles became subject of intensive research because of their potential antibiotic properties. Nanoparticles such as silver, gold or zinc oxide particles are easily and cost-effectively synthesized by blending metal salts with plant extracts that reduce the metal. Different extracts, varying in the plant or the part of the plant used for the extract, are currently investigated in regard to their capacity to form nanoparticles and their antimicrobial efficacy. The formation of nanoparticles can be verified by UV-Vis spectroscopy due to surface plasmon resonance of the particles that lead to a characteristic spectrum defined by the underlying metal and particle size. Subsequent analysis of nanoparticles on microbial growth is typically tested by methods based on absorbance changes.
Here, we present how the spectrometer-based BMG LABTECH instruments are used to quickly confirm Ag and ZnO nanoparticle formation and their inhibitory effect on the diarrhea-causing bacteria Vibrio cholerae and enterotoxic Escherichia coli (ETEC).
The fluorescent probe NR12S detects changes in plasma membrane cholesterol levelsWendy S. Smith , Sopsamorn U. Flavell and David J. Flavell, The Simon Flavell Leukaemia Research Laboratory , Southampton General Hospital , Southampton , Hampshire , SO16 6YD, 02/2017
The cell membrane is a bilayer of phospholipids with embedded proteins. It contains cholesterol that determines the membrane's fluidity, permeability and activity of membrane proteins. Changes in membrane cholesterol are implicated in diseases such as Alzheimer's and cancer, demanding its investigation.
NR12S is a fluorophore that exhibits emission maxima at 560 and 630 nm dependent on the liquid order of the membrane. Incorporation of the dye in a liquid ordered phase (increased cholesterol) shifts the emission to 560 nm, whereas in a liquid disordered phase (lower cholesterol) it emits at 630 nm. Hence, the 560/630 ratio correlates with the membrane cholesterol content.
Inhibition of cholesterol synthesis by lovastatin or incubation with methyl-§-cyclodextrin decreased membrane cholesterol in hematological cells and was reported by decreases in the 560/630 ratio. Both emissions were conveniently measured on the filter-based FLUOstar® Omega microplate reader and could be analyzed with one-click using the MARS analysis software.