Abstract
BackgroundPlasmodium falciparum in vitro growth inhibition assays are widely used to evaluate and quantify the functional activity of acquired and vaccine-induced antibodies and the anti-malarial activity of known drugs and novel compounds. However, several constraints have limited the use of these assays in large-scale population studies, vaccine trials and compound screening for drug discovery and development.MethodsThe D10 P. falciparum line was transfected to express green fluorescent protein (GFP). In vitro growth inhibition assays were performed over one or two cycles of P. falciparum asexual replication using inhibitory polyclonal antibodies raised in rabbits, an inhibitory monoclonal antibody, human serum samples, and anti-malarials. Parasitaemia was evaluated by microscopy and flow cytometry.ResultsTransfected parasites expressed GFP throughout all asexual stages and were clearly detectable by flow cytometry and fluorescence microscopy. Measurement of parasite growth inhibition was the same when determined by detection of GFP fluorescence or staining with ethidium bromide. There was no difference in the inhibitory activity of samples when tested against the transfected parasites compared to the parental line. The level of fluorescence of GFP-expressing parasites increased throughout the course of asexual development. Among ring-stages, GFP-fluorescent parasites were readily separated from uninfected erythrocytes by flow cytometry, whereas this was less clear using ethidium bromide staining. Inhibition by serum and antibody samples was consistently higher when tested over two cycles of growth compared to one, and when using a 1 in 10 sample dilution compared to 1 in 20, but there was no difference detected when using a different starting parasitaemia to set-up growth assays. Flow cytometry based measurements of parasitaemia proved more reproducible than microscopy counts.ConclusionsFlow cytometry based assays using GFP-fluorescent parasites proved sensitive and highly reproducible for quantifying the growth-inhibitory activity of antibodies and anti-malarials, with superior reproducibility to light microscopy, and are suitable for high-throughput applications.
Highlights
Plasmodium falciparum in vitro growth inhibition assays are widely used to evaluate and quantify the functional activity of acquired and vaccine-induced antibodies and the anti-malarial activity of known drugs and novel compounds
Cultures were maintained in airtight boxes at 37°C in an atmosphere of 1% O2, 4% CO2 and 95% N2. Parasites used in these studies include the D10 clonal line, an isogenic D10-P. chabaudi MSP1-19 parasite line in which the P. chabaudi orthologue replaces the region of P. falciparum msp1 encoding MSP1-19, and an isogenic D10-P. falciparum MSP1-19 parasite line generated in the same manner as PcMEGF to act as a transfection control (PfM3')
The AMA1 monoclonal antibodies (MAb) which recognizes a region of P. falciparum AMA1 domain 1 common to both the 3D7 and D10 parasite lines showed similar levels of growth inhibition of the PfMSP1-19 and PcMSP1-19 lines at a concentration of 0.5 mg/ml
Summary
Plasmodium falciparum in vitro growth inhibition assays are widely used to evaluate and quantify the functional activity of acquired and vaccine-induced antibodies and the anti-malarial activity of known drugs and novel compounds. A number of studies suggest an association between antibody levels to merozoite antigens measured by enzyme linked immunosorbent assays (ELISA) and protection from malaria disease [415]. Plasmodium falciparum asexual stage growth inhibition assays have been used extensively in malaria research to measure the levels of growth inhibitory antibodies in clinical and pre-clinical studies [16,28,29,30,31,32,33,34,35]. Growth inhibition assays are used extensively for testing the sensitivity of isolates to antimalarial drugs or screening compounds for anti-malarial activity in drug development studies
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