Abstract
BackgroundThe standard in vitro test to assess anti-malarial activity of chemical compounds is the [3H]hypoxanthine incorporation assay. It is a radioactivity-based method to measure DNA replication of Plasmodium in red blood cells. The method is highly reproducible, however, the handling of radioactive material is costly, hazardous and requires the availability of appropriate technology and trained staff. Several other ways to evaluate in vitro anti-malarial activity do exist, all with their own assets and limitations.MethodsThe newly developed double-antibody sandwich ELISA described here is based on the properties of a non-overlapping pair of monoclonal antibodies directed against Plasmodium falciparum aldolase. This glycolytic enzyme possesses some unique nucleotide sequences compared to the human isoenzymes and has been highly conserved through evolution. Out of twenty possibilities, the most sensitive antibody pair was selected and used to quantitatively detect parasite aldolase in infected blood lysates.ResultsA total of 34 compounds with anti-malarial activity were tested side-by-side by ELISA and the [3H]hypoxanthine incorporation assay. The novel ELISA provided IC50s closely paralleling those from the radioactivity-based assay (R = 0.99, p < 0.001). At the investigated assay conditions (72 h incubation time, parasitaemia = 0.3%), the assay was found to be reproducible and easy to perform.ConclusionThe newly developed ELISA presents several advantages over the comparative method, the [3H]hypoxanthine incorporation assay. The assay is highly reproducible, less hazardous (involves no radioactivity) and requires little and cheap technical equipment. Relatively unskilled personnel can conduct this user-friendly assay. All this makes it attractive to be employed in resource-poor laboratories.
Highlights
The standard in vitro test to assess anti-malarial activity of chemical compounds is the [3H]hypoxanthine incorporation assay
When P. falciparum cultures (NF54) of e.g. initial parasitaemias of 0.3% or 0.03% were incubated for 72 hours and lysed and analysed by enzyme-linked immunosorbent assay (ELISA), the observed optical densities (ODs) were 2.3 and 0.23, with a background signal of about 0.04
Having obtained such a solid signal to background ratio of > 50 at the higher starting parasitaemia (0.3%), and since 0.3% is the typical parasitaemia employed for the hypoxanthine assay, an ELISA with five anti-malarial compounds was performed under these conditions, side-byside with a [3H]hypoxanthine incorporation assay
Summary
The standard in vitro test to assess anti-malarial activity of chemical compounds is the [3H]hypoxanthine incorporation assay. It is a radioactivity-based method to measure DNA replication of Plasmodium in red blood cells. The most commonly used method, especially in well-equipped laboratories, is the [3H]hypoxanthine incorporation assay [1] This method is highly reproducible, the handling of radioactive material is costly, hazardous and quite complex and, problematic for resource-poor locations. The same group reports that the aldolase protein sequence shows no insertions by analysing 36 of the original 75 P. falciparum isolates [10] This prompted us to develop a suitable double-antibody sandwich ELISA detecting P. falciparum aldolase to evaluate anti-malarial drug sensitivity. The newly developed aldolase ELISA was compared to the [3H]hypoxanthine incorporation assay, testing anti-malarial compounds such as OZ277 [11,12], artesunate (AS), chloroquine (CQ), pyrimethamine (PYR) and mefloquine (MEF)
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