A new double-antibody sandwich ELISA targeting Plasmodium falciparum aldolase to evaluate anti-malarial drug sensitivity

Abstract

Background: 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 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.

Methods: The 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.

Results: A 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 IC 50s 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.

Conclusion: The 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.

Figure 1

Correlation of log IC₅₀s obtained from the aldolase ELISA and the [³H]hypoxanthine method with the P. falciparum strain NF54. 34 compounds were tested by ELISA and [³H]hypoxanthine incorporation (n = 1) with the P. falciparum strain NF54. The log IC₅₀ in nM (or ng/ml for the standard compounds) from [³H]hypoxanthine incorporation is plotted against the log IC₅₀ from ELISA, for each compound. (n = 34, correlation coefficient (R) = 0.991 and p-value < 0.001).

Figure 2

Difference in log IC₅₀s obtained from the the aldolase ELISA and the [³H]hypoxanthine method. Bland-Altman plot of the difference in log IC₅₀s in nM (or ng/ml for the standard compounds) for 34 anti-malarial compounds, determined by ELISA and [³H]hypoxanthine assay plotted against their mean. The mean difference is -0.261. The limits of agreement are represented by ± 2SD (-0.85 to 0.32).