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Background   Plasmodium berghei  rodent malaria is a well-known model for the investigation of anti-malarial drug efficacy  in vivo . However, the availability of drug  in vitro  assays in  P. berghei  is reduced when compared with the spectrum of techniques existing for  Plasmodium falciparum . New alternatives to the current manual or automated methods described for  P. berghei  are attractive. The present study reports a new ELISA drug  in vitro  assay for  P. berghei  using two monoclonal antibodies against the parasite lactate dehydrogenase (pLDH).    Methods  This procedure includes a short- in vitro  culture, the purification of schizonts and the further generation of synchronized mice infections. Early stages of the parasite are then incubated against different concentrations of anti-malarial drugs using micro-plates. The novelty of this procedure in  P. berghei  relies on the quantification of the drug activity derived from the amount of pLDH estimated by an ELISA assay using two monoclonal antibodies: 14C1 and 19G7. The IC 50 s obtained through the ELISA assay were compared with those from the micro-test.    Results  The initial parameters of the synchronized samples used in the  in vitro  assays were a parasitaemia of 0.5% and haematocrit of 1%, with an incubation period of 22 hours at 36.5°C. pLDH detection using a 14C1 coating at 10 μg/ml and 19G7 at 2.5 × 10 -3  μg/ml provided good readouts of optical densities with low background in negative controls and specific detection levels for all parasite stages. IC 50 s values derived from the ELISA assay for artesunate, chloroquine, amodiaquine and quinine were: 15, 7, 2, and 144 nM, respectively. When artesunate and chloroquine IC 50 s were evaluated using the micro-test similar values were obtained.    Conclusion  This ELISA-based  in vitro  drug assay is easy to implement, fast, and avoids the use radioisotopes or expensive equipment. The utility of this simple assay for screening anti-malarial drug activity against  P. berghei in vitro  is demonstrated.

A lactate dehydrogenase ELISA-based assay for the in vitro determination of Plasmodium berghei sensitivity to anti-malarial drugs