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01.12.2012 | Methodology | Ausgabe 1/2012 Open Access

Malaria Journal 1/2012

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

Zeitschrift:
Malaria Journal > Ausgabe 1/2012
Autoren:
Pamela Orjuela-Sánchez, Erika Duggan, John Nolan, John A Frangos, Leonardo JM Carvalho
Wichtige Hinweise

Electronic supplementary material

The online version of this article (doi:10.​1186/​1475-2875-11-366) contains supplementary material, which is available to authorized users.

Competing interests

Authors do not have any competing interests.

Authors’ contributions

The work was carried out in collaboration between all authors. POS and LJMC designed experiments. POS carried out the laboratory experiments, analysed the data, interpreted the results and wrote the manuscript. ED assisted in FACS experiments and FCS Express software analyses. LJMC conceived the study, interpreted results and revised the manuscript. JAF and JN discussed the results and revised the manuscript. All authors have approved the final manuscript.

Abstract

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 IC50s 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. IC50s values derived from the ELISA assay for artesunate, chloroquine, amodiaquine and quinine were: 15, 7, 2, and 144 nM, respectively. When artesunate and chloroquine IC50s 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.
Zusatzmaterial
Authors’ original file for figure 1
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Authors’ original file for figure 2
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Authors’ original file for figure 3
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Authors’ original file for figure 4
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Authors’ original file for figure 5
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Literatur
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