title: A lactate dehydrogenase ELISA-based assay for the in vitro determination of Plasmodium berghei sensitivity to anti-malarial drugs.
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.
  
  
  CONCLUSIONS
  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.
authors.affiliation: La Jolla Bioengineering Institute, 3535 General Atomics Court, Suite 210, San Diego, California 92121, USA. posanchez@ljbi.org
authors.forename: Pamela
authors.initials: P
authors.lastname: Orjuela-Sánchez
authors.forename: Erika
authors.initials: E
authors.lastname: Duggan
authors.forename: John
authors.initials: J
authors.lastname: Nolan
authors.forename: John A
authors.initials: JA
authors.lastname: Frangos
authors.forename: Leonardo Jm
authors.initials: LJ
authors.lastname: Carvalho
mesh_terms: D000818:Animals; D000911:Antibodies, Monoclonal; D000913:Antibodies, Protozoan; D000962:Antimalarials / Q000494:pharmacology*; D004353:Drug Evaluation, Preclinical; D004351:Drug Resistance; D004797:Enzyme-Linked Immunosorbent Assay / Q000379:methods*; D049452:Green Fluorescent Proteins / Q000235:genetics; D007770:L-Lactate Dehydrogenase / Q000032:analysis* / Q000276:immunology; D008288:Malaria / Q000188:drug therapy / Q000469:parasitology; D051379:Mice; D008810:Mice, Inbred C57BL; D018512:Parasitemia / Q000188:drug therapy / Q000469:parasitology; D010962:Plasmodium berghei / Q000187:drug effects* / Q000201:enzymology* / Q000235:genetics; D011994:Recombinant Proteins / Q000235:genetics
chemical_list: D000911:Antibodies, Monoclonal; D000913:Antibodies, Protozoan; D000962:Antimalarials; D011994:Recombinant Proteins; D049452:Green Fluorescent Proteins; D007770:L-Lactate Dehydrogenase