title: An improved method for undertaking limiting dilution assays for in vitro cloning of Plasmodium falciparum parasites.
abstract: BACKGROUND
  Obtaining single parasite clones is required for many techniques in malaria research. Cloning by limiting dilution using microscopy-based assessment for parasite growth is an arduous and labor-intensive process. An alternative method for the detection of parasite growth in limiting dilution assays is using a commercial ELISA histidine-rich protein II (HRP2) detection kit.
  
  
  METHODS
  Detection of parasite growth was undertaken using HRP2 ELISA and compared to thick film microscopy. An HRP2 protein standard was used to determine the detection threshold of the HRP2 ELISA assay, and a HRP2 release model was used to extrapolate the amount of parasite growth required for a positive result.
  
  
  RESULTS
  The HRP2 ELISA was more sensitive than microscopy for detecting parasite growth. The minimum level of HRP2 protein detection of the ELISA was 0.11 ng/ml. Modeling of HRP2 release determined that 2,116 parasites are required to complete a full erythrocytic cycle to produce sufficient HRP2 to be detected by the ELISA. Under standard culture conditions this number of parasites is likely to be reached between 8 to 14 days of culture.
  
  
  CONCLUSIONS
  This method provides an accurate and simple way for the detection of parasite growth in limiting dilution assays, reducing time and resources required in traditional methods. Furthermore the method uses spent culture media instead of the parasite-infected red blood cells, enabling culture to continue.
authors.affiliation: Clinical Tropical Medicine Laboratory, Queensland Institute of Medical Research, Herston, Brisbane, 4006, Australia. alice.butterworth@qimr.edu.au
authors.forename: Alice S
authors.initials: AS
authors.lastname: Butterworth
authors.forename: Alan J
authors.initials: AJ
authors.lastname: Robertson
authors.forename: Mei-Fong
authors.initials: MF
authors.lastname: Ho
authors.forename: Michelle L
authors.initials: ML
authors.lastname: Gatton
authors.forename: James S
authors.initials: JS
authors.lastname: McCarthy
authors.forename: Katharine R
authors.initials: KR
authors.lastname: Trenholme
mesh_terms: D019976:Cloning, Organism / Q000379:methods; D004797:Enzyme-Linked Immunosorbent Assay / Q000379:methods*; D004912:Erythrocytes / Q000469:parasitology; D006801:Humans; D007201:Indicator Dilution Techniques / Q000295:instrumentation*; D016778:Malaria, Falciparum / Q000097:blood / Q000469:parasitology; D008853:Microscopy / Q000295:instrumentation; D010274:Parasitology / Q000379:methods*; D010963:Plasmodium falciparum / Q000166:cytology* / Q000254:growth & development / Q000378:metabolism; D011506:Proteins / Q000032:analysis*; D015800:Protozoan Proteins / Q000032:analysis; D000099053:Histidine-Rich Glycoprotein
chemical_list: D011506:Proteins; D015800:Protozoan Proteins; D000099053:Histidine-Rich Glycoprotein