An improved method for undertaking limiting dilution assays for in vitro cloning of Plasmodium falciparum parasites

Butterworth, Alice S., Robertson, Alan J., Ho, Mei-Fong, Gatton, Michelle L., McCarthy, James S., & Trenholme, Katherine R. (2011) An improved method for undertaking limiting dilution assays for in vitro cloning of Plasmodium falciparum parasites. Malaria Journal, 10(95).

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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.


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.


The HRP2 ELISA was more sensitive than microscopy for detecting parasite growth. The minimum level of HRP2 protein detection of the ELISA was 0.11ng/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.


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.

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8 citations in Scopus
7 citations in Web of Science®
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ID Code: 75110
Item Type: Journal Article
Refereed: Yes
Additional Information: Butterworth, Alice S
Robertson, Alan J
Ho, Mei-Fong
Gatton, Michelle L
McCarthy, James S
Trenholme, Katharine R
Evaluation Studies
Malaria journal
Malar J. 2011 Apr 18;10:95.
Additional URLs:
Keywords: Cloning, Organism/methods, Enzyme-Linked Immunosorbent Assay/*methods, Erythrocytes/parasitology, Humans, Indicator Dilution Techniques/*instrumentation, Malaria, Falciparum/blood/parasitology, Microscopy/instrumentation, Parasitology/*methods, Plasmodium falciparum/*cytology/growth & development/metabolism, Proteins/*analysis, Protozoan Proteins/analysis
DOI: 10.1186/1475-2875-10-95
ISSN: 1475-2875
Subjects: Australian and New Zealand Standard Research Classification > BIOLOGICAL SCIENCES (060000) > MICROBIOLOGY (060500) > Infectious Agents (060502)
Divisions: Current > QUT Faculties and Divisions > Faculty of Health
Copyright Owner: Copyright 2011 Butterworth et al; licensee BioMed Central Ltd.
Copyright Statement: This is an Open Access article distributed under the terms of the Creative Commons Attribution License (, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.
Deposited On: 18 Aug 2014 01:45
Last Modified: 22 Jun 2017 08:01

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