Characterization of Enzyme Linked Immunosorbent Assay for Plasmodium falciparum Histidine-Rich Protein 2 in Blood, Plasma and Serum

Walter Reed Project, Kenya Medical Research Institute, Kisumu, Kenya; Cellabs Pty Ltd, Brookvale, NSW 2001, Australia; Department of Molecular Microbiology and Immunology, Johns Hopkins University, Bloomberg School of Public Health, 615 N. Wolfe St., Baltimore, MD, USA; Division of Malaria Vaccine Development, Walter Reed Army Institute of Research, Silver Spring, MD, USA; Malaria Vaccine Development Program, United States Agency for International Development, Washington, DC, USA

^* To whom correspondence should be addressed. Email: jwaitumbi{at}wrp-ksm.org.

	Abstract

Microscopy, the gold standard for detection and quantification of malaria parasitemia, is in many aspects deficient for this purpose. The method is poorly reproducible, and can be inaccurate because falciparum parasites sequester for a portion of each asexual cycle. Due to these deficiencies, biomarkers such as P. falciparum Histidine Rich Protein 2 (PfHRP2) are increasingly being used. In this study, we evaluated the use of a commercial PfHRP2 ELISA kit with some procedural modifications. We determined the linear range of the assay, including the lower limits of detection and quantitation, using recombinant PfHRP2 (rPfHRP2). In ten repeat experiments, the linear range was from an optical density _450-650 nm (OD) of 0.05±0.002 to 2.28±0.042, corresponding to 3.91-250 ng/mL of rPfHRP2. The coefficient of variation (CV) at each target concentration was 1.93-8.07%. Using cultured parasites, we confirmed the linear range of optical densities, and the association between the PfHRP2 ELISA results and microscopic parasite density. For whole blood spiked with cultured washed ring-stage infected red cells (iRBC), the linear range was 11.7-750 iRBC/µL, with CVs of 0.29-7.56%. The same spiked samples evaluated by microscopists had a similar sensitivity, but CVs were unacceptably high (20.7-161.6%). Stock rPfHRP2 was stable through 4 freeze thaw cycles (P < 0.05, paired T-test). When comparing different patient sample types at different concentrations within the linear range of the assay, the recovery of PfHRP2 from blood and serum was within ±20%, whereas the recovery for plasma ranged between +35 to -41%. We conclude that PfHRP2 ELISA in whole blood and serum is a suitable adjunct to microscopy, which ultimately could benefit malaria intervention trials.