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Clinical and Vaccine Immunology, June 2008, p. 1012-1018, Vol. 15, No. 6
1071-412X/08/$08.00+0     doi:10.1128/CVI.00385-07
Copyright © 2008, American Society for Microbiology. All Rights Reserved.

Enzyme-Linked Immunosorbent Assay for Detection of Plasmodium falciparum Histidine-Rich Protein 2 in Blood, Plasma, and Serum{triangledown}

Carolyne M. Kifude,1 Halli G. Rajasekariah,2 David J. Sullivan Jr.,3 V. Ann Stewart,1 Evelina Angov,4 Samuel K. Martin,1 Carter L. Diggs,5 and John N. Waitumbi1*

Walter Reed Project/Kenya Medical Research Institute, Kisumu, Kenya,1 Cellabs Pty. Ltd., Brookvale, NSW 2001, Australia,2 Department of Molecular Microbiology and Immunology, Johns Hopkins University, Bloomberg School of Public Health, 615 N. Wolfe St., Baltimore, Maryland,3 Division of Malaria Vaccine Development, Walter Reed Army Institute of Research, Silver Spring, Maryland,4 Malaria Vaccine Development Program, United States Agency for International Development, Washington, DC5

Received 7 September 2007/ Returned for modification 6 November 2007/ Accepted 12 March 2008

Microscopy, the gold standard for the detection and quantification of malaria parasites in blood, is in many aspects deficient for this purpose. The method is poorly reproducible and can be inaccurate because Plasmodium 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 enzyme-linked immunosorbent assay (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 10 repeat experiments, the linear range of optical densities (ODs) at 450 to 650 nm was from 0.05 ± 0.002 to 2.28 ± 0.042, corresponding to 3.91 to 250 ng/ml of rPfHRP2. The coefficient of variation (CV) at each target concentration ranged from 1.93 to 8.07%. Using cultured parasites, we confirmed the linear range of ODs as well as the association between the PfHRP2 ELISA results and the microscopic parasite densities. For whole-blood samples spiked with cultured, washed, ring-stage-infected red blood cells (iRBCs), the linear range was 11.7 to 750 iRBCs/µl, with CVs of 0.29 to 7.56%. The same spiked samples evaluated by microscopists had similar sensitivities, but the CVs were unacceptably high (20.7 to 161.6%). Stock rPfHRP2 was stable through four freeze-thaw cycles (P < 0.05; paired t test). When different patient sample types at different concentrations within the linear range of the assay are compared, the recoveries of PfHRP2 from blood and serum were within ±20%, whereas the recoveries from plasma ranged between +35 and –41%. We conclude that PfHRP2 ELISA using whole-blood and serum samples is a suitable adjunct to microscopy and could ultimately benefit malaria intervention trials.

* Corresponding author. Mailing address: Walter Reed Project/Kenya Medical Research Institute, Kisumu, United Nations Avenue Gigiri, Village Market, Nairobi 00621, Kenya. Phone: 254(0) 733 616548. Fax: 254(0) 572 022903. E-mail: jwaitumbi{at}wrp-ksm.org

{triangledown} Published ahead of print on 26 March 2008.

Clinical and Vaccine Immunology, June 2008, p. 1012-1018, Vol. 15, No. 6
1071-412X/08/$08.00+0     doi:10.1128/CVI.00385-07
Copyright © 2008, American Society for Microbiology. All Rights Reserved.





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