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. 2008 Jun;15(6):1012-8.
doi: 10.1128/CVI.00385-07. Epub 2008 Mar 26.

Enzyme-linked immunosorbent assay for detection of Plasmodium falciparum histidine-rich protein 2 in blood, plasma, and serum

Carolyne M Kifude  1 , Halli G Rajasekariah, David J Sullivan Jr, V Ann Stewart, Evelina Angov, Samuel K Martin, Carter L Diggs, John N Waitumbi
Affiliations

Enzyme-linked immunosorbent assay for detection of Plasmodium falciparum histidine-rich protein 2 in blood, plasma, and serum

Carolyne M Kifude et al. Clin Vaccine Immunol. 2008 Jun.

Abstract

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/microl, 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.

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Figures

FIG. 1.
FIG. 1.
Log versus log scale plot of a standard curve for results from 10 independent assay repetitions using rPfHRP2 in PBS/T. The dose response is linear for concentrations up to 250 ng/ml rPfHRP2, which corresponds to an OD of 2.28, above which the dose response becomes nonlinear. The lower LOD, i.e., the point at which the analysis becomes just feasible, is 3.91 ng/ml (arrow). The curve fitted to a true line without any data transformation.
FIG. 2.
FIG. 2.
Relationship of ODs to numbers of parasites plotted on a log versus log scale of results from five assay repetitions using washed iRBCs diluted in uninfected whole blood. The upper LOQ measured is 750 iRBCs/μl, which corresponds to an OD of 1.97. The lower LOD is 5.86 iRBCs/μl (inset table, horizontal arrow), while the lower LOQ, i.e., the point at which the analysis becomes linear and remains reproducible, is 11.7 iRBCs/μl (vertical arrow). The inset graph shows the quantities of PfHRP2 in spiked samples.
FIG. 3.
FIG. 3.
CVs of results for microscopy (•) and PfHRP2 ELISA (▪) as a function of parasite density plotted on a log scale. The LODs for both assays were similar (5.8 iRBCs/μl; arrow), but the reproducibility for microscopy results (CVs, 20.7 to 161.6%) was poor compared to that for PfHRP2 ELISA results (CVs, <10%).
FIG. 4.
FIG. 4.
Effect of multiple freeze-thaw cycles on a 10-μg/ml stock of rPfHRP2 antigen. The dashed line represents 100% concentration at cycle 1. The scatter plots represent percent recoveries at target concentrations between 7.8 and 250 ng/ml. The percent recoveries were significantly different from the fifth freeze-thaw cycle onward (P ≤ 0.05; paired t test). •, 7.8; formula image, 15.1; ◭, 31.3; ⬘, 62.5; ⬒, 125.0; ◓, 250.0.
FIG. 5.
FIG. 5.
Percent recovery of PfHRP2 in diluted patient samples. Sample types include whole blood (✖), plasma (formula image), and serum (▪), assessed at different dilutions, and were taken from patients with low, medium, and high levels of PfHRP2. Analyte recovery was determined by comparing observed values with expected values. The precision of the assay was within the acceptable ±20% range (area between the dashed lines marked “a” and “b”) between the lower LOQ (3.91 ng/ml; arrow) and the upper LOQ (250 ng/ml) for serum and whole-blood samples. Plasma samples had higher CVs (+35 to −41%) at PfHRP2 concentrations of ≤25 ng/ml.
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