Detection of histidine rich protein & lactate dehydrogenase of Plasmodium falciparum in malaria patients by sandwich ELISA using in-house reagents

Abstract

Background & objectives: Despite major control efforts, malaria remains a major public health problem that still causes high mortality rate worldwide especially in Africa and Asia. Accurate and confirmatory diagnosis before treatment initiation is the only way to control the disease. The present study was undertaken to develop reagents using sandwich ELISA for simultaneous detection of PfHRP2 (Plasmodium falciparum histidine rich protein) and PfLDH (P. falciparum lactate dehydrogenase) antigens in the proven malaria cases.

Methods: The antibodies were raised against two epitopes of PfHRP2 protein and three unique and unexplored epitopes of PfLDH protein. These antibodies were able to detect PfHRP2 and PfLDH antigens in culture supernatant and parasitized RBC lysate of P. falciparum, respectively up to 50 parasites/μl. The in-house reagents were tested in 200 P. falciparum positive patients residing in Baghpat district of Uttar Pradesh in northern India.

Results: Microsphere (PLGA) with CpG ODN were used to generate high titre and high affinity antibodies against selected peptides of PfHRP-2 and pLDH antigen in mice and rabbit. The peptide specific peak titre varied from 12,800 - 102,400 with an affinity ranging 0.73 - 3.0 mM. The indigenously developed reagents are able to detect PfHRP2 and PfLDH antigens as low as 75 parasites/μl of blood with a very high sensitivity (96-100%) and specificity (100%).

Interpretation & conclusions: The study highlight the identification of unique epitopes of PfHRP2 and PfLDH, and the generated antibodies against these antigens were used for quantitative estimation of these two antigens using sandwich ELISA. No corresreactivity with P. vivax infected patients was observed with the sera.

Fig. 1

EMBOSS stretcher alignment of Plasmidium lactate dehydrogenase amino acid sequences. The unique plasmodial epitope differentiating PfLDH and PvLDH appear below the highlighted region. Accession numbers are PfLDH: PlasmoDBid: PF3D7_1324900), PVLDH: PlasmoDBid: PVX_116630.

Fig. 2

Schematic diagram showing the localization of P. falciparum specific LDH epitope in the pLDH protein at (A) 0° rotation and (B) 180° rotation along the Y axis (PDB Id: 2A94). The peptide localization of all Pf LDH peptide was indicated in magenta. The Figure was drawn by PyMOL [DeLano WL (2002) The PyMOL molecular graphic system on world wide Web: http://www.pymol.org, for this study.

Fig. 3

Quantification of PfHRP2 antigen in culture supernatants of four different isolates of P. falciparum (Isolate 1 FDL-B, Isolate 2 FDL-NG, Isolate 3 FSH-4 and Isolate 4 FSH-11). Values are mean + SD of 3 observations.

Fig. 4

Quantification of PfHRP2 antigen (A) and PfLDH antigen (B) in parasitized RBC lysates of four different isolates of P. falciparum (Isolate 1 FDL-B, Isolate 2 FDL-NG, Isolate 3 FSH-4 and Isolate 4 FSH-11). Values are mean + SD of 3 observations.

Fig. 5

(A) Association between mean parasitaemia and parasite count in different groups of P. falciparum positive patients. Parasitaemia is expressed as parasites/μl and data expressed as mean +4 SD. (B) Relationship between parasite count and mean PfHRP2 (O) and mean PfLDH antigen concentration (Δ) in P. falciparum positive. Parasitemia is expressed as parasites/μl and data is expressed as mean + 4 SD.