New developments in malaria diagnostics

Abstract

Currently available rapid diagnostic tests (RDTs) for malaria show large variation in sensitivity and specificity, and there are concerns about their stability under field conditions. To improve current RDTs, monoclonal antibodies (mAbs) for novel malaria antigens have been developed and screened for their possible use in new diagnostic tests. Three antigens, glutamate rich protein (GLURP), dihydrofolate reductase-thymidylate synthase (DHFR-TS) and heme detoxification protein (HDP), were selected based on literature searches. Recombinant antigens were produced and used to immunize mice. Antibody-producing cell lines were subsequently selected and the resulting antibodies were screened for specificity against Plasmodium falciparum and Plasmodium vivax. The most optimal antibody couples were selected based on antibody affinity (expressed as dissociation constants, KD) and detection limit of crude antigen extract from P. falciparum 3D7 culture. The highest affinity antibodies have KD values of 0.10 nM ± 0.014 (D5) and 0.068 ± 0.015 nM (D6) for DHFR-TS mAbs, 0.10 ± 0.022 nM (H16) and 0.21 ± 0.022 nM (H18) for HDP mAbs and 0.11 ± 0.028 nM (G23) and 0.33 ± 0.093 nM (G22) for GLURP mAbs. The newly developed antibodies performed at least as well as commercially available histidine rich protein antibodies (KD of 0.16 ± 0.13 nM for PTL3 and 1.0 ± 0.049 nM for C1–13), making them promising reagents for further test development.

Keywords: :

• Plasmodium falciparum
• Dihydrofolate reductase-thymidylate synthase
• Plasmodium
• diagnostics
• glutamate rich protein
• heme detoxification protein
• malaria
• monoclonal antibodies
• rapid diagnostic test

Acknowledgments

This project is part of a collaborative effort for the improvement of malaria diagnosis with the Foundation for Innovative New Diagnostics (FIND). Support for this project was provided through funding from FIND. HRP2 recombinant protein was provided by ReaMetrix India Pvt., Ltd.; anti-HRP2 antibodies by National Bioproducts Institute in South Africa; and the purified recombinant heme-detoxification protein (HDP) by Virginia Polytechnic Institute and State University (Virginia Tech.,) in the USA. We thank Dionicia Gamboa from the Instituto de Medicina Tropical “Alexander von Humboldt”—Universidad Peruana Cayetano de Heredia in Peru for sending samples from patients infected with P. vivax parasites. Those samples were collected in the frame of collaborative projects with FIND with the required approval of a local ethical committee. We thank MR4 for providing us with 3D7 malaria parasites contributed by Daniel Carucci and Alister Craig. We thank the Centre for clinical malaria studies of the University Medical Centre St. Radboud in Nijmegen for providing us with the NF54 malaria parasites.

Figures and Tables

Figure 1 Detection of native antigens in a crude parasite extract dilution from P. falciparum culture with selected monoclonal antibodies. (A) HRP2 antibodies (C1–13 and PTL3, triangles) compared with strongest HDP (marked with H, solid lines) and DHFR-TS (marked with D, open squares) antibodies, (B) DHFR-TS antibodies, (C) HDP antibodies and (D) GLURP antibodies.

Table 1 Antibody response of mouse sera to the recombinant antigen in ELISA at different days before each (booster) immunization during the immunization procedure

Table 2 Overview and characteristics of hybridoma clones during immunization and selection process

Table 3 Dissociation constants (K_D) of monoclonal antibodies as determined by ELISA

Table 4 Detection limit of crude parasite antigen in ELISA by selected antibodies compared with HRP2 antibodies

Table 5 Primer sequences for recombinant antigens and encoding nucleotide range