Development of diagnostic reagents: Raising antibodies against synthetic peptides of PfHRP-2 and LDH using microsphere delivery

Abstract

Malaria causes significant morbidity and mortality worldwide, including countries with mainly imported malaria. In developing nations, scarce resources lead to inadequate diagnostic procedures. Microscopy of Giemsa-stained thick and thin films remains the current gold standard for diagnosis. Although it has good sensitivity and allows species identification, it is time consuming, requires microscopical expertise and maintenance of equipment. Antigen tests are promising tools for the diagnosis of malaria. Two such antigens are Plasmodium falciparum histidine rich protein (pfHRP-2) and lactate dehydrogenase (LDH). The present study was aimed to develop indigenous, rapid and sensitive immunodiagnostic method based on detection of PfHRP-2 and LDH antigen in the blood. Unique peptide sequences of PfHRP-2 (two regions) and LDH (three regions) antigen were synthesized by solid phase technique and purified to homogeneity. The antibodies raised against these sequences were raised in mice as well as rabbit using microspheres (PLGA) to generate high titre and affinity antibodies. The peptide-specific peak titres varied from 25,000 to 50,000 and affinity of the antibodies produced was found to be in order of 0.73–5.3 nM. The antibodies generated using microspheres were able to detect the PfHRP-2 and LDH antigen in the culture supernatant and parasitized RBC lysate of P. falciparum respectively by sandwich ELISA up to 0.002% parasitaemia level. The assay allowed the detection of parasite infections of 0.08–2.68% parasitaemia with a sensitivity of 100% in the whole blood of P. falciparum positive patients. No cross-reactivity was observed with P. vivax infected patients.