RESUMEN
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.
RESUMEN
Objective] To explore the humoral and cellular immune responses in mice to eukaryotic expression recombinant plasmid encoding histidine rich protein 2 (HRP\|Ⅱ) of Plasmodium falciparum. [Methods] The start and stop codes were introduced into HRP\|Ⅱ gene fragment, the reading frame and the position of start and stop codes in HRP\|Ⅱ were identified by sequencing. HRP\|Ⅱ fragment containing the start and stop codes was cloned into pcDNA3 1(\|) to form pcDNA3 1(\|)/HRP\|Ⅱ. The BALB/c mice were immunized i.m. with the plasmids for 3 times in 3 weeks intervals. Two weeks after the last immunization, the sera and splenocytes were collected to investigate anti\|HRP\|Ⅱ antibodies by ELISA and the splenocytes proliferation response to HRP\|Ⅱ. [Results] Sequence data show that the reading frame and the position of start and stop codes are correct. Restriction enzyme digestion indicated that the HRP\|Ⅱ gene fragment containing start and stop codes was successfully cloned into pcDNA3 1(\|). Mice raised significant anti\|HRP\|Ⅱ antibodies after pcDNA3 1(\|)/HRP\|Ⅱ immunization, and the splenocytes proliferated prominently when stimulated with HRP\|Ⅱ protein. [Conclusion] Eukaryotic expression recombinant plasmid \{encoding\} HRP\|Ⅱ gene can induce significantly humoral and cellular immune response in mice. HRP\|Ⅱ gene may be a good candidate for P.falciparum blood\|stage multiple DNA vaccine.