4A)

4A). high affinity antibodies against selected peptides ofPfHRP-2 andpLDH Oxybenzone 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 Oxybenzone developed reagents are able to detectPfHRP2 andPfLDH antigens as low as 75 parasites/l of blood with a very high sensitivity (96-100%) and specificity (100%). == Interpretation & conclusions: == The study highlight the identification of unique epitopes ofPfHRP2 andPfLDH, and the generated antibodies against these antigens were used for quantitative estimation of these two antigens using sandwich ELISA. No corresreactivity withP. vivaxinfected patients was observed with the sera. Keywords:Diagnosis, ELISA, histidine rich protein 2, lactate dehydrogenase, malaria,Plasmodium falciparum (Pf) Malaria is potentially a life-threatening disease, which predominantly occurs in tropical and subtropical regions. It continues to be a major public health problem in endemic countries lacking adequate health care and malaria control programme. The clinical diagnosis based on symptoms is the most common method used but it shows less specificity due to overlapping of FGF-13 symptoms with other tropical diseases. Microscopic examination of stained blood films is another cornerstone for diagnosis and estimation of parasite load in malaria victims. Although it is cost effective and simple, but it shows poor reproducibility, variable sensitivity and requires skilled operators for accurate diagnosis. Importantly, in remote areas where malaria commonly occurs, it is hard to maintain good quality microscopy and the result based on microscopic examination is sometimes unreliable. In many cases co-infection withPlasmodium falciparumandP. vivaxoccur, therefore, accurate and prompt parasitological confirmation of malaria infection is essential for effective disease management. Many of the new technologies for malaria diagnosis incorporate immunochoromatographic procedure, where conjugated monoclonal antibodies are the key reagents. Currently many rapid diagnostic tests (RDTs) are widely used for the diagnosis of malaria. These RDTs are simple lateral-flow immunochromatographic tests that detect parasite specific antigens released from red blood cells. Two of the tests, the ICT MalariaPf/PvandParaSight-F detect histidine rich protein 2 (HRP-2), a protein produced by asexual stages and young gametocyte ofP. falciparum1,2. Oxybenzone The third test OptiMAL detectsPlasmodiumlactate dehydrogenase (PLDH), a marker protein for the intraerythrocytic form of the malaria parasite. HRP-2 is an abundant protein produced by all blood stages ofP. falciparum3. However, there are certain limitations of these rapid tests, including the decrease in sensitivity4(70 in parasitaemia <50/l) and also these tests cannot give Oxybenzone quantitative (parasite/l) results with malaria positive serum samples. The HRP-2 antigen is expressed by allP. falciparumparasites regardless of knob-phenotype, and can be recovered from culture supernatants as a secreted soluble protein5. HRP-2 can be detected in erythrocytes, serum, plasma, cerebro-spinal fluid and even in urine6,7. Sequencing of the genomic DNA has shown that HRP-2 antigen contains 35 per cent histidine as well as alanine and aspartate (40 and 12%, respectively). It is characterized by the presence of tandem repeats of Oxybenzone AHH and AHHAAD. Since HRP-2 antigen is only produced byP. falciparum, these tests cannot be used for the detection ofP. vivaxor other human malarial parasites. In endemic areasP. falciparumhas been reported to lack HRP-2 or HRP-3 or both in positive patients8. In India,P. falciparumlackingPfHRP-2 and thePfHRP-3 gene has so far not been reported. However, the HRP-2 antigen remains detectable for several weeks after parasite clearance which causes false- positive RDT results in the test samples9. Distribution ofP. falciparumisolates lackingPfHRP-2 andPfHRP-3 is a major challenge for RDTs. In these areas parasite specificpLDH is the major target for the detection of malaria.pLDH is an intracellular glycolytic enzyme, which catalyses the oxidation of lactate to pyruvate. InP. falciparum, the coenzyme for LDH is preferably 3-acetyl pyridine adenine dinucleotide, (APAD), whereas the activity of human LDH requires nicotinamide adenine dinucleotide10indicating thatpLDH could be a good marker following active malarial infections11.pLDH is expressed at high levels during asexual stage or blood-stage in all four malaria parasites12and also correlated with the number of parasites present in the plasma of infected patients13.pLDH fromP. vivax,P. malariae, andP. ovaleexhibit 87 per cent sequence identity withpLDH fromP. falciparum14. Genetic diversity may be particularly important forPfHRP-2-based RDTs since the antigen consists of a number of alanine and histidine-rich amino acid repeats and varies in size between parasite strains15. In the present study we developed a simple antigen capture.