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Fulltext Detection of Plasmodium knowlesi in whole blood samples with sandwich enzyme-linked immunosorbent assay (ELISA) using rhoptry-associated protein 1 specific polyclonal antibodies

Wan Nazri WSM, Ling LY, Wen CF

J Vector Borne Dis, 2024 Apr 01;61(2):203-210.
PMID: 38922654 DOI: 10.4103/jvbd.jvbd_55_23

BACKGROUND OBJECTIVES: Plasmodium knowlesi, a simian malaria species, is now known to infect humans. Due to disadvantages in the current diagnosis methods, many efforts have been placed into developing new methods to diagnose the disease. This study assessed the ability of the PkRAP-1 sandwich enzyme-linked immunosorbent (ELISA) to detect P knowlesi antigens in whole blood specimens.
METHODS: Western blot assay was conducted to evaluate the ability of raised mouse and rabbit anti-PkRAP-1 polyclonal antibodies to bind to the native proteins in P. knowlesi lysate. The polyclonal antibodies were then used in sandwich ELISA to detect P. knowlesi. In the sandwich ELISA, mouse and rabbit polyclonal antibodies were used as the capture and detection antibodies, respectively. The limit of detection (LOD) of the assay was determined using P. knowlesi A1H1 culture and purified recombinant PkRAP-1.
RESULTS: Western blot results showed positive reactions towards the proteins in P. knowlesi lysate. The LOD of the assay from three technical replicates was 0.068% parasitaemia. The assay performance in detecting P. knowlesi was 83% sensitivity and 70% specificity with positive and negative predictive values of 74% and 80%, respectively. The anti-PkRAP-1 polyclonal antibodies did not cross-react with P. falciparum and healthy samples, but P. vivax by detecting all 12 samples.
INTERPRETATION CONCLUSION: PkRAP-1 has the potential as a biomarker for the development of a new diagnostic tool for P. knowlesi detection. Further studies need to be conducted to establish the full potential of the usage of anti-PkRAP-1 antibodies for P. knowlesi detection.
Potential use of anti-thrombospondin-related apical merozoite protein (TRAMP) polyclonal antibodies in sandwich enzyme-linked immunosorbent assay (ELISA) for detection of Plasmodium knowlesi

Wan Nazri WSM, Lau YL, Cheong FW

Trop Biomed, 2024 Sep 01;41(3):356-362.
PMID: 39548790 DOI: 10.47665/tb.41.3.016

Plasmodium knowlesi, primarily a zoonotic malaria species is the most common malaria pathogen in the Southeast Asia especially in Malaysian Borneo, Malaysia. Due to morphological resemblance of P. knowlesi to other human Plasmodium, the sensitivity for microscopic detection of P. knowlesi, which is the gold standard, is compromised. Thus, efforts have been made in finding alternatives for the disease diagnosis. This study described the potential use of anti-PkTRAMP polyclonal antibodies in sandwich ELISA for P. knowlesi detection. Anti-PkTRAMP polyclonal antibodies raised from mice and rabbit were first evaluated for their binding capability towards native proteins in P. knowlesi lysates using Western blot. These mice and rabbit polyclonal antibodies were then used in the sandwich ELISA as capture and detection antibodies, respectively. P. knowlesi A1H1 culture was utilised to determine the limit of detection (LOD) of this assay. Its clinical performance was determined by testing with archived human malaria and uninfected samples. Western blot analysis affirmed the polyclonal antibodies reactivity to P. knowlesi. The LOD obtained from three replicated assays was at 0.015% parasitaemia. The assay has 76% sensitivity and 75% specificity for P. knowlesi. Its positive and negative predictive values were 76% and 75%, respectively. No cross reactivity with P. falciparum and healthy samples was observed, except for P. vivax where 10 out of 12 samples were detected. In conclusion, anti-PkTRAMP polyclonal antibodies can be useful in detecting P. knowlesi. Regardless, the full potential of anti-PkTRAMP antibodies for diagnostic purposes need to be explored further.

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