Potential use of anti-thrombospondin-related apical merozoite protein (TRAMP) polyclonal antibodies in sandwich enzyme-linked immunosorbent assay (ELISA) for detection of Plasmodium knowlesi

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.