Background

The growth inhibition assay examines the re-invasion and growth of the Plasmodium spp. over two cycles of parasite replication (one cycle is approximately 24 h for P. knowlesi and 48 h for P. vivax, P. cynomolgi, and P. falciparum). After egress, asexual merozoites are released to continuously invade and multiply within host erythrocytes, increasing parasitaemia. During merozoite invasion, a cascade of parasitic proteins interacts with host cell receptors to manipulate host cell entry. In asymptomatic P. vivax patients, monoclonal antibodies recognise a wide range of parasitic ligands such as the duffy binding proteins (DBPs), reticulocyte binding proteins (RBPs), merozoite surface proteins (MSPs), and apical membrane antigen (AMA) (Han et al., 2019; Rawlinson et al., 2019). These antibodies have been shown to be strain-transcendent, confer protective immunity, and reduce merozoite invasion to reticulocytes in vitro short-term culture (Russell et al., 2011). However, the lack of a successfully multiplying continuous culture system of P. vivax reduces the detection signal in the growth inhibition assay (GIA) (Gunalan et al., 2020). To overcome this limitation, past P. vivax studies utilized surrogate species such as P. knowlesi, P. cynomolgi, and P. falciparum to screen inhibitory antibodies (Mitran et al., 2019; Collins et al., 1999; Muh et al., 2020).

The conventional GIA quantifies parasite viability and density using the SYBR green dye and lactate dehydrogenase (LDH) enzyme via flow cytometry and spectrophotometry, respectively (Mohring et al., 2020). Our approach explores thiol reactivity quantification in P. cynomolgi, which reliably assesses P. falciparum parasitaemia in antibody-dependent cellular inhibition assays (Jogdand et al., 2012). The quantification is based on the accumulation and fluorescent covalent biconjugates formed by the thiol-reactive probe carbocyanin, which acts as an indicator for the parasite’s viability in the sarcoplasmic reticulum and mitochondria of eukaryotes (Habicht and Brune 1980; Amaratunga et al., 2014). As the Mitotracker product is available in a wide range of fluorescent dyes and probes, this method also presents an opportunity to investigate other fluorescent channels in flow cytometry. Furthermore, our approach adapts the mean inhibitory concentration approach commonly used in drug assays, allowing researchers to select amongst highly effective inhibitory antibodies with low inhibitory concentrations.