METHODS: Two hybridoma clones (Aa3c10, b10c1) were used for the production of ascites in BALB/c mice. Purification of monoclonal antibodies from the ascites was carried out using affinity columns. The thermal stability study of monoclonal antibodies was done by storing it at 37°C and 45°C for thirty days. The stored antibodies were analyzed using SDS-PAGE and flow-through device where the antigenantibody interaction was visualized by Protein A colloidal gold solution. Sensitivity was determined by endpoint dilution ELISA and the dissociation constant by competitive ELISA. Sensitive pair optimization was done by sandwich ELISA using biotinylated antibodies. Prototype preparation for lateral flow assay had a colloidal gold-based detection system.
RESULTS: Thermal stability experiments showed that both mAbs (Aa3c10; b10c1) are stable up to thirty days at 45°C while the commercially available mAbs were stable up to fifteen days only. Compared to commercial antibodies, the mAb Aa3c10, showed the highest sensitivity in end-point titre. In sensitive pair optimization, it was observed that the mAb, b10c1, as a detector and the mAb, Aa3c10, as a capture antibody showed the highest absorbance to detect 50pg/ml PfHRP2 antigen. The prototype formulation of lateral flow assay using the mAbs (Aa3c10; b10c1) showed good reactivity with WHO panel and no false-positive results were observed with twenty clinically negative samples and five P. vivax positive samples.
CONCLUSIONS: The novel monoclonal antibodies (Aa3c10, b10c1) against truncated PfHRP2, could be a strong potential candidates that can be included in making RDTs with better sensitivity and stability.
RESULTS: In the erythrocyte-binding assay, binding level was determined by scoring the number of rosettes that were formed by erythrocytes surrounding transfected mammalian COS-7 cells which expressed PkDBPαII. The assay result revealed a significant difference in the binding level. The number of rosettes scored for Fya+/b+ was 1.64-fold higher than that of Fya+/b- (155.50 ± 34.32 and 94.75 ± 23.16 rosettes, respectively; t(6) = -2.935, P = 0.026).
CONCLUSIONS: The erythrocyte-binding assay provided a simple approach to quantitatively determine the binding level of PkDBPαII to the erythrocyte Duffy antigen. Using this assay, PkDBPαII was found to display higher binding to Fya+/b+ erythrocytes than to Fya+/b- erythrocytes.
METHODS: Ten RDTs were evaluated: nine to detect clinical P. knowlesi infections from Malaysia, and nine assessing limit of detection (LoD) for P. knowlesi (PkA1-H.1) and P. falciparum (Pf3D7) cultures. Targets included Plasmodium-genus parasite lactate dehydrogenase (pan-pLDH) and P. vivax (Pv)-pLDH.
RESULTS: Samples were collected prior to antimalarial treatment from 127 patients with microscopy-positive PCR-confirmed P. knowlesi mono-infections. Median parasitaemia was 788/µL (IQR 247-5,565/µL). Pan-pLDH sensitivities ranged from 50.6% (95% CI 39.6-61.5) (SD BIOLINE) to 87.0% (95% CI 75.1-94.6) (First Response® and CareStart™ PAN) compared to reference PCR. Pv-pLDH RDTs detected P. knowlesi with up to 92.0% (95% CI 84.3-96.7%) sensitivity (Biocredit™). For parasite counts ≥200/µL, pan-pLDH (Standard Q) and Pv-pLDH RDTs exceeded 95% sensitivity. Specificity of RDTs against 26 PCR-confirmed negative controls was 100%. Sensitivity of six highest performing RDTs were not significantly different when comparing samples taken before and after (median 3 hours) antimalarial treatment. Parasite ring stages were present in 30% of pre-treatment samples, with ring stage proportions (mean 1.9%) demonstrating inverse correlation with test positivity of Biocredit™ and two CareStart™ RDTs.For cultured P. knowlesi, CareStart™ PAN demonstrated the lowest LoD at 25 parasites/µL; LoDs of other pan-pLDH ranged from 98 to >2000 parasites/µL. Pv-pLDH LoD for P. knowlesi was 49 parasites/µL. No false-positive results were observed in either P. falciparum-pLDH or histidine-rich-protein-2 channels.
CONCLUSION: Selected RDTs demonstrate sufficient performance for detection of major human malaria species including P. knowlesi in co-endemic areas where microscopy is not available, particularly for higher parasite counts, although cannot reliably differentiate among non-falciparum malaria.