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.
METHODS: Plasma from children and adults with P. vivax malaria in Sabah, Malaysia, were collected during acute infection, 7 and 28 days after drug treatment. Complement-fixing antibodies and immunoglobulin M and G (IgM and IgG), targeting 3 distinctive regions of PvMSP3α, were measured by means of enzyme-linked immunosorbent assay.
RESULTS: The seroprevalence of complement-fixing antibodies was highest against the PvMSP3α central region (77.6%). IgG1, IgG3, and IgM were significantly correlated with C1q fixation, and both purified IgG and IgM were capable of mediating C1q fixation to PvMSP3α. Complement-fixing antibody levels were similar between age groups, but IgM was predominant in children and IgG3 more prevalent in adults. Levels of functional antibodies increased after acute infection through 7 days after treatment but rapidly waned by day 28.
CONCLUSION: Our study demonstrates that PvMSP3α antibodies acquired during P. vivax infection can mediate complement fixation and shows the important influence of age in shaping these specific antibody responses. Further studies are warranted to understand the role of these functional antibodies in protective immunity against P. vivax malaria.