Development Of Plasmodium Vivax-specific Aldolase Antigen And Monoclonal Antibodies:Applications In Rapid Diagnostic Testing Of Malaria

Malaria is a major cause of death worldwide. Most rapid diagnostic tests (RDTs) currently used for malaria diagnosis cannot distinguish between the various Plasmodium infections. The development of a P. vivax specific RDTs with high sensitivity to sufficiently differentiate the two most common Plasmodium infections would be very crucial for disease treatment and control. P. vivax aldolase gene (PvALDO) was amplified from the extracted genomic DNA and constructed into pET30a vector. Recombinant P. vivax aldolase protein was successfully expressed in E. coli BL21(DE3) in soluble form and the overall purity was over95%after one-step affinity chromatography purification. The purified product was used for mice and rabbit immunization. Rabbit polyclonal antibodies generated were deployed to develop a novel antibody-capture ELISA for hybridoma screening. Nine stable monoclonal antibodies (1C3,1G3,1H3,3F1,9A1,9B5,9G6,11G7,12F10) were selected as either capture or detector antibodies in the establishment of immunochromatographic assay.The best antibody-pair (1C3-12F10) was evaluated for the possibility of use in developing commercial rapid diagnostic test kits for P. vivax detection. P. vivax positive and negative clinical blood samples (n=503) collected from Yunnan and Guangzhou (China) were evaluated. Our RDTs showed excellent specificity for P. vivax detection (100%at95%Confidence Interval (CI):99.12to100.00%) and a sensitivity of98.75%(95%CI:93.20to99.79%) using microscopic examination results as standard. Only one false negative result was obtained from the P. vivax positive samples screened (1/80). All P. falciparum samples (n=33) and healthy uninfected samples (n=390) were negative. Overall performance of this RDT was excellent with positive predictive value (PPV) and negative predictive value (NPV) of100.00%and99.76%, respectively, at95%CI and a very good correlation with microscopic examination (kappa value, K=0.992530) was observed. The immunochromatographic assay showed good sensitivity to recombinant P. vivax aldolase (rPvALDO) at a minimum concentration of6.25ng/ml. This study further elucidates the possibility of developing aldolase-specific RDTs which can differentiate the different Plasmodium infections and improve accurate diagnosis of malaria. This RDT could adequately differentiate between P. vivax and P. falciparum infections. The novel monoclonal antibody screening method developed here could find application in the screening of highly specific antibodies against other antigens.