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Background Multiplex ELISA that detect antibodies against more than one Plasmodium species while allowing species differentiation would be highly valuable for epidemiology and vaccine studies in areas of mixed infections and identification of malaria-exposed blood donors in non-endemic countries. Here we report a highly sensitive, multiplex ELISA based on recombinant proteins from Plasmodium falciparum, P. vivax and P. malariae malaria for panspecies and species-differentiating detection of antibodies in malaria-positive reference samples and in samples from individuals living in a malaria endemic region in Ghana, Africa. Materials and methods Multiplex ELISA was developed utilizing the Luminex xMAP technology that allows the simultaneous detection of antibodies of different specificities that react with antigenic epitopes on multiple beads (microspheres) of different dye intensity. Seven recombinant Plasmodium antigens (P. falciparum: CSP, AMA-1, LSA-1 and MSP119, P. vivax :A MA-1 and MSP119 and P. malariae: MSP119) were covalently coupled to carboxylated magnetic beads. The dilutions of human plasma/serum were incubated with 3000-5000 antigen-conjugated beads in 96-well plate. Following incubation with a biotin-labeled human antiIgG conjugate, a streptavidin-PE conjugated fluorescent substrate was added and the plates were read on Bio-Rad BioPlex 200 reader. The reader was set to read a minimum of 50 beads with identical unique detection signal, the results were expressed as median-fluorescent intensity and cut-off titers were established using a pool of normal human serum samples from the US blood donors. Results Multiplex ELISA detected 100% of the confirmed malaria reference samples belonging to P. falciparum, P. vivax and P. malariae infected patients. The inclusion of multiple antigens in the multiplex assay makes the test more sensitive than the conventional plate ELISA. The assay was capable to detect differential antibody reactivity to seven Plasmodium antigens in serum samples from 75 adults from malaria endemic area in Ghana who had no demonstrable parasitemia by microscopy. The assay also successfully distinguished between the mixed P. falciparum and P. malariae infections in imported malaria samples obtained in United States. Conclusions

Multiplex multi-antigen, multi-species, microsphere-based ELISA to detect antibodies to three human Plasmodium species