Production of monoclonal antibodies for Plasmodium vivax lactate dehydrogenase and patient sera screening using sandwich ELISA

Abstract

Malaria is a worldwide infectious disease. There are many diagnostic kits to detect malaria infection. However, the sensitivity of these diagnostic kits remains a problem. To develop a diagnostic kit for malaria that has high sensitivity, it is necessary to produce monoclonal antibodies (McAbs) with high affinity. The present study was undertaken to produce hybridoma cells that can be used to generate McAbs with high affinity and specificity against Plasmodium vivax lactate dehydrogenase (pvLDH). In this study, BALB/c mice were immunized with purified recombinant polypeptides that encode pvLDH. McAbs against pvLDH were produced according to the protocol of hybridoma technique using myeloma cells (SP2/0 cell lines). The McAbs were characterized by isotyping and by Western blot analysis. Two McAbs (D2H and D7E) against pvLDH antigen were obtained. The isotypes of D2H and D7E were IgG2b. They recognize 33 kDa proteins that were defined as pvLDH by Western blot analysis. In the affinity test, D2H and D7E showed positively optical density value until each McAbs were serially diluted at concentrations of 0.156 and 0.078 μg/ml, respectively. To evaluate sensitivity and specificity against clinical specimens of P. vivax, purified McAbs were tested with alkaline phosphatase-conjugated monoclonal antibodies and blood samples (n = 180) of P. vivax patients using the sandwich enzyme-linked immunosorbent assay, showing the 98% sensitivity. We suggest that McAbs produced in this study may be used for developing efficient and rapid diagnostic kits.