Development of Monoclonal Antibodies for Diagnosis of Plasmodium vivax

Abstract

Plasmodium lactate dehydrogenase (pLDH) is a strong target antigen for the determination of infection with Plasmodium species specifically. However, a more effective antibody is needed because of the low sensitivity of the current antibody in many immunological diagnostic assays. In this study, recombinant Plasmodium vivax LDH (PvLDH) was experimentally constructed and expressed as a native antigen to develop an effective P. vivax-specific monoclonal antibody (mAb). Two mAbs (2CF5 and 1G10) were tested using ELISA and immunofluorescence assays (IFA), as both demonstrated reactivity against pLDH antigen. Of the 2 antibodies, 2CF5 was not able to detect P. falciparum, suggesting that it might possess P. vivax-specificity. The detection limit for a pair of 2 mAbs-linked sandwich ELISA was 31.3 ng/ml of the recombinant antigen. The P. vivax-specific performance of mAbs-linked ELISA was confirmed by in vitro-cultured P. falciparum and P. vivax-infected patient blood samples. In conclusion, the 2 new antibodies possessed the potential to detect P. vivax and will be useful in immunoassay.

Keywords: Plasmodium lactate dehydrogenase; Plasmodium vivax; monoclonal antibody.

Fig. 1

Homology of P. vivax LDH to other species and generation of recombinant protein. (A) The percentage identity between P. vivax LDH and other species. (B) SDS-PAGE analysis of recombinant LDH protein expression and purification. Lane 1, protein marker; lane 2, PvLDH before IPTG induction in E. coli; lanes 3–4, PvLDH after IPTG induction, pellet and lysate, respectively; lane 5, E. coli PfLDH before IPTG induction; lanes 6–7, E. coli PfLDH after IPTG induction, pellet and lysate, respectively; lanes 8–9, purified PvLDH and PfLDH, respectively. (C) Western blot of purified protein with 6×His tag antibody. Lane 1, protein marker; lane 2, BSA; lane 3, PfLDH; lane 4, Pv-LDH.

Fig. 2

Development of monoclonal antibody (mAb). (A) Antibody formation in 75T culture flask after final limiting dilution was confirmed by ELISA. Eight monoclones for PvLDH (left) and 7 monoclones for PfLDH (right) were selected. (B) Two mAbs were purified from ascites fluid and confirmed by SDS-PAGE. 2CF5 stands for 2CF1-BF5. (C) Left panel shows the Coomassie blue staining for the presence of antigen. Western blot analysis for mAbs confirmed the antigen and mAb. Lane 1, protein marker; lane; 2, BSA; lane 3, purified PfLDH; lane 4, purified PvLDH. (D) The relative affinity of both antibodies against pLDH antigens were confirmed by direct ELISA). **P<0.01; ***P<0.001.

Fig. 3

Immunofluorescence of selected mAbs for P. falciparum. (A) P. falciparum-infected RBCs were stained with Giemsa to confirm the blood stage. (B) IFA image was obtained by under a 100× oil immersion objective lens for 2CF5 (upper panel) and 1G10 (middle panel) for those infected RBC. The IFA result of 1G10 was also taken by 200× oil immersion objective lens (bottom panel).

Fig. 4

Evaluation of diagnostic performance of mAbs-linked ELISA. (A) Indirect ELISA of selected mAbs for P. falciparum (10⁶ parasites/mL). (B) Sandwich ELISA of selected antibodies with serial dilution of PvLDH antigen. (C) Sandwich ELISA of selected antibodies with P. falciparum parasitemia from 10⁴–10⁶ parasites/ml. (D) Sandwich ELISA of selected antibodies with P. vivax-infected patient’s bloods (n=7) and negative bloods (n=7). *P<0.05; ***P<0.001.