Designing and Evaluation of a Recombinant Multiepitope Protein by Using ELISA for Diagnosis of Leishmania infantum Infected in Dogs

Abstract

Background: Visceral leishmaniasis (VL) is the most severe form of leishmaniasis. Correct identification of infected patients and reservoirs is vital to control the spread of VL. One important step in the control of Zoonotic Visceral leishmaniasis (ZVL) is the identification of infected dogs, which are the main domestic reservoir hosts of Leishmania infantum. We aimed to prepare and evaluate a new recombinant antigen using Bioinformatics tools for diagnosis of ZVL in domestic dogs.

Methods: The present study was carried out in Cellular and Molecular Biology Research Center, Shahid Beheshti University of Medical Sciences, Tehran, Iran  during 2015- 2018. Three L. infantum (JPCM5 strain) proteins were analyzed as follows: Nucleotide sequences of the surface proteins, putative amastin-like surface protein (P1), surface antigen protein 2 precursor (P2) and surface antigen-like protein (P3). The epitopes were predicted by several different bioinformatics servers using different methods. The predicted epitopes were selected with the highest immunogenic potential (P1P2P3) linked to each other with linkers (Gly, Se) and synthesized. Then the expression and protein purification were performed. In total, 114 serum samples were collected at 7 months. Positive and negative sera were confirmed using direct agglutination test (DAT). These recombinant antigens from L. infantum were used by indirect ELISA.

Results: Considering the cut-off point of 0.23, the test showed a sensitivity of 98% (95%CI=89.50%-99.90%) and a specificity of 95.31% (95%CI=87.10%-98.72%). Kappa analysis indicated very good agreement (kappa=0.831) between ELISA and DAT (P<0.05).

Conclusion: ELISA using the recombinant protein P1P2P3 has great potential for the diagnosis of canine visceral leishmaniasis (CVL).