Molecular characterization of Babesia microti seroreactive antigen 5-1-1 and development of rapid detection methods for anti-B. microti antibodies in serum

Babesiosis has become a new global threat impacting human health, and most human babesiosis cases are caused by Babesia microti. Until now few antigens of B. microti have been described which can be used for the diagnosis of human babesiosis. In the present study, we report on the bioinformatic analysis, cloning and expression of the sequence encoding the B. microti seroreactive antigen 5-1-1 to investigate its potential incorporation in serologic diagnostic tools for babesiosis. Bioinformatic analysis and recombinant gene expression were performed to molecularly characterize seroreactive antigen 5-1-1. Enhanced chemiluminescence (ECL)-Western blot methods were used to detect specific antibodies in infected mice. Immunofluorescence antibody assays (IFA) were performed to detect the localization of BmSA5-1-1 in B. microti parasites. ELISA and immunochromatographic (ICT) tests were developed using recombinant BmSA5-1-1 to evaluate its potential use in rapid detection methods for B. microti antibodies and for the diagnosis of babesiosis. A recombinant expression plasmid was constructed by inserting the target gene fragment in the pET28a vector after double digestion with BamHI and XhoI restriction enzymes. The recombinant BmSA5-1-1 protein was expressed in Escherichia coli (rBmSA5-1-1) and purified by means of Ni-nitrilotriacetic acid (NTA) agarose columns. Polyclonal antibodies were generated against rBmSA5-1-1. Based on indirect immunofluorescence assay results, BmSA5-1-1 appeared to localize on the surface of B. microti. ELISA tests using the rBmSA5-1-1 antigen detected specific antibodies from infected mice as early as 4 days post-infection. Our results indicate that the two methods we developed can detect specific antibodies in mice at different stages of infection with sensitivities of 100% (rBmSA5-1-1 ELISA) and 90% (ICT). The specificity of the two methods was 100%. Sera of patients suffering from other closely related parasitic diseases, such as malaria and toxoplasmosis, produced negative results. In conclusion, seroreactive antigen 5-1-1, a member of the BMN1 protein family, is expressed on the outer surface of B. microti and is a promising candidate antigen for the early diagnosis of babesiosis. rBmSA5-1-1 ELISA and ICT methods show good potential for detecting specific antibodies in mice at different stages of infection.