Use of Full-Length Recombinant Calflagin and Its C Fragment for Improvement of Diagnosis ofTrypanosoma cruziInfection

Abstract

Serological diagnosis of Trypanosoma cruzi infection is hampered by issues related to test specificity due to the cross-reactivity of most antigens with proteins of related parasites such as Leishmania spp. The recombinant calflagins are considered relevant antigens for the diagnosis of infection by Trypanosoma cruzi. In the present work, we describe two genes coding for putative calflagins in Leishmania major with the N-terminal moieties presenting high similarity with T. cruzi genes. This fact raised questions about their role in some cross-recognition of this antigen by sera from Leishmania spp.-infected individuals. The complete T. cruzi calflagin and two fragments of the protein, consisting of 146 amino acids of the N-terminal and 65 amino acids of the C-terminal regions, were expressed and evaluated against a panel of sera, which included wellcharacterized samples from T. cruzi, and Leishmania-infected patients. We were able to show that sera from Leishmania (Viannia) braziliensis-infected individuals recognized the recombinant full-length calflagin. Both the N-terminal and the complete protein presented the same high sensitivity (98.5% of sera from T. cruziinfected patients was detected) but different specificities (94% and 98%, respectively, when evaluated against sera from people not infected by T. cruzi, including 15 sera from people infected with L. braziliensis). The C-terminal fragment presented low sensitivity (70%) but 100% specificity. We propose the use of these antigens in two sequential assays to optimize the serological diagnosis of T. cruzi infection in humans in geographic areas where Leishmania spp. infection is coendemic.

Design, Construction, and Evaluation of a Specific Chimeric Antigen To Diagnose Chagasic Infection

“…Regions that are responsible for undesired cross-reactivity can be removed (28), and regions showing high specificity can be grafted one to another to raise sensitivity (16,17). In line with this, the construction of the chimeric protein herein reported constitutes a promising land to obtain Ags that allows for standardization of diagnosis techniques and provides a novel platform to obtain new immunogens.…”

“…1 A). The T. cruzi cDNA encoding the full-length calflagin (C29 FL ) subcloned in the EcoRI site of the vector pMALC2 (pMALC2/C29 FL ) was obtained as previously described (28). The construction was digested with HindIII, and the 3Ј fragment of 400 bp was purified by GFX PCR (Amersham), ligated in the HindIII site of the pET-32a/TcP2␤ N vector, and used to transform BL21(DE3) competent cells.…”

“…The recombinant C-terminal region of the 29-kDa calfalgin protein (C29 C ) was expressed and purified as previously described (28). Serum against C29 C was obtained from rabbits inoculated twice subsequently, as previously described (37).…”

“…All of these results are summarized in Table 1. The obtained results prompted us to explore the possibility of expressing this protein fused to C29 C , which presents lower sensitivity than C29 FL but higher specificity toward chagasic Abs (28).…”

“…This is the case for the T. cruzi calflagin, in which a fragment responsible for cross-reactivity with sera from Leishmania spp. has been identified, and the Ag has been successfully optimized for diagnostic purposes by the excision of this low-specificity fragment (28).…”