F(ab')2 antibody fragments against Trypanosoma cruzi calreticulin inhibit its interaction with the first component of human complement

Trypanosoma cruzi calreticulin: A novel virulence factor that binds complement C1 on the parasite surface and promotes infectivity

“…Given the C1q role in parasite infectivity (Rimoldi et al 1989), the fact that TcCRT interacts with both C1 and C1q (Ferreira et al 2004;Valck et al 2010) and since T. cruzi infection elicits the production of specific anti-TcCRT antibodies in both humans and experimental animals (Aguilar et al, 2005;Aguillon et al 1997Aguillon et al , 2000bMarcelain et al, 2000), we asked whether these antibodies, upon binding TcCRT, could increase parasite infectivity. In vitro, the human C1q/rTcCRT interaction is strongly and indirectly enhanced by whole Igs against the parasite molecule (Fig.…”

“…with 10 3 Dm28c trypomastigotes (control group). Other three groups were infected with the trypomastigotes, alternatively pre-incubated, for 30 min at room temperature, with 10 g human C1q, 30 g of antiTcCRT polyclonal IgG or 30 g of anti-TcCRT F(ab ) 2 polyclonal IgG fragments, both reagents generated in our laboratories (Aguilar et al 2005). Parasitemia was measured every 2 days, starting at day 5 post-infection.…”

“…After washing, wells were coated with rabbit polyclonal antirTcCRT antibodies (Aguilar et al 2005) in 1% (w/v) PBS-BSA or with media alone. Different amounts of human C1q (Complement Technology, Tyler, Texas, USA), diluted in 1% (w/v) PBS-BSA, were added and incubated for 1 h at 37 • C. C1q specific binding to immobilized rTcCRT, in the presence or absence of anti-rTcCRT, was detected with goat anti-human C1q (Sigma-Aldrich, USA), followed by affinity purified horseradish peroxidase (HRP)-conjugated antigoat antibodies (Calbiochem, USA).…”

“…It codes for a 45 kDa protein containing the "KDEL"-ER retention sequence and presents more than 50% identity to the human counterpart (Aguillon et al 1995(Aguillon et al , 2000a. Most important, natural and experimental infections expose TcCRT to B cells, since the production of anti-TcCRT IgG is readily detected in infected humans (Aguillon et al 1997;Marcelain et al 2000), mice (Aguillon et al 2000b;Ribeiro et al 2009) and rabbits (Aguilar et al 2005;Ribeiro et al 2009). These antibodies, upon binding to cell surface TcCRT, should promote additional C1q capture by the parasite, a reflection of the capacity of the collagenous and globular C1q domains to simultaneously bind TcCRT and the Fc portions of antigen aggregated Igs, respectively (Ferreira et al 2004;Painter 1984).…”

Trypanosoma cruzi Calreticulin Topographical Variations in Parasites Infecting Murine Macrophages

“…A rabbit polyclonal antiserum was generated against rTcCRT. 6,21 Previous to the immunization, a preimmune serum was obtained. By serial dilutions, the immune serum had a titer of 10…”