Western Blot Analysis of Sera from Leishmania major-Infected BALB/c and C۵۷BL/۶ Mice

In Leishmania (L.) major-infected BALB/c mice Th۲-type cells results in disease progression, whereas C۵۷BL/۶-infected mice mount a Th۱-type response, which leads to control of the infection. Th۲ response correlates with IgG۱ whereas, Th۱ response supports switching to IgG۲a subclasses. Since IgG isotype-dominated response depends on different CD۴ + T cell subsets, we studied the antigenic profile of L. major promastigotes that induce IgG۱, IgG۲a and IgG۲b isotypes in BALB/c and C۵۷BL/۶ mice to provide insight into Th۱ and Th۲ inducing antigens. Humoral immune responses were studied in sera from L. major-infected BALB/c and C۵۷BL/۶ mice by ELISA and immunoblot analysis. These techniques were used to detect IgG, IgG۱, IgG۲a, and IgG۲b antibodies against crude promastigote antigens. BALB/c mice showed higher IgG, IgG۱ and IgG۲a antibody levels compared to C۵۷BL/۶ mice (p<۰.۰۱). The IgG۲a/IgG۱ ratio was higher in C۵۷BL/۶ mice. There was no significant difference in IgG۲b level between susceptible (BALB/c) and resistant (C۵۷BL/۶) mice. Western blot analysis revealed that ۲۷, ۳۲, ۱۱۷, ۱۲۵ and ۱۵۳ kDa bands reacted only with IgG۱ from both mice strains. The ۴۱, ۴۵, ۴۷, ۵۲ and ۵۵ kDa bands reacted only with IgG۲a from C۵۷BL/۶. A very similar pattern of immunostaining was observed for IgG۲a and IgG۲b in each mouse strain. The results from both mouse strains indicate that some antigens react exclusively with IgG۱, the antibody, which may be involved in generation of a predominant Th۲ response. The antigens that reacted strongly with IgG۲a in C۵۷BL/۶ are potential candidates for eliciting protective Th۱ response.

Keywords:

Leishmania major , Promastigote , Western blot analysis