T. solium and T. crassiceps: Characterization of the antigens and production of monoclonal antibodies

Author(s)

Noeli Maria Espindola

• Document type: Master's Dissertation
• Press: São Paulo.
• Institution: Universidade de São Paulo (USP). Conjunto das Químicas (IQ e FCF) (CQ/DBDCQ)
• Defense date: 1999-12-09

Examining board members

Adelaide Jose Vaz; Regina Ayr Florio da Cunha; Mirthes Ueda

• Advisor: Adelaide Jose Vaz

Abstract

The objective of this study was to perform the production of monoclonal antibodies (MAb) using BALB/c mice immunized with T. crassiceps (Tcra) antigen for identification of Taenia solium (Tso) antigens in human infection. Both antigens vesicular fluid of Tcra (VF-Tcra) and total of Tso (T-Tso) were submitted to analysis by means of SDS-PAGE: Tso antigen presented several peptides from 8 to 158kD, while VF-Tcra antigen was rich in <20kD peptides. Female BALB/c mice were divided into seven groups: GI-control; GII immunized with 20&#181;g of VF-Tcra; GIII and GV with 50&#181;g of VF-Tcra; GIV with ~50&#181;g de gel<30kD-Tcra; GVI with 50 &#181;g antigen eluate <30kD-Tcra, and GVII with 50&#181;g of T-Tso, in order to study antibody production kinetic. IgM, IgG and IgA antibodies were detected by means of ELISA-Tcra, starting on 15th day in serum samples from all groups of mice immunized with Tcra, except from GIV which presented no detectable IgM. On western blot-Tcra (blot-Tcra), these antibodies presented reactivity with <20kD Tcra peptides. Analysis of cross-reactivity showed that groups anti-Tcra antibodies presented anti-Tso cross-reactivity, with exception of GVI which presented no IgG antibodies; and GIV presented no IgM and IgG antibodies when analyzed on ELISA-Tso. On blot-Tso, anti-Tcra IgM antibodies in samples from GII and GV weakly recognized peptide of 30kD, and peptides 60, 43, 30 and 12kD were reactive in samples from GIV. Samples from all groups of mice, IgG anti-Tcra antibodies identified 12kD peptide of Tso. IgA antibodies in sera from GIII, GIV and GV identified 65, 50, 30 and 12kD peptides of Tso. Samples from GVII group immunized with T-Tso identified IgM antibodies on ELISA-Tso starting on 5th day, and IgG antibodies starting on 15th day with increasing levels after booster. This group presented high IgA levels in assayed samples. On blot-Tso, IgM antibodies identified peptides of 60, 43, 30, and 12kD, IgG antibodies reacted with 95, 75, 60, 67, 45, 40-35, 25, 18, and 12kD peptides, and IgA antibodies identified peptides of 45 , 40-35, 28-25 and 18kD. These antibodies presented cross-reactivity with Tcra antigen: peptides of 72, 35, 30, 24 and 14kD were identified by IgM antibodies, 95, 70, 50, 30, 18 and 14kD by IgG antibodies, and 70, 37, 35, 30, 18 and 14kD of Tcra by IgA antibodies. For spleen cells fusion BALB/c mice immunized with VF-Tcra antigen and mieloma cells P3X63-Ag8.653 were used. After cell fusion 36 IgM-clones and 117 IgG-clones were detected. These clones specifically reacted with Tcra antigen. It was also detected 40 IgM-clones and 10 IgG-clones which recognized Tso antigen. By immunoblotting, two clones identified 8-14 and 18kD peptides of VF-Tcra. (AU)