Characterization and biological activities of anti- Brugia pahangi tubulin monoclonal antibodies

Abstract

Three monoclonal antibodies (mAb) specific to β-tubulin were used to investigate the heterogeneity of tubulins from nematodes and mammals. Western blot analysis of one-dimensional SDS-PAGE showed that anti- Brugia pahangi tubulin mAb 1B6 and P3D react with epitope(s) specific to nematode β-tubulin and recognize tubulin from adults and microfilariae of B. pahangi, adult B. malayi and Dirofilaria immitis, eggs of Haemonchus contortus and adult Ascaris suum. However, the same mAb did not recognize tubulin from trophozoites of Giardia lamblia, pig brain or 3T3 mouse fibroblast cells. In two-dimensional SDS-PAGE, mAb 1B6 recognized one isoform of β-tubulin and mAb P3D recognized two β-tubulin isoforms. Limited proteolysis showed that mAb l B6 reacted with the amino-terminal fragments of β-tubulin. In contrast, mAb P3D recognized the carboxy-terminal fragments of β-tubulin. In ELISA, mAb P3D reacted with an 18 amino acid peptide corresponding to residues 430–448 of B. pahangi β-tubulin. These observations confirm that the epitope of mAb P3D is located on the extreme carboxy-terminal region. Immunogold labelling of adult B. pahangi sections with mAb P3D revealed the presence of β-tubulin isoforms in the cuticle, hypodermal layer and somatic muscle blocks of B. pahangi. Under in vitro conditions, mAb P3D caused 80% reduction in worm viability, during exposure over 48 h.