CHARACTERIZATION AND BIOLOGICAL-ACTIVITIES OF ANTI-BRUGIA PAHANGI TUBULIN MONOCLONAL-ANTIBODIES

Three monoclonal antibodies (mAb) specific to beta-tubulin were used t o investigate the heterogeneity of tubulins from nematodes and mammals . Western blot analysis of one-dimensional SDS-PAGE showed that anti-B rugia pahangi tubulin mAb 1B6 and P3D react with epitope(s) specific t o nematode beta-tubulin and recognize tubulin from adults and microfil ariae 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 r ecognized one isoform of beta-tubulin and mAb P3D recognized two beta- tubulin isoforms. Limited proteolysis showed that mAb 1B6 reacted with the amino-terminal fragments of beta-tubulin. In contrast, mAb P3D re cognized the carboxy-terminal fragments of beta-tubulin. In ELISA, mAb P3D reacted with an 18 amino acid peptide corresponding to residues 4 30-448 of B. pahangi beta-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 reveal ed the presence of beta-tubulin isoforms in the cuticle, hypodermal la yer and somatic muscle blocks of B. pahangi. Under in vitro conditions , mAb P3D caused 80% reduction in worm viability, during exposure over 48 h.