Canine IgE monoclonal antibody specific for a filarial antigen: production by a canine x murine heterohybridoma using B cells from a clinically affected lymph node

Abstract

Canine popliteal lymph node cells taken at the onset of clinical disease from a rear limb infected with the filarial nematode Brugia pahangi were fused with mouse myeloma cell line P3X63.Ag8.653 cells. Of the several canine immunoglobulin-producing clones from this fusion, one was found to produce canine IgE specific for a filarial nematode antigen. The cell line has undergone limiting dilution cloning six times over the past 3 years and continues to produce monoclonal antibody of the IgE subclass at a rate of greater than 3 mg/l. Sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE) of the cell culture supernatant protein that bound to protein A beads, showed bands at molecular weights (MW) of approximately 75,000 and 25,000 that were characteristic of epsilon and kappa or lambda chains, respectively. A mouse monoclonal antibody specific for canine IgE bound the 75,000 MW band, as demonstrated by Western blot. Western blots of aqueous extracts of adult filarial nematodes demonstrated binding of the canine IgE monoclonal antibody to a single 35,000 MW peptide from B. pahangi but not Dirofilaria immitis; immunochemistry using frozen sections of adult worms, microfilariae and fourth stage larvae revealed focal binding of the monoclonal IgE to worm tissue adjacent to dorsal and ventral cords of only Brugia adults.