Elucidation and partial purification of the inducible humoral factor in the American cockroach responsible for selective immune binding activity

The goal of this research was to elucidate and purify specific antigen (Ag) binding proteins in immune hemolymph from the American cockroach, Periplaneta americana. Early results indicated that non-reducing polyacrylamide gel electrophoresis (NR-PAGE) bands 2 and 4, and their putative 102 kDa subunit, were consistently enhanced after immunization with several unrelated protein Ags. Time course studies implied that these bands may be in vivo Ag-binding proteins since they were selectively absorbed out of the hemolymph after immunization, followed by a strong enhancement several days later. Rabbit affinity chromatography and precipitation experiments utilizing whole unfractionated hemolymph demonstrated that both NR-PAGE bands 2 and 4 were in vitro Ag-binding molecules. Partial purification of these bands by CM Affi-Gel Blue ion exchange chromatography or non-reducing preparative electrophoresis demonstrated that fractionated bands 2 and 4 were also capable of binding Ag in an in vitro Western Blot Binding assay. These binding studies utilizing whole hemolymph or partially fractionated hemolymph proteins demonstrated that both bands had observable Ag-binding activity. However, it was observed that NR-PAGE band 4 appeared to be more selective in the uptake of Ag. Passive transfer experiments further implied that these bands were involved in mediating the roach's in vivo humoral immune response, since in vivo protection was conferred to naive recipients receiving isolated NR-PAGE bands 2 and 4. In addition, SDS-PAGE analysis of the native binding proteins indicated that binding activity was strongly associated with isolated protein species that contained the 102 kDa subunit previously found to be enhanced in immune samples. The results from these studies indicates that the American cockroach generates an unique invertebrate, Ag-binding molecule in response to immunization with soluble proteins. Further purification and characterization of this inducible defense protein will expand our knowledge of the diversity and evolution of adaptive immunity.