Development and partial characterization of monoclonal antibodies to the hemolymph juvenile hormone binding protein of Manduca sexta

Evidences for a stage-specific juvenile hormone binding protein in the hemolymph of the silkworm,Bombyx mori L.: Identification and characterization by photoaffinity labeling and immunological analyses

“…These results indicated that both JH binding proteins have the same affinities for JH I. In another case, Goodman et al (1990) observed multiple forms of JHBP in the hemolymph of Manduca sexta using two-dimensional Western blot and suggested that these cross-reacting proteins were either precursors or degradation products of native JHBP. In the recent study, Park and Goodman (1993) identified multiple electromorphs upon melanization of the hemolymph.…”

Effects of stress on the hemolymph juvenile hormone binding protein titers of Manduca sexta

“…Serial dilutions of hemolymph (to yield 8, 16, 24 nl of hemolymph per well) were plated on a 96 well assay plate (76-381-04; Linbro/Titertek ICN Biomedical, Aurora, OH) in duplicate. Plates were blocked and then incubated with an anti-hJHBP monoclonal antibody (737A5C10, Ascites 2; Goodman et al, 1990) diluted 1:10,000, followed by incubation with secondary antibody, goat anti-mouse IgG conjugated to horseradish peroxidase (GAM-HRP; KPL Laboratories, Gaithersburg, MD) diluted 1:2000. The samples were then treated with HRP substrate (o-phenylenediamine, Sigma Chemical), developed, and read on a microtiter plate reader.…”

“…Hemolymph (15 ml) proteins were separated on an 11% SDS-polyacrylamide gel (Laemmli, 1970). The proteins were then transferred to nitrocellulose paper (45 m; Osmonics, Westborough, MA) and developed using an anti-hJHBP monoclonal antibody (737A5C10; Ascites 2; 1:1000 dilution) and a GAM-HRP secondary antibody (1:2000 dilution; KPL Laboratories) as described by Goodman et al (1990). The blot was stained with 3,3 0 -diaminobenzidine tetrahydrochloride (Sigma Chemical).…”

Juvenile Hormone Binding Protein Titers During Adult Metamorphosis in Manduca sexta

“…Nitrocellulose sheets were treated (blocked) with a l% casein solution dissolved in buffer 1 (15 nM Na2HPO4, 5 mM NaH2PO4, 150 mM NaCl，pH 7.7) for 20 min. After blocking, the nitrocellulose sheets were then incubated for 2 h with ascites fluid(monoclonal antibody 6, Goodman et al, 1990) diluted 1 : 1000 in 0.5% casein solution dissolved in buffer 1. The nitrocellulose sheets were washed twice 10 min in buffer 2 (buffer 1 containing 0.05% Tween 20), then incubated 2 h with peroxidase-labeled, affinitypurified goat antimouse IgG, heavy and light chain (Kirkegaard and Perry Laboratories) diluted 1 : 2000 in 0.5% casein solution dissolved in buffer l.…”

“…JHBP titers were determined by enzyme immunoassay (EIA) as described by Goodman et al (1990). Radial immunodiffusion (RID) analyses of both hemolymph and egg insecticyanin were performed as described by Trost and Goodman (1986).…”