“…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.…”