Immunoassays to study prevalence of antibody against GB virus C in blood donors.

Immunoassays were developed to determine the seroprevalence of antibody against human GB virus C (GBV-C). The antigenic target in each assay was a 44.6-kDa glycosylated protein representing the first 315 amino acids encoded by the GBV-C E2 gene. Sera or plasma were assayed for E2 antibody using an anti-human EIA format in which antigen-coated polystyrene beads were reacted with sample, and bound antibody was detected by addition of enzyme labelled goat anti-human IgG. The presence of anti-E2 antibody was confirmed using a sandwich EIA format in which samples were reacted with antigen coated polystyrene beads, followed by addition of solution phase biotinylated antigen. Detection of antibody captured biotinylated E2 was accomplished by addition of enzyme-conjugated anti-biotin antibody. Antibody against the E2 antigen was detected in 7.4 and 7.8% of 500 sera and 500 plasma, respectively, from US volunteers donating to a Wisconsin blood center, and in approximately 10.7% of hepatitis and retrovirus marker-negative volunteer blood donors from a Missouri blood center. The rate in 1018 sera from US commercial donors at multiple US blood centers was 36.7%. These results indicated a relatively high prevalence of GBV-C exposure in US volunteer donors, and particularly in commercial donors. The clinical implication of the high exposure rate is unclear. These immunoassays are being combined with nucleic acid detection to assess prevalence of GBV-C world wide and to determine if GBV-C plays a role as an etiologic agent.

Purification and characterization of the carrier protein for juvenile hormone from the hemolymph of the tobacco hornworm Manduca sexta Johannson (Lepidoptera: Sphingidae).

The larval hemolymph of the tobacco hornworm, Manduca sexta, contains a carrier protein that binds specifically and with high affinity the juvenile hormone, an important regulator of insect development. This protein serves to transport the hormone and to protect it from the action of degradative enzymes during early larval stages. Using hemolymph from the last larval stage, we have isolated a pure carrier protein using acetone precipitation, gel filtration, ion exchange chromatography, and preparative isoelectric focusing. Gel filtration, polyacrylamide gel electrophoresis in sodium dodecyl sulfate, and equilibrium ultracentrifugation established that the carrier protein is a single chain polypeptide of approximately 28,000 daltons. The amino acid composition is unexceptional, and no evidence for hexosamine has been obtained. An ion exchange filter disc assay method was used to determine the formation of the complex between the carrier protein and isotopically labeled juvenile hormone. With this technique it was shown that each carrier protein binds one hormone molecule with a dissociation constant of 4.4 +/- 0.2 X 10(-7) M at 0 degrees.