title: A novel method to assay proteins in blood plasma after intravenous injection of plasmid DNA.
abstract: Gene therapy is expected to lead to new and useful methods to treat diseases. The development of assays to quantitate gene-therapy-derived proteins circulating in blood will be essential to investigate the effects and side effects of the introduced proteins. The purpose of this study is to evaluate whether a protein circulating at trace concentrations in blood can be measured by tagging a peptide corresponding to glucagon residues 19-29 onto its C-terminal end. We constructed plasmids encoding chimeric proteins and transferred them into rats by hydrodynamics-based delivery. When plasmids encoding human IL8-glucagon 19-29 chimeric protein were injected into rats to evaluate the accuracy of this method, there was a high correlation between chimeric proteins measured by an enzyme-linked immunosorbent assay for human IL8 and one by a radioimmunoassay for glucagon. Furthermore, when plasmids coding rat IFN gamma receptor IgG-Fc glucagon 19-29 chimeric protein were injected to evaluate the time course of chimeric proteins in blood plasma, we could calculate the concentrations in blood from 10 microl plasma samples using glucagon 19-29 tag as follows: 2815+/-2318 ng/ml after 4 hours (mean+/-s.D.), 6061+/-2789 ng/ml after 8 hours, 5752+/-2270 ng/ml after 12 hours, 2870+/-1062 ng/ml after one day, 1440+/-334 ng/ml after three days, 1120+/-433 ng/ml after seven days, and 281+/-162 ng/ml after 16 days. Blood sugar levels which might have been increased by glucagon did not increase even at peak chime- ric protein concentrations. These results demonstrate a useful and convenient method to assay gene therapy products circulating in blood using a glucagon 19-29 tagging vector.
authors.affiliation: Division of Cardiology, Niigata University Graduate School of Medical and Dental Science, Niigata 951-8510, Japan. hanawa@med.niigata-u.ac.jp
authors.forename: Haruo
authors.initials: H
authors.lastname: Hanawa
authors.forename: Ritsuo
authors.initials: R
authors.lastname: Watanabe
authors.forename: Manabu
authors.initials: M
authors.lastname: Hayashi
authors.forename: Tsuyoshi
authors.initials: T
authors.lastname: Yoshida
authors.forename: Satoru
authors.initials: S
authors.lastname: Abe
authors.forename: Satoru
authors.initials: S
authors.lastname: Komura
authors.forename: Hui
authors.initials: H
authors.lastname: Liu
authors.forename: Raafat
authors.initials: R
authors.lastname: Elnaggar
authors.forename: He
authors.initials: H
authors.lastname: Chang
authors.forename: Yuji
authors.initials: Y
authors.lastname: Okura
authors.forename: Kiminori
authors.initials: K
authors.lastname: Kato
authors.forename: Makoto
authors.initials: M
authors.lastname: Kodama
authors.forename: Hiroki
authors.initials: H
authors.lastname: Maruyama
authors.forename: Junichi
authors.initials: J
authors.lastname: Miyazaki
authors.forename: Yoshifusa
authors.initials: Y
authors.lastname: Aizawa
mesh_terms: D000818:Animals; D001786:Blood Glucose / Q000378:metabolism; D018014:Gene Transfer Techniques; D015316:Genetic Therapy / Q000379:methods; D005934:Glucagon / Q000235:genetics / Q000378:metabolism; D006801:Humans; D007118:Immunoassay / Q000379:methods*; D016209:Interleukin-8 / Q000235:genetics / Q000378:metabolism; D008297:Male; D010446:Peptide Fragments / Q000235:genetics / Q000378:metabolism; D010957:Plasmids / Q000235:genetics*; D011506:Proteins / Q000737:chemistry* / Q000378:metabolism*; D051381:Rats; D017471:Receptors, Interferon / Q000235:genetics / Q000378:metabolism; D011993:Recombinant Fusion Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D000096743:Interferon gamma Receptor
chemical_list: D001786:Blood Glucose; D005934:Glucagon; D016209:Interleukin-8; D010446:Peptide Fragments; D011506:Proteins; D017471:Receptors, Interferon; D011993:Recombinant Fusion Proteins; D000096743:Interferon gamma Receptor; C039265:glucagon (19-29)