A novel method to assay proteins in blood plasma after intravenous injection of plasmid DNA.
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.
Hanawa
Haruo
H
Division of Cardiology, Niigata University Graduate School of Medical and Dental Science, Niigata 951-8510, Japan. hanawa@med.niigata-u.ac.jp
Watanabe
Ritsuo
R
Hayashi
Manabu
M
Yoshida
Tsuyoshi
T
Abe
Satoru
S
Komura
Satoru
S
Liu
Hui
H
Elnaggar
Raafat
R
Chang
He
H
Okura
Yuji
Y
Kato
Kiminori
K
Kodama
Makoto
M
Maruyama
Hiroki
H
Miyazaki
Junichi
J
Aizawa
Yoshifusa
Y
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
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)