A Novel Method to Assay Proteins in Blood Plasma after Intravenous Injection of Plasmid DNA

A Novel Method to Assay Proteins in Blood Plasma after Intravenous Injection of Plasmid DNA

HARUO HANAWA, RITSUO WATANABE, MANABU HAYASHI, TSUYOSHI YOSHIDA, SATORU ABE, SATORU KOMURA, HUI LIU, RAAFAT ELNAGGAR, HE CHANG, YUJI OKURA, KIMINORI KATO, MAKOTO KODAMA, HIROKI MARUYAMA,¹ JUNICHI MIYAZAKI² and YOSHIFUSA AIZAWA

Division of Cardiology, ¹Division of Clinical Nephrology and Rheumatology, Niigata University Graduate School of Medical and Dental Science, Niigata 951-8510, and
²Divison of Stem Cell Regulation, Osaka University Graduate School of Medical Science, Osaka 565-0872

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 ml 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 chimeric 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.

keywords —— gene therapy; glucagon; pCAGGS; radioimmunoassay

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Tohoku J. Exp. Med., 2004, 202, 155-161

Address for reprints: Haruo Hanawa, Division of Cardiology, Niigata University Graduate School of Medical and Dental Science, 1-754 Asahimachi, Niigata 951-8510, Japan.

e-mail: hanawa@med.niigata-u.ac.jp