Preparation of a conjugate of 2',5'-triadenylate 5'-triphosphate and biotinylated firefly luciferase and its use for sensitive bioluminescent enzyme immunoassay.
A bioluminescent enzyme immunoassay (BLEIA) for 2',5'-oligoadenylate 5'-triphosphate (pppA2'p5'A2'Ap5'A, 2-5A) was developed using a conjugate of 2-5A and firefly luciferase as a probe. The conjugate was prepared from a 2-5A derivative bearing a thiol group at the 2'(or 3') end, and streptavidin (SA) bearing maleimide via a linker-arm and biotinylated luciferase. The thiol group of the 2-5A derivative was relatively stable under aerobic conditions and remained 100% and 56% intact at 25 degrees C after 1 and 96 h, respectively, without dimerization by aerobic oxidation. The thiol-modified 2-5A was coupled with the SA-maleimide conjugate, followed by complex formation with biotinylated firefly luciferase. BLEIA using this conjugate allowed for the direct analysis of 2-5A in a range of 5-500 fmol (0.1-10 nM in a 50 microL sample) and in a reaction time of 15 min. The measurement of microquantities of 2-5 A from various sources is easy to perform by this BLEIA, because no radioisotope labeled 2-5A was required as a probe.
Seto
Yoshiaki
Y
Department of Chemistry, Faculty of Engineering, Gunma University, 1-5-1 Tenjin-cho, Kiryu, Gunma, 376-8515, Japan. yoshiakiseto@eiken.co.jp
Abe
Katsushi
K
Itoh
Masao
M
Sawai
Hiroaki
H
D002851:Chromatography, High Pressure Liquid; D002853:Chromatography, Liquid; D007124:Immunoenzyme Techniques / Q000379:methods*; D008156:Luciferases / Q000032:analysis / Q000737:chemistry* / Q000378:metabolism*; D008163:Luminescent Measurements; D015394:Molecular Structure; D009843:Oligoribonucleotides / Q000737:chemistry*; D011863:Radioimmunoassay; D012680:Sensitivity and Specificity; D013054:Spectrophotometry, Atomic; D013997:Time Factors
D009843:Oligoribonucleotides; D008156:Luciferases