Preparation of a Conjugate of 2‘,5‘-Triadenylate 5‘-Triphosphate and Biotinylated Firefly Luciferase and Its Use for Sensitive Bioluminescent Enzyme Immunoassay

• Published in: Bioconjugate chemistry Vol. 13; no. 2; pp. 303 - 308
• Main Authors: Seto, Yoshiaki, Abe, Katsushi, Itoh, Masao, Sawai, Hiroaki
• Format: Journal Article
• Language: English
• Published: United States American Chemical Society 01.03.2002
• Subjects: Chromatography, High Pressure Liquid; Chromatography, Liquid; Immunoenzyme Techniques - methods; Luciferases - analysis; Luciferases - chemistry; Luciferases - metabolism; Luminescent Measurements; Molecular Structure; Oligoribonucleotides - chemistry; Radioimmunoassay; Sensitivity and Specificity; Spectrophotometry, Atomic; Time Factors
• ISSN: 1043-1802; 1520-4812
• DOI: 10.1021/bc015554l

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 °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 μL 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.