A FRET Assay for Celiac Disease

• Published in: Journal of experimental & biomedical sciences Vol. 22; no. 4; pp. 160 - 166
• Main Authors: Lee, Sae A, Cho, Chul Min, Jang, Il Ho, Kang, Jung Sook
• Format: Journal Article
• Language: Korean
• Published: 2016
• Subjects: Celiac disease; Anti-tTG antibodies; Tissue transglutaminase; Fluorescence resonance energy transfer
• ISSN: 1738-3226; 2288-7415

To provide a basis for a homogeneous fluorescence resonance energy transfer (FRET) immunoassay for celiac disease, we carried out a FRET experiment using guinea pig tissue transglutaminase (tTG) and antibodies to tTG (anti-tTG) purified from rat serum. Fluorescein was utilized as the probe, and a nonfluorescent dye, QSY 7 served as the quencher. We labeled anti-tTG and tTG with fluorescein isothiocyanate and QSY 7 succinimidyl ester, respectively. Fluorescein-labeled anti-tTG was the donor, and QSY 7-labeled tTG was the acceptor of the FRET experiment. When we titrated fluorescein-labeled anti-tTG with QSY 7-labeled tTG, we observed a large decrease in the steady-state fluorescence intensity, which was due to strong FRET from fluorescein-labeled anti-tTG to QSY 7-labeled tTG. Using time-resolved fluorescence spectroscopy, we could also observe a decrease in the fluorescence lifetime, which confirms the steady-state data. We expect that these results might be useful in the development of a novel fluorescence immunoassay for an easy screening and follow-up of celiac patients.