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1) Polycarboxylates
DTPA anhydrate (A) (2) and N1(p-isothiocyanatobenzyl)-diethylenetriamine-N1,N2,N3,N3-tetraacetic acid (B) --- The polycarboxylate-type chelates of Eu normally emit little fluorescence. However, after immuno-reactions, Eu is dissociated from the labeled compound using an enhancement solution (trioctylphosphine oxide, 2-naphthoyltrifluoroacetone, Triton X-100) to form a new and highly fluorescent chelate (See C). This technology is referred to as dissociation enhanced lanthanide fluoroimmunoassay (DELFIA).
2) beta-Diketons
BHHCT (D) (3)------beta-Diketon-type chelates emit high fluorescence, and BHHCT can react directly with proteins.
3) Aryl amines
Quantum-dye (QD) (E)-------Fluorescence of Eu in QD is strong enough for TRF, but it can be further amplified by addition of the enhancement solution.
2. Applications in glycobiology
1) Assay of carbohydrate-related enzymes
There are some reports on glycosyltransferase assays using TR-FIA (4). Activities of glycosyltransferases (5), endoglycosidase and glycopeptidase (6) can also be measured using Eu-labeled lectins.
2) Interactions between carbohydrates and proteins
The use of Eu-labeled glycoproteins and neoglycoproteins in TRF makes it possible to analyze binding specificity of lectins. Lee et al. (5) reported that binding and inhibition assays of carbohydrate-recognition domain of rat serum monnose-binding protein coated on microtiter plates using QD-labeled neoglycoprotein, QD-Man-BSA, yielded results comparable to those obtained using 125I-Man-BSA. TRF is applicable to the assay of hepatic Gal/GalNAc receptor using QD-Gal-BSA. Recently, Oda et al. (7) determined binding specificity of Crocus sativus lectin (CSL) with solid phase method with Eu-labeled (Man)3GlcNAc-glycopepide as well as flow injection, fluorescence polarization and SPR.
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References |
(1) |
Hemmlila, I, Dakubu, S, Mukkala, V.-M, Siitari, H, Lovgren, T, Anal. Biochem. 137, 335-343,1984
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(2) |
Kawasaki, N, Lee, YC, Anal. Biochem. 250, 260-262, 1997 |
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(3) |
Yuan, J, Matsumoto, K, Kimura, H, Anal. Chem. 70, 596-601,1998 |
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(4) |
Taki, T, Nishiwaki, S, Handa, N, Hattori, N, Handa, S, Anal. Biochem. 219, 104-108 (1994)
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(5) |
Lee, YC, Kawasaki, N, Lee, RT, Suzuki, N, Glycobiology 8, 849-856 (1998)
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(6) |
Deras, I L, Sano, M, Kato, I, Lee, YC, Anal. Biochem. In press. |
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(7) |
Oda, Y, Nakayama, K, Kinoshita, M, Kawasaki, N, Hayakawa, T, Kakehi, K, Abdul-Rahman, B, Lee, YC, Glycobiology(abstract), 9, 1132 (1999)
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