Synthesis of a terbium fluorescent chelate and its application to time-resolved fluoroimmunoassay

Article abstract of DOI:10.1021/ac0013305

A new nonadentate ligand, N, N, N^l, N¹-[2,6-bis(3′aminomethyl-1′-pyrazolyl)-4- phenylpyridine]tetrakis(acetic acid) (BPTA) for a Tb³⁺ fluorescent complex was synthesized. The Tb³⁺ complex is strongly fluorescent, having a large fluorescence quantum yield of 1.00 and very long fluorescence lifetime of 2.681 ms in 0.05 M borate buffer of pH 9.1. Streptavidin (SA) was labeled with BPTA by using its succinimidyl monoester, and the BPTA-Tb³⁺-labeled SA was used in sandwich-type time-resolved fluoroimmunoassay (TR-FIA) of α-fetoprotein (AFP) and carcinoembryonic antigen (CEA) in human sera. The Tb³⁺-labeled SA was also used in competitive-type TR-FIA of bensulfuron-methyl (BSM) in water. The detection limits of these assays are 42 pg/mL for AFP, 70 pg/mL for CEA, and 0.4 ng/mL for BSM. In addition, a new simultaneous measurement method for AFP and CEA in a human serum sample was developed by using 4,4′-bis(l″,1″,1″,2″,2″,3″,3″ -heptafluoro-4″,6″-hexanedion-6″-yl)chlorosulfo-o-terphenyl (BHHCT)-Eu³⁺-labeled anti-AFP antibody, biotinylated anti-CEA antibody, and BPTA-Tb³⁺-labeled SA. The concentrations of AFP and CEA in 39 human serum samples were determined, and the results were compared with those of the independently determined AFP and CEA by TR-FIA with a single-label method. A good correlation was obtained with the correlation coefficients of 0.991 for AFP and 0.994 for CEA.

Full text of DOI:10.1021/ac0013305

Anal. Chem. 2001, 73, 1869-1876
Synthesis of a Terbium Fluorescent Chelate and
Its Application to Time-Resolved
Fluoroimmunoassay
Jingli Yuan,† Guilan Wang,† Keisuke Majima,‡ and Kazuko Matsumoto*,‡
Department of Chemistry, Waseda University, Japan Science and Technology Corporation, Shinjuku-ku,
Tokyo 169-8555, Japan, and Department of Analytical Chemistry, Dalian Institute of Chemical Physics,
Chinese Academy of Sciences, Dalian, China
A new nonadentate ligand, N, N, N1 , N1 -[2 ,6 -bis(3 ′-
most important advantage of the method is that it allows easy
distinction of the specific fluorescence signal of the long-lived
fluorescent lanthanide probe from the short-lived background
fluorescence present in most biological samples and also obviates
the problems associated with the scattering light of the optical
components. The representative lanthanide fluorescent chelates
used in the current TR-FIA systems include europium, terbium,
and samarium fluorescent chelates, such as the Eu3+-â-diketonate
(2-naphthoyltrifluoroacetonate)-trioctylphosphine oxide ternary
fluorescent complex in the “DELFIA” system,3 Eu3+ chelate with
4,7-bis(chlorosulfodiphenyl)-1,10-phenanthroline-2,9-dicarboxylic
acid in the“FIAgen” system,5 Eu3+-tris(bipyridine) cryptate in the
“time-resolved amplified cryptate emission (TRACE)” system,9,10
and 5-fluorosalicylate-Tb3+-EDTA in the “enzyme-amplified time-
resolved fluoroimmunoassay” system.11
aminomethyl-1 ′-pyrazolyl)-4 -phenylpyridine]tetrakis(ace-
tic acid) (BP TA) for a Tb3 + fluorescent complex was
synthesized. The Tb3 + complex is strongly fluorescent,
having a large fluorescence quantum yield of 1 .0 0 and
very long fluorescence lifetime of 2 .6 8 1 ms in 0 .0 5 M
borate buffer of pH 9.1. Streptavidin (SA) was labeled with
BP TA by using its succinimidyl monoester, and the
BP TA-Tb3 +-labeled SA was used in sandwich-type time-
resolved fluoroimmunoassay (TR-FIA) of r-fetoprotein
(AFP ) and carcinoembryonic antigen (CEA) in human
sera. The Tb3 +-labeled SA was also used in competitive-
type TR-FIA of bensulfuron-methyl (BSM) in water. The
detection limits of these assays are 4 2 pg/ mL for AFP ,
7 0 pg/ mL for CEA, and 0 .4 ng/ mL for BSM. In addition,
a new simultaneous measurement method for AFP and
CEA in a human serum sample was developed by using
4 ,4 ′-bis(1 ′′,1 ′′,1 ′′,2 ′′,2 ′′,3 ′′,3 ′′ -heptafluoro-4 ′′,6 ′′-hex-
Recently, TR-FIA has been directed to multiple labeling for
use in simultaneous detection of multiple biomolecules, and Eu3+
,
Sm3+, Tb3+, and Dy3+ chelates were used as fluorescent labels
having long-lived fluorescence at different wavelengths.7 Dual-label
TR-FIA using Eu3+ and Sm3+ chelates has been studied for many
multiple-component simultaneous measurements.12-17 Although
the dual-label TR-FIA has been used successfully, the Sm3+
chelates need some improvement; i.e., their fluorescence is weaker
and the fluorescence lifetime is shorter compared to those of Eu3+
chelates. Since the fluorescence quantum yields and lifetimes of
anedion-6 ′′-yl)chlorosulfo-o-terphenyl (BHHCT)-Eu3 +
-
labeled anti-AFP antibody, biotinylated anti-CEA antibody,
and BP TA-Tb3 +-labeled SA. The concentrations of AFP
and CEA in 3 9 human serum samples were determined,
and the results were compared with those of the inde-
pendently determined AFP and CEA by TR-FIA with a
single-label method. A good correlation was obtained with
the correlation coefficients of 0 .9 9 1 for AFP and 0 .9 9 4
for CEA.
Sm3+ chelates are smaller than those of Eu3+ chelates, the Eu3+
-
Sm3+ pair is suitable only for the simultaneous measurement of a
low-concentration component and a high-concentration component
in a sample. Recently we developed a new TR-FIA method for
Lanthanide fluorescent chelates have been successfully devel-
oped as fluorescence probes for highly sensitive time-resolved
fluoroimmunoassay (TR-FIA) and DNA hybridization assay with
microsecond time-resolved fluorometric measurement.1-8 The
(8) Elbanowski, M.; Makowska, B. J. Photochem. Photobiol. A: Chem. 1 9 9 6 ,
99, 85-92.
(9) Mathis, G. Clin. Chem. 1 9 9 5 , 41, 1391-1397.
(10) Mathis, G. J. Clin. Ligand Assay 1 9 9 7 , 20, 141-147.
(11) Christopoulos, T. K.; Diamandis, E. P. Anal. Chem. 1 9 9 2 , 64, 342-346.
(12) Hemmila¨, I.; Holttinen, S.; Pettersson, K.; Lo¨ vgren, T. Clin. Chem. 1 9 8 7 ,
32, 2281-2283.
* To whom correspondence should be addressed. (tel) +81-3-5286-3108; (fax)
+81-3-5273-3489; (e-mail) kmatsu@mn.waseda.ac.jp.
† Dalian Institute of Chemical Physics.
(13) Vuori, J.; Rasi, S.; Takala, T.; Vaananen, K. Clin. Chem. 1 9 9 1 , 37, 2087-
2092.
‡ Waseda University.
(1) Hemmila¨, I. Clin. Chem. 1 9 8 5 , 31, 359-370.
(14) Pettersson, K.; Alfthan, H.; Stenman, U.; Turpeinen, U.; Suonpa¨a¨, M.;
So¨ derholm, J.; Larsen, S. O.; Nørgaard-Pedersen, B. Clin. Chem. 1 9 9 3 , 39,
2084-2089.
(2) Soini, E.; Lo¨ vgren, T. CRC Crit. Rev. Anal. Chem. 1 9 8 7 , 18, 105-154.
(3) Diamandis, E. P. Clin. Biochem. 1 9 8 8 , 21, 139-150.
(4) Hemmila¨, I. Scand. J. Clin. Lab. Invest. 1 9 8 8 , 48, 389-400.
(5) Diamandis, E. P.; Christopoulos, T. K. Anal. Chem. 1990, 62, 1149A-1157A.
(6) Dickson, E. F. G.; Pollak, A.; Diamandis, E. P. Pharmacol. Ther. 1 9 9 5 , 66,
207-235.
(15) Qin, Q.; Christiansen, M.; Lo¨ vgren, T.; Nørgaard-Pedersen, B.; Pettersson,
K. J. Immunol. Methods 1 9 9 7 , 205, 169-175.
(16) Barnard, G.; Kohen, F. Clin. Chem. 1 9 9 8 , 44, 1520-1528.
(17) Ito, K.; Oda, M.; Tsuji, A.; Maeda, M. J. Pharm. Biomed. Anal. 1 9 9 9 , 20,
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10.1021/ac0013305 CCC: $20.00 © 2001 American Chemical Society
Published on Web 03/09/2001
Analytical Chemistry, Vol. 73, No. 8, April 15, 2001 1869

the simultaneous measurement of two low-concentration compo-
nents, R-fetoprotein (AFP) and carcinoembryonic antigen (CEA)
in human serum, by using the Eu3+ and Sm3+ fluorescence
chelates as labels,18 in which the fluorescence of the Sm3+ label
was amplified by multiple labeling using Sm-labeled streptavidin
bound to biotinylated protein.
Compared with the Sm3+ fluorescent chelates, some Tb3+
fluorescent chelates are reported to have higher fluorescence
quantum yields and very long lifetimes. Therefore, dual-label TR-
FIA by using Eu3+ and Tb3+ chelates would be more preferable.
Although Eu3+-Tb3+ dual-label TR-FIA is reported for the simul-
taneous measurement of free and total prostate-specific antigen
(PSA),19 the Eu3+-Tb3+ combination is not as widely used as the
Eu3+-Sm3+ dual-label.
samples were determined, and the results were compared with
those of the usual single-assay method. Good correlations were
obtained between the simultaneous and single assays with the
correlation coefficients of 0.991 for AFP and 0.994 for CEA.
EXPERIMENTAL SECTION
Synthesis of BP TA. The new ligand BPTA was synthesized
following the six-step reaction shown in Figure 1. The details of
the procedure are described in the following.
(i) Synthesis of 2 ,6 -Dibromo-4 -phenylpyridine (2 ). The
starting compound 4-amino-2, 6-dibromopyridine (1 ) was synthe-
sized from 2,6-dibromopyridine by using the literature meth-
ods.24,25 Anal. Calcd for C5H4Br2N2: C, 23.84; H, 1.60; N, 11.16.
1
Found: C, 24.00; H, 1.47; N, 11.02. H NMR (acetone-d6) δ 6.82
Recently, several Tb3+ fluorescent chelates having large
fluorescence quantum yields and long fluorescence lifetimes have
been synthesized.20-22 The amino-active forms of these chelates
have also been developed for protein labeling.23 These ligands
include polyacid derivatives of 2,6-bis(pyrazol-1-yl)pyridine, 2,6-
bis(pyrazol-1-yl)pyrazine, and 1,3-bis(pyridin-2-yl)pyrazole, whereas
polyacid derivatives of 2,2′:6′,2′′-terpyridine are not effective for
Tb3+ strong fluorescence. In the present work, a new ligand for
Tb3+, N,N,N1,N1-[2,6-bis(3′-aminomethyl-1′-pyrazolyl)-4-phenylpy-
ridine]tetrakis(acetic acid) (BPTA) was synthesized, and the
fluorescence properties of the Eu3+ and Tb3+ chelates were
examined as well as the application of the BPTA-Tb3+ as a
fluorescence label for TR-FIA. In the new ligand, a phenyl group
is introduced to the pyridine ring of the formerly prepared
N,N,N1,N1-[2,6-bis(3′-aminomethyl-1′-pyrazolyl)pyridine]tetra-
kis(acetic acid),22 to increase the molar absorption coefficient. An
additional usefulness of this phenyl group is that an amino-active
group can be introduced to the phenyl group for protein labeling.
The fluorescence property of the BPTA-Tb3+ complex shows that
both the molar absorption coefficient and the fluorescence
quantum yield are increased remarkably by introduction of the
phenyl group.
To evaluate the usefulness of BPTA-Tb3+ as a fluorescence
label for TR-FIA, human AFP in serum, CEA in serum, and
bensulfuron-methyl (BSM) in water were measured by sandwich-
type (AFP and CEA) and competitive-type (BSM) TR-FIA, by using
BPTA-Tb3+-labeled streptavidin (SA). The methods gave detec-
tion limits of 42 pg/ mL for AFP, 70 pg/ mL for CEA, and 0.4 ng/
mL for BSM. In addition, a new TR-FIA method for simultaneous
measurement of AFP and CEA in human serum was developed,
in which 4,4′-bis(1′′,1′′,1′′,2′′,2′′,3′′,3′′-heptafluoro-4′′,6′′-hexanedion-
6′′-yl)chlorosulfo-o-terphenyl (BHHCT)-Eu3+-labeled anti-AFP
antibody, biotinylated anti-CEA antibody, and BPTA-Tb3+-labeled
SA were used. Compared with the previous method using the Eu3+
and Sm3+ chelates as labels,18 the new method is simpler and more
sensitive. The AFP and CEA concentrations in 39 human serum
(s, 2H), 6.16 (b, 2H). To 180 mL of benzene containing 3.7 g (15
mmol) of 1 was added 30 mL of trifluoroacetic acid and 3.3 g (28
mmol) of isopentyl nitrite with stirring. After stirring for 20 min
at room temperature, the solution was refluxed for 2.5 h. To the
solution was added 50 mL of benzene, and the solution was
washed with 3 × 100 mL of water and 100 mL of 5% Na2CO3.
After the solution was dried with Na2SO4, the solvent was
evaporated. The residue was purified by silica gel column
chromatography using benzene as eluent and then was recrystal-
lized from methanol. Pale yellow crystals of 2 were obtained (2.3
g, 49.0% yield). Anal. Calcd for C11H7Br2N: C, 42.21; H, 2.25; N,
1
4.47. Found: C, 41.76; H, 2.11; N, 4.65. H NMR (acetone-d6) δ
7.95 (s, 2H), 7.88-7.84 (m, 2H), 7.58-7.53 (m, 3H).
(ii) Synthesis of 2,6-Bis(3 ′-methoxycarbonyl-1 ′-pyrazolyl)-
4 -phenylpyridine (3 ). Potassium (1.56 g, 40 mmol) was added
in small portions to a solution of 5.04 g (40 mmol) of 3-methoxy-
carbonylpyrazole in 120 mL of dry THF at 60-70 °C with stirring.
After the metal was dissolved, 3.13 g (10 mmol) of 2 was added,
and the mixture was refluxed for 1 week. The solvent was
evaporated, and the residue was extracted with 3 × 150 mL of
CH2Cl2. After CH2Cl2 was evaporated, the residue was purified
by silica gel column chromatography using CH2Cl2-CH3OH (98:
2, w/ w) as eluent and then was recrystallized from benzene. White
crystals of 3 were obtained (0.55 g, 13.6% yield). Anal. Calcd for
C21H17N5O4: C, 62.53; H, 4.25; N, 17.36. Found: C, 62.48; H, 4.01;
N, 17.28. 1H NMR (CDCl3) δ 8.65 (d, J, 2.64 Hz, 2H), 8.31 (s, 2H),
7.86-7.82 (m, 2H), 7.57-7.50 (m, 3H), 7.05 (d, J, 2.64 Hz, 2H),
4.00 (s, 6H).
(iii) Synthesis of 2 ,6 -Bis(3 ′-hydroxymethyl-1 ′-pyrazolyl)-
4 -phenylpyridine (4 ). To 250 mL of dry THF containing 500
mg LiAlH4 was added 1.0 g (2.48 mmol) of 3 . After stirring for
3.5 h at room temperature, 0.45 mL of water, 0.45 mL of 15%
NaOH, and 1.5 mL of water were added dropwise to the solution.
The solution was filtered to remove precipitate, and the solvent
was evaporated. The product was washed with CH3CN and then
dried. A white powder of 4 was obtained (0.66 g, 76.6% yield). 1H
NMR (DMSO-d6) δ 8.91 (d, J, 2.31 Hz, 2H), 7.96 (s, 2H), 7.91-
7.87 (m, 2H), 7.64-7.56 (m, 3H), 6.61 (d, J, 2.31 Hz, 2H), 5.31 (t,
J, 5.94 Hz, 2H), 4.57 (d, J, 5.94 Hz, 4H).
(18) Matsumoto, K.; Yuan, J.; Wang, G.; Kimura, H. Anal. Biochem. 1 9 9 9 , 276,
81-87.
(19) Merio¨ , L.; Pettersson, K.; Lo¨ vgren, T. Clin. Chem. 1 9 9 6 , 42, 1513-1517.
(20) Remuin˜ a´n, M. J.; Roma´n, H.; Alonso, M. T.; Rodr´ıguez-Ubis, J. C. J. Chem.
Soc., Perkin Trans. 2 1 9 9 3 , 1099-1102.
(iv) Synthesis of 2 ,6 -Bis(3 ′-bromomethyl-1 ′-pyrazolyl)-4 -
phenylpyridine (5 ). To 100 mL of dry THF containing 0.66 g of
(21) Rodr´ıguez-Ubis, J. C.; Sedano, R.; Barroso, G.; Juanes, O.; Brunet, E. Helv.
Chim. Acta 1 9 9 7 , 80, 86-96.
(22) Latva, M.; Takalo, H.; Mukkala, V.-M.; Matachescu, C.; Rodr´ıguez-Ubis, J.
C.; Kankare, J. J. Lumin. 1 9 9 7 , 75, 149-169.
(24) Evans, R. F.; van Ammers, M.; den Hertog, H. J. Recl. Trav. Chim. Pays-Bas
1 9 5 9 , 78, 408-411.
(23) Rodr´ıguez-Ubis, J. C.; Sedano, R.; Juanes, O.; Brunet, E. Helv. Chim. Acta
(25) van Ammers, M.; den Hertog, H. J. Recl. Trav. Chim. Pays-Bas 1 9 5 8 , 77,
1 9 9 7 , 80, 372-387.
340-345.
1870 Analytical Chemistry, Vol. 73, No. 8, April 15, 2001

Figure 1. Synthesis of BPTA (7).
4 (1.9 mmol) was added 1.45 g of PBr3 (5.36 mmol), and the
solution was refluxed for 4 h with stirring. After the solvent was
evaporated, the residue was washed with 10% Na2CO3 and then
dissolved in 20 mL of benzene with slight warming. After filtering,
the solvent was evaporated. The product was washed with hexane
and then dried. A white powder of 5 was obtained (0.81 g, 90.0%
yield). H NMR (CDCl3) δ 8.54 (d, J, 2.64 Hz, 2H), 8.08 (s, 2H),
7.84-7.80 (m, 2H), 7.60-7.50 (m, 3H), 6.58 (d, J, 2.64 Hz, 2H),
4.59 (s, 4H).
precipitate of BPTA was collected by filtration and washed with
1% HCl. After drying, 429 mg of BPTA (90.0% yield) was obtained.
Anal. Calcd for C27H29N7O9 (BPTA‚H2O): C, 54.45; H, 4.91; N,
16.45. Found: C, 54.12; H, 5.03; N, 16.18. 1H NMR (DMSO-d6) δ
8.92 (d, J, 2.64 Hz, 2H), 8.20 (s, 2H), 7.90-7.86 (m, 2H), 7.60-
7.55 (m, 3H), 6.59 (d, J, 2.31 Hz, 2H), 4.00 (s, 4H), 3.53 (s, 8H).
Labeling of SA with BP TA. SA was labeled with BPTA by
using succinimidyl monoester of BPTA (NHS-BPTA). The
preparation of N-hrdroxysuccinimide (NHS)-BPTA and the
labeling procedure are described in the following.
(i) P reparation of NHS-BP TA. Before preparation, BPTA
and NHS were vacuum-dried for 24 h in a desiccator in the
presence of P2O5. To a solution of 173 mg of BPTA (0.3 mmol)
dissolved in 5 mL of dry DMF, 34.5 mg of NHS (0.3 mmol) and
61.9 mg of N,N′-dicyclohexylcarbodiimide (0.3 mmol) were added.
The solution was stirred for 24 h at room temperature. After
undissolved white powder was removed by filtration, the DMF
solution was evaporated to dryness by vacuum evaporation, and
the product was washed with 2-propanol and dried. The product
was directly used for SA labeling without further purification. Since
the esterification between BPTA and NHS is equimolar, and four
carboxyl groups of BPTA are chemically equivalent, it can be
considered that only one carboxyl group is esterified in NHS-
BPTA.
1
(v) Synthesis of Tetraethyl N,N,N1 ,N1 -[2 ,6 -Bis(3 ′-ami-
nomethyl-1 ′-pyrazolyl)-4 -phenylpyridine] Tetrakis(acetate)
(6 ). To a solution of 100 mL of dry CH3CN-20 mL of dry THF
containing 0.75 g of 5 (1.58 mmol), 608 mg of diethyl iminodiac-
etate (3.2 mmol) dissolved in 20 mL of CH3CN and 2.2 g of K2-
CO3 were added. The mixture was refluxed for 24 h with stirring.
After filtering, the solvent was evaporated. The residue was
dissolved in 200 mL of CHCl3, and then the solution was washed
with 2 × 100 mL of water. After the organic phase was dried with
Na2SO4, the solvent was evaporated. The oily residue was purified
by silica gel column chromatography using ethyl acetate as eluent
1
to yield 0.57 g (52.3% yield) of 6 . H NMR (CDCl3) δ 8.54 (d, J,
2.31 Hz, 2H), 8.05 (s, 2H), 7.84-7.80 (m, 2H), 7.55-7.45 (m, 3H),
6.56 (d, J, 2.64 Hz, 2H), 4.18 (q, J, 7.26 Hz, 8H), 4.08 (s, 4H), 3.64
(s, 8H), 1.26 (t, J, 7.26 Hz, 12H).
(vi) Synthesis of N,N,N1 ,N1 -[2 ,6 -Bis(3 ′-aminomethyl-1 ′-
pyrazolyl)-4 -phenylpyridine] Tetrakis(acetic acid). To a solu-
tion of 1.2 g of KOH dissolved in 40 mL of ethanol and 5 mL of
water was added 550 mg of 6 (0.80 mmol), and then the resultant
solution was refluxed for 3 h with stirring. After the solvent was
evaporated, the residue was dissolved in 40 mL of water. To the
solution was added dropwise 3 M HCl to adjust the pH to ∼1,
and the solution was stirred for 3 h at room temperature. The
(ii) Labeling of SA with NHS-BP TA. To a solution of 5 mg
of SA dissolved in 10 mL of 0.1 M carbonate buffer of pH 9.1 was
added 10 mg of NHS-BPTA. After stirring for 4 h at room
temperature, the solution was dialyzed three times against 3 L of
0.1 M NaHCO3 containing 0.25 g of NaN3, at 4 °C each for 24 h.
The concentration of BPTA in the labeled SA solution was
measured by TbCl3 fluorescence titration, and the labeling ratio
of BPTA to SA was calculated to be ∼26 (26 BPTA per SA). The
Analytical Chemistry, Vol. 73, No. 8, April 15, 2001 1871

solution of labeled SA was stored at -20 °C after 25 mg of NaN3
was added. To the BPTA-labeled SA solution was added TbCl3
(Tb3+:BPTA ) 1.5:1) and the resultant mixture was diluted 1000-
fold with 0.05 M Tris-HCl buffer, pH 7.8, containing 0.2% BSA,
0.1% NaN3, and 0.9% NaCl, before use in immunoassay.
previous report.18 When BHHCT-labeled anti-AFP antibody was
used in a single-label assay of AFP, the stock solution was diluted
750-fold with 0.05 M Tris-HCl buffer, pH 7.8 containing 1.0 × 10-6
M EuCl3, 0.2% BSA, 0.1% NaN3, and 0.9% NaCl. When biotinylated
anti-CEA antibody was used in a single assay of CEA, the stock
solution was diluted 1000-fold with 0.05 M Tris-HCl buffer of pH
7.8 containing 0.2% BSA, 0.1% NaN3, and 0.9% NaCl. When
BHHCT-labeled anti-AFP antibody and biotinylated anti-CEA
antibody were used for the simultaneous measurement of AFP
and CEA, a mixture of 1/ 750 BHHCT-labeled anti-AFP antibody
solution and 1/ 1000 biotinylated anti-CEA antibody solution was
prepared with 0.05 M Tris-HCl buffer, pH 7.8, containing 1.0 ×
10-6 M EuCl3, 0.2% BSA, 0.1% NaN3, and 0.9% NaCl.
P hysical Measurements. The 1H NMR spectra were mea-
sured on a JEOL JNM-LA270 spectrometer. The fluorescence
quantum yields (φ1) of BPTA-Eu3+ and BPTA-Tb3+ were
measured in 0.05 M borate buffer, pH 9.1, and calculated22 by
using the equation φ1 ) I1ꢀ2C2φ2/ I2ꢀ1C1 with a standard quantum
yield of φ2 ) 0.110 for [Eu(2,2′:6′,2′′-terpyridine-6,6′′-dicarboxy-
late)2] (molar absorption coefficient ꢀ327 nm ) 1.75 × 104 cm-1 M-1
)
or φ2 ) 0.090 for [Tb(2,2′:6′,2′′-terpyridine-6,6′′-dicarboxylate)2]
(ꢀ327 nm ) 1.75 × 104 cm-1 M-1).22 In the equation, I1 and I2 are the
fluorescence intensities for the measured chelate and the standard
chelate, ꢀ1 and ꢀ2 are molar absorption coefficients of the measured
chelate and the standard, and C1 and C2 are concentrations of the
chelate and the standard, respectively. The fluorescence excitation
and emission spectra were measured on a Hitachi F-4500 fluo-
rospectrometer. The fluorescence lifetime was measured on a
Perkin-Elmer LS 50B luminescence spectrometer. The time-
resolved fluorometric immunoassay was performed on a DELFIA
1234 time-resolved fluorometer (Wallac). For the terbium fluo-
rescence measurement, the measurement conditions were as
follows: excitation wavelength, 340 nm; emission wavelength, 545
nm; delay time, 0.2 ms; window time, 0.4 ms; cycling time, 1.0
ms. The europium fluorescence measurement was carried out
under the following conditions: excitation wavelength, 340 nm;
emission wavelength, 615 nm; delay time, 0.2 ms; window time,
0.4 ms; cycling time, 1.0 ms.
P reparation of Biotinylated BSM-BSA Conjugate. The
preparation was carried out by using the same method as
described in the preparation of biotinylated anti-AFP antibody. In
the reaction, 1.0 mL of the BSM-BSA conjugate solution (BSA
concentration is ∼1 mg/ ml, Iatron Laboratories, Inc.) and 5 mg
of NHS-LC-biotin were used. After purification by dialysis, the
biotinylated BSM-BSA was diluted to 2.0 mL with water, and 10
mg of BSA and 10 mg of NaN3 were added. The solution was
stored at 4 °C before use. When the biotinylated BSM-BSA
solution was used for immunoassay, it was diluted 1000-fold with
0.05 M Tris-HCl buffer, pH 7.8, containing 0.4% BSA, 0.2% NaN3,
and 1.8% NaCl.
Immunoassay of Human AFP by Using BP TA-Tb3 +
-
Labeled SA. The standard solutions of human AFP were prepared
by diluting human AFP (Nippon Bio-Test Laboratories, Inc.) with
0.05 M Tris-HCl buffer, pH 7.8, containing 5% BSA, 0.9% NaCl,
and 0.1% NaN3.
Application of BP TA-Tb3 +-Labeled SA to TR-FIA. The
BPTA-Tb3+-labeled SA was tested for both sandwich-type TR-
FIA and competitive-type TR-FIA. By using BPTA-Tb3+-labeled
SA, human AFP and CEA in serum and BSM in water were
measured by sandwich-type TR-FIA (AFP and CEA) and competi-
tive-type TR-FIA (BSM). In addition, BHHCT-Eu3+-labeled anti-
After anti-AFP monoclonal antibody (Oemconcepts Co., diluted
to 5 µg/ mL with 0.1 M carbonate buffer, pH 9.6) was coated on
the wells (100 µL/ well) of a 96-well microtiter plate (FluoroNunc
plate) by physical adsorption,18 50 µL of human AFP standard
solution was added to each well. After incubation at 37 °C for 1 h,
the wells were washed twice with 0.05 M Tris-HCl buffer, pH 7.8,
containing 0.05% Tween 20 (buffer 1) and once with 0.05 M Tris-
HCl buffer, pH 7.8 (buffer 2). Then 50 µL of biotinylated goat
anti-AFP antibody was added to each well, and the plate was
incubated at 37 °C for 1 h. After the wells were washed with buffer
1 and buffer 2, 50 µL of BPTA-Tb3+-labeled SA was added to
each well and the plate was incubated at 37 °C for 1 h. After the
wells were washed four times with buffer 1, the plate was subjected
to solid-phase terbium time-resolved fluorometric measurement.
AFP antibody, biotinylated anti-CEA antibody, and BPTA-Tb3+
-
labeled SA were used for simultaneous determination of AFP and
CEA in human serum. Labeling of the antibodies and each assay
were carried out according to the following procedures.
P reparation of Biotinylated Anti-AFP Antibody. After two
dialyses of 1.0 mL of goat anti-human AFP antibody solution (0.35
mg/ mL, Nippon Bio-Test Laboratories, Inc.) for 24 h at 4 °C
against 3 L of saline-water, 8.4 mg of NaHCO3 and 5 mg of sul-
fosuccinimidyl-6-(biotinamido)hexanoate (NHS-LC-biotin, Pierce
Chemical Co.) were added with stirring. After stirring for 1 h at
room temperature, the solution was incubated for 24 h at 4 °C.
The solution was twice dialyzed each for 24 h at 4 °C against 3 L
of 0.1 M NaHCO3 containing 0.25 g of NaN3, and then 10 mg of
NaN3 was added. The solution was stored at -20 °C before use.
When the biotinylated antibody solution was used for immunoas-
say, it was diluted 2000-fold with 0.05 M Tris-HCl buffer, pH 7.8,
containing 0.2% BSA, 0.1% NaN3, and 0.9% NaCl.
Immunoassay of Human CEA by Using BP TA-Tb3 +
-
Labeled SA. The standard solutions of human CEA were pre-
pared by diluting human CEA (Seikagaku Co.) with 0.05 M Tris-
HCl buffer, pH 7.8, containing 5% BSA, 0.9% NaCl and 0.1% NaN3.
After anti-CEA monoclonal antibody (Nippon Bio-Test Labo-
ratories, Inc., diluted to 5 µg/ mL with 0.1 M carbonate buffer of
pH 9.6) was coated on the wells (100 µL/ well) of a 96-well
microtiter plate by physical adsorption, 50 µL of human CEA
standard solution was added to each well. After incubation at 37
°C for 1 h, the wells were washed twice with buffer 1 and buffer
2. Then 50 µL of biotinylated rabbit anti-CEA antibody was added
to each well and the plate was incubated at 37 °C for 1 h. After
the wells were washed with buffer 1 and buffer 2, 50 µL of BPTA-
Tb3+ labeled SA was added to each well and the plate was
P reparation of Biotinylated Anti-CEA Antibody and BH-
HCT-Labeled Anti-AFP Antibody. Biotinylated rabbit anti-
human CEA antibody (Dako-immunoglobulins) and BHHCT-
labeled goat anti-human AFP antibody (Nippon Bio-Test Labora-
tories, Inc.) were prepared using the methods described in a
1872 Analytical Chemistry, Vol. 73, No. 8, April 15, 2001

incubated at 37 °C for 1 h. After wells were washed four times
with buffer 1, the plate was subjected to solid-phase terbium time-
resolved fluorometric measurement.
Immunoassay of BSM by Using BP TA-Tb3 +-Labeled SA.
The standard solutions of BSM were prepared by diluting 1 mg/
mL BSM-acetonitrile solution with 0.05 M Tris-HCl buffer, pH
7.8.
After anti-BSM monoclonal antibody (Iatron Laboratories, Inc.,
diluted to 4 µg/ mL with 0.1 M carbonate buffer, pH 9.6) was
coated on the wells (100 µL/ well) of a 96-well microtiter plate by
physical adsorption, 50 µL of a 1:1 mixture of the BSM standard
solution and the biotinylated BSM-BSA was added to each well.
The plate was incubated at 37 °C for 2 h and washed with buffer
1 and buffer 2. Then 50 µL of the BPTA-Tb3+-labeled SA was
added to each well, and the plate was incubated at 37 °C for 1 h.
After wells were washed four times with buffer 1, the plate was
subjected to solid-phase terbium time-resolved fluorometric mea-
surement.
Figure 2. Excitation and emission spectra of BPTA-Tb3+ (1 µM,
s) and BPTA-Eu3+ (20 µM, - - -) in 0.05 M borate buffer, pH 9.1.
Table 1. Fluorescence Properties of BPTA-Tb3+ and
BPTA-Eu3+ in 0.05 M Borate Buffer, pH 9.1a
ꢀ
ꢀφ
(cm-1
Simultaneous Measurement of AFP and CEA Using
b
c
λex,max (cm-1 λem,max
τ
τ
D2O
H2O
chelate
(nm) M-1
)
(nm)
φ
M-1
)
(ms) (ms)
BHHCT-Eu3 +-Labeled Anti-AFP Antibody and BP TA-Tb3 +
-
Labeled SA. The standard solutions containing AFP and CEA
were prepared by diluting human AFP and CEA with 0.05 M Tris-
HCl buffer, pH 7.8, containing 5% BSA, 0.1% NaN3, and 0.9% NaCl.
After a solution containing anti-AFP monoclonal antibody and
anti-CEA monoclonal antibody (5 µg/ mL anti-AFP and 5 µg/ mL
anti-CEA in 0.1 M carbonate buffer, pH 9.6) was coated on the
wells (100 µL/ well) of a 96-well microtiter plate by physical
adsorption, 50 µL of a human AFP-CEA standard solution (or
human sera) was added to each well. After incubation at 37 °C
for 1 h, the wells were washed with buffer 1 and buffer 2. Then
a 50-µL mixture of BHHCT-Eu3+-labeled anti-AFP and biotinylated
anti-CEA was added to each well, and the plate was incubated at
37 °C for 1 h. After the wells were washed four times with 0.05 M
Tris-HCl, pH 9.1, containing 0.05% Tween 20, the plate was
subjected to solid-phase europium time-resolved fluorometric
measurement. After measurement, 50 µL of BPTA-Tb3+-labeled
SA was added to each well and the plate was incubated at 37 °C
for 1 h. The plate was washed four times with buffer 1 and was
subjected to solid-phase terbium time-resolved fluorometric mea-
surement.
BPTA-Tb3+
BPTA-Eu3+
BPTA-Tb3+
(bound to SA)
BPTA-Eu3+
(bound to SA)
325
325
322
9290
9290
543 1.00
9290 2.681 3.031
620 0.134 1245 1.353 2.427
543
6460 2.410 2.920
322
619
731 1.213 2.285
a
The experimental uncertainty for quantum yield is <15%. b Mea-
sured in 0.05 M NaHCO3-H2O buffer. c Measured in 0.05 M NaHCO3-
D2O buffer.
To compare the correlation between the simultaneous mea-
surement and the independent single measurement of AFP and
CEA in human serum, AFP and CEA concentrations in 39 human
serum samples were determined by using both the dual-label
method and single-label independent assay for AFP and CEA. In
the single-label independent assay of AFP, the anti-AFP antibody-
coated plate, AFP standard solutions (or serum samples) and
BHHCT-Eu3+-labeled anti-AFP antibody were used for measure-
ment. After the anti-AFP antibody-coated wells were incubated
with the AFP standard solutions or serum samples at 37 °C for 1
h, BHHCT-Eu3+-labeled anti-AFP antibody was added, and the
solution was incubated at 37 °C for 1 h. After the wells were
washed four times with 0.05 M Tris-HCl, pH 9.1, containing 0.05%
Tween 20, the plate was subjected to solid-phase europium time-
resolved fluorometric measurement, and the AFP concentrations
in the samples were calculated. The method of CEA independent
Figure 3. Log(fluorescence count) vs log[BPTA-Tb3+] in 0.05 M
Tris-HCl buffer, pH 8.0: (A) free BPTA-Tb3+. (B) BPTA-Tb3+ bound
to SA. The concentration of BPTA-Tb3+ is in molar and the
fluorescence count is in arbitrary units.
RESULTS AND DISCUSSION
Fluorescence P roperties of BP TA Chelates with Eu3 + and
Tb3 +. The excitation and emission spectra of BPTA-Tb3+ and
BPTA-Eu3+ are shown in Figure 2, while the fluorescence
properties are shown in Table 1. Both BPTA-Tb3+ and BPTA-
Eu3+ show the excitation maximum wavelength at 325 nm (ꢀ )
9.29 × 103 cm-1 M-1 for both). The BPTA-Tb3+ complex shows
four sharp emissions at 489, 543, 582, and 620 nm with the
intensities in the order of 543 > 489 > 582 > 620 nm. The BPTA-
Eu3+ complex shows two sharp emissions at 589 and 620 nm with
assay by using biotinylated anti-CEA antibody and BPTA-Tb3+
-
labeled SA has been described above.
Analytical Chemistry, Vol. 73, No. 8, April 15, 2001 1873

Table 2. Measurement Precision and Analytical
Recovery of AFP Added to Serum Samples
added
(ng/ mL)
found
(ng/ mL)
CV (%)
recovery
(%)
(n ) 4)a
1.81
28.6
10.9
7.06
4.02
2.72
3.28
26.2
14.2
8.22
5.57
4.19
5.12
26.4
16.2
10.1
7.41
6.05
9.19
6.92
5.82
7.80
6.98
9.85
4.15
7.81
5.82
4.15
7.21
7.12
5.55
5.18
4.19
4.15
7.20
3.15
25.0
10.0
5.00
2.50
1.00
107.2
91.9
105.0
88.4
91.0
25.0
10.0
5.00
2.50
1.00
91.7
109.2
98.8
91.6
91.0
25.0
10.0
5.00
2.50
1.00
85.1
110.8
99.6
91.6
93.0
a
Intraassay using four different wells in a plate.
and 58% (Eu chelate) and 2.410 and 1.213 ms, respectively. Using
the fluorescence lifetimes of BPTA-Eu3+ (free and bound to SA)
in D2O and in H2O, the average number (q) of coordinated water
molecules in the first coordination sphere of Eu3+ was calculated
from the equation22 of q ) 1.05(1/ τH O - 1/ τD O) to be 0.34 (free
2
2
chelate) and 0.41 (bound to SA).
The effect of pH on the fluorescence intensity and the lifetime
was measured by using a solution of 1.0 µM BPTA-Tb3+ (free
and bound to SA) in 0.05 M Tris-HCl buffer with several pHs (from
5.5 to 9.5). The results show that both fluorescence intensity and
lifetime are not affected (the change of fluorescence intensity <5%)
by the pH in the range of pH 6.0-8.5. In addition, Tris-HCl,
carbonate, and borate buffers did not change the fluorescence
intensity and lifetime of BPTA-Tb3+. These experiments also
proved that BPTA chelates of Tb3+ and Eu3+ are highly stable in
these buffers.
The calibration curve of BPTA-Tb3+ (free and bound to SA)
in 0.05 M Tris-HCl buffer, pH 8.0, was measured and is shown in
Figure 3. The detection limits of free BPTA-Tb3+ and BPTA-
Tb3+ bound to SA were calculated as the concentration corre-
sponding to 2 standard deviations (SD) of the background
intensity and are 5.8 × 10-13 (free BPTA-Tb3+) and 1.6 × 10-12
M (BPTA-Tb3+ bound to SA) (which are 8.7 × 10-13 and 2.4 ×
10-12 M when 3 SD of the background intensity was used).
Figure 4. Calibration curves of TR-FIA by using BPTA-Tb3+
labeled SA for AFP (A), CEA (B), and BSM (C). bg, background. The
inset curves in (A) and (B) are the plot of the background-subtracted
signals vs the concentrations of AFP and CEA.
-
TR-FIA of AFP , CEA, and BSM by Using BP TA-Tb3 +
-
the intensity order of 620 > 589 nm. Compared with the Tb3+
complex, the Eu3+ complex is weakly fluorescent. In 0.05 M borate
buffer, pH 9.1, the fluorescence quantum yield and lifetime of free
BPTA-Tb3+ are 1.00 and 2.681 ms, and those of BPTA-Eu3+ are
0.134 and 1.353 ms, respectively. These fluorescence quantum
yield and molar absorption coefficient of BPTA-Tb3+ are clearly
larger than those of N,N,N1,N1-[2,6-bis(3′-aminomethyl-1′-pyra-
zolyl)pyridine]tetrakis(acetic acid) chelate with Tb3+ (the fluo-
rescence quantum yield and the molar absorption coefficient of
the Tb3+ chelate are 0.580 and 8100 cm-1 M-1, respectively).22
After the two BPTA chelates were bound to SA, their fluorescence
intensities (ꢀφ) and lifetimes decreased to about 70 (Tb chelate)
Labeled SA. The calibration curves of AFP, CEA, and BSM are
shown in Figure 4. The detection limits defined as the concentra-
tion corresponding to 2 SD of the background intensity were 42
pg/ mL for AFP, 70 pg/ mL for CEA, and 0.4 ng/ mL for BSM
(which are 65 pg/ mL for AFP, 106 pg/ mL for CEA, and 0.68 ng/
mL for BSM when 3 SD of the background intensity is used).
These detection limits are low enough to measure AFP and CEA
in human sera and BSM in environmental samples. The standard
deviations of the background signals were calculated by using
eight date points measured in eight different wells in a 96-well
microtiter plate. As panels A and B of Figure 4 show, the plot of
the background-subtracted signals versus antigen concentrations
1874 Analytical Chemistry, Vol. 73, No. 8, April 15, 2001

Figure 5. Schematic representation of the simultaneous measure-
ment system for AFP and CEA. Eu, BHHCT-Eu3+; Tb, BPTA-Tb3+
.
For the details of the procedure, see the Experimental Section.
Figure 7. Correlation between the independent measurement and
the simultaneous measurement of AFP (A) and CEA (B) in 39 human
serum samples.
measured for AFP are summarized in Table 2. The CVs of all the
assays are below 10%, and the analytical recoveries are in the range
of 85-110%. These results indicate that TR-FIA by using BPTA-
Tb3+-labeled SA is highly sensitive with good accuracy and
precision.
Simultaneous Measurement of AFP and CEA by Using
BHHCT-Eu3 +-Labeled Anti-AFP Antibody and BP TA-Tb3 +
-
Labeled SA. The assay format for the simultaneous measurement
of AFP and CEA in sera is shown in Figure 5, in which BHHCT-
Eu3+-labeled anti-AFP antibody, biotinylated anti-CEA antibody,
and BPTA-Tb3+-labeled SA are employed. As shown in Figure
2, the emission of BPTA-Tb3+ at 620 nm overlaps the emission
of the Eu3+ chelate (the fluorescence intensity of BPTA-Tb3+
measured with the Eu3+ fluorescence measurement mode (615
nm) of DELFIA 1234 is ∼1/ 30 of the intensity obtained with the
Tb3+ fluorescence measurement mode (545 nm)), and therefore,
the Eu3+ fluorescence intensity at 615 nm is affected by the
presence of the Tb3+ chelate. To overcome this problem, a
separated assay format as shown in Figure 5 was used for the
simultaneous measurement of AFP and CEA by sequential
addition of the tracers and by Tb3+ fluorescence measurement at
545 nm since Tb3+ fluorescence measurement at this wavelength
is not affected by the presence of the Eu3+ fluorescence label.
Figure 6. Calibration curves of the simultaneous measurement of
AFP (A) and CEA (B) using BHHCT-Eu3+-labeled anti-AFP antibody,
biotinylated anti-CEA antibody, and BPTA-Tb3+-labeled SA; bg,
background. The inset curves are the plot of the background-
subtracted signals vs the concentrations of AFP and CEA.
shows straight lines expressed as log(signal) ) 0.919 log[AFP]
+ 3.535 (r ) 1.000) for AFP and log(signal) ) 0.979 log[CEA] +
3.353 (r ) 0.998) for CEA. The upper limits of the three calibration
curves are ∼100 ng/ mL for AFP, CEA, and BSM. The analytical
precision and recovery of TR-FIA using BPTA-Tb3+-labeled SA
Analytical Chemistry, Vol. 73, No. 8, April 15, 2001 1875

Using this assay format, the sensitivity of CEA measurement is
also increased by the multiple labeling via the biotin-streptavidin
binding.
simultaneous measurement; x, the results of single-label assay),
respectively. Although the slopes of the two statistical straight
lines are somewhat higher than 1.00, and their intercepts are not
zero, these might be caused by some unexpected errors of the
two measurement methods, and the results are in the range of
normal measurement errors. These results indicate that the two
antigens can be simultaneously determined in the present method
with enough accuracy and precision. Based on the above results,
it is concluded that the TR-FIA by using BPTA-Tb3+ is highly
sensitive and can be practically applied to the general clinical
analysis. Although the sensitivity of the measurement is lower
The calibration curves for the simultaneous measurement of
AFP and CEA are shown in Figure 6. The plot of the background-
subtracted signals versus antigen concentrations shows almost
linear calibration curves for both AFP and CEA. The detection
limits of the present method are calculated26 to be 44 pg/ mL for
AFP and 76 pg/ mL for CEA. These detection limits are low
enough to measure AFP and CEA in human sera. Compared with
our previous method for simultaneous determination of AFP and
CEA by using Eu3+ and Sm3+ chelates as the labels,18 the present
method is simpler and more sensitive, especially for CEA, for
which the previous Sm3+ label has been replaced by the Tb3+ label.
The AFP and CEA concentrations in 39 human serum samples
were determined with both the simultaneous measurement
method and the single-label assay method. Correlation of the two
methods is shown in Figure 7. The correlation coefficients of AFP
and CEA are 0.991 and 0.994 with the correlation equations of y
) 1.081x - 0.184 and y ) 1.092x + 0.646 (y, the results of
than that of our previous label, BHHCT-Eu3+ 27,28 the present Tb3+
,
label is more sensitive than BHHCT-Sm3+ chelate.18
ACKNOWLEDGMENT
The present work is supported financially by CREST (Core
Research for Evolutional Science and Technology) of Japan
Science and Technology Corporation (JST) and Advanced Re-
search Center for Science and Engineering of Waseda University.
(26) Kropf, J.; Quitte, E.; Gressner, A. M. Anal. Biochem. 1 9 9 1 , 197, 258-265.
(27) Yuan, J.; Matsumoto, K.; Kimura, H. Anal. Chem. 1 9 9 8 , 70, 596-601.
(28) Yuan, J.; Wang, G.; Kimura, H.; Matsumoto, K. Anal. Sci. 1 9 9 9 , 15, 125-
128.
Received for review November 10, 2000. Accepted
January 18, 2001.
AC0013305
1876 Analytical Chemistry, Vol. 73, No. 8, April 15, 2001