Biolabeling with 2,4-dichlorophenoxyacetic acid derivatives: The 2,4-D tag

Article abstract of DOI:10.1021/ac901900n

Many bioanalytic and diagnostic procedures rely on labels with which the molecule of interest can be tracked in or discriminated from accompanying like substances. Herein, we describe a new labeling and detection system based on derivatives of 2,4-dichlorophenoxyacetic acid (2,4-D) and anti-2,4-D antibodies. The 2,4-D system is highly sensitive witha KDof7×10 ^-11Mforthehapten-antibody pair, can be used on a large variety of biomolecules such as proteins, peptides, carbohydrates, and nucleic acids, is not hampered by endogenous backgrounds because 2,4-D is a xenobiotic, and is robust because 2,4-D is a very stable compound that withstands the conditions of most reactions usually performed on biomolecules. With this unique blend of properties, the 2,4-D system compares favorably with its rivals digoxigenin (DIG)/anti-DIG and biotin/(strept)avidin and provides an interesting and powerful tool in biomolecular labeling.

Full text of DOI:10.1021/ac901900n

Anal. Chem. 2009, 81, 9695–9702
Biolabeling with 2,4-Dichlorophenoxyacetic Acid
Derivatives: The 2,4-D Tag
Steffen Bade,† Niels Ro¨ ckendorf,† Milan Franek,| Hans H. Gorris,†,⊥ Buko Lindner,‡
Verena Olivier,†,# Klaus-Ju¨ rgen Schaper,§ and Andreas Frey*,†
Division of Mucosal Immunology, Department of Pneumology, Division of Immunochemistry, and Division of Structural
Biochemistry, Department of Infection Biology, Research Center Borstel, Parkallee 22, 23845 Borstel, Germany, and
Department of Analytical Biotechnology, Veterinary Research Institute, Hudcova 70, 621 32 Brno, Czech Republic
Many bioanalytic and diagnostic procedures rely on labels
with which the molecule of interest can be tracked in or
discriminated from accompanying like substances. Herein,
we describe a new labeling and detection system based
on derivatives of 2,4-dichlorophenoxyacetic acid (2,4-D)
and anti-2,4-D antibodies. The 2,4-D system is highly
sensitivewithaKD of7×10-11 Mforthehapten-antibody
pair, can be used on a large variety of biomolecules
such as proteins, peptides, carbohydrates, and nucleic
acids, is not hampered by endogenous backgrounds
because 2,4-D is a xenobiotic, and is robust because
2,4-D is a very stable compound that withstands the
conditions of most reactions usually performed on
biomolecules. With this unique blend of properties, the
2,4-D system compares favorably with its rivals digoxi-
genin (DIG)/anti-DIG and biotin/(strept)avidin and
provides an interesting and powerful tool in biomo-
lecular labeling.
detection reagents can be used and self-quenching, a common
problem of direct fluorophore labels, can be avoided in indirect
setups.
Biotin/(strept)avidin and digoxigenin (DIG)/anti-DIG are the
most commonly used indirect labeling systems in bioanalytical
applications.1-4 Nevertheless, these systems have several draw-
backs that limit their use, for example, the endogenous occurrence
of biotin in various biological sources (serum, plasma, tissues,
cells, organelles, e.g., mitochondria),4,5 the uptake of biotin or DIG
by epithelial transporters,6,7 or the metabolic modification of DIG
by intestinal bacteria.8 Because multiple labeling is frequently
required in modern analytical and diagnostic procedures9,10 it is
necessary to improve the bioanalytical toolbox with new labeling
and detection reagents that are versatile and robust and can be
applied in combination with established systems.
Here, we describe a novel indirect labeling/detection system
based on 2,4-dichlorophenoxyacetic acid (2,4-D, (1); see Figure
S-1 in the Supporting Information) and anti-2,4-D antibodies. 2,4-D
has been used as a herbicide since the 1950s,11 and antibodies
against 2,4-D have been developed12,13 to determine the 2,4-D
burden in the environment, e.g., in drinking water, food or body
fluids.14,15 In the competition-type immunoassays used for this
purpose, the 2,4-D-specific antibodies displayed a satisfactory
In the course of many analytical and diagnostic procedures,
the sensitive and specific detection of biomolecules, such as
peptides, proteins, oligosaccharides, or DNA, is of critical impor-
tance. Hence, reagents and methodologies that are suitable for
the labeling, detection, and quantification of target-molecules
present in complex biological samples are urgently required. In
recent years, numerous nonradioactive labeling methods have
been developed. Target molecules can be equipped either with
“indirect labels” (e.g., biotin, digoxigenin) or with “direct labels”
(fluorophores or luminophores). While direct labels allow im-
mediate visualization of derivatized target molecules, indirect
labels require the use of secondary detection reagents. Sensitivity
of detection of indirect labels is often superior as signal amplifying
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* To whom correspondence should be addressed. Phone: +49-4537-188-562.
Fax: +49-4537-188-693. E-mail: afrey@fz-borstel.de.
† Division of Mucosal Immunology, Department of Pneumology, Research
Center Borstel.
.
(10) Saban, M. R.; Towner, R.; Smith, N.; Abbott, A.; Neeman, M.; Davis, C. A.;
Simpson, C.; Maier, J.; Memet, S.; Wu, X. R.; Saban, R. BMC Cancer 2007,
7, 219
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1999, 364, 584–589
.
‡ Division of Immunochemistry, Research Center Borstel.
§ Division of Structural Biochemistry, Department of Infection Biology,
Research Center Borstel.
.
| Veterinary Research Institute.
.
⊥ Present address: Institute of Analytical Chemistry, Chemo- and Biosensors,
University of Regensburg, 93040 Regensburg, Germany.
# Present address: Intercell AG, Campus Vienna Biocenter 3, 1030 Vienna,
Austria.
.
.
.
10.1021/ac901900n CCC: $40.75  2009 American Chemical Society
Published on Web 10/29/2009
Analytical Chemistry, Vol. 81, No. 23, December 1, 2009 9695

sensitivity, with lower detection limits of 1 × 10-7 g/L 2,4-D.16
However, the use of 2,4-D/anti-2,4-D as an indirect labeling
system has not been pursued since any derivatization of
substrates with 2,4-D had to be performed in situ in the
presence of condensing agents and organic solvents.17
Our new 2,4-D derivatives allow the straightforward labeling
of numerous biological substrates, such as peptides, proteins, and
DNA, under physiological conditions. Moreover, a modification
of the 2,4-D derivatives with aliphatic spacers improves their
binding toward the 2,4-D-specific antibodies resulting in a greatly
increased sensitivity of detection. The 2,4-D-based system, there-
fore, represents a valuable addition to the field of indirect labeling
systems, comparing favorably with established systems and
facilitating multiple labeling applications.
washing with Dulbecco’s phospate-buffered saline (D-PBS: 2.7 mM
KCl, 1.5 mM KH2PO4, 136 mM NaCl, 8.1 mM Na2HPO4, pH
7.4) and blocking with 1% (w/v) casein in D-PBS (casein-PBS),
the labeled proteins were detected with either monoclonal
antibodies directed against 2,4-D or DIG followed by Alexa-
Fluor680-labeled goat anti-mouse IgG or with AlexaFluor680-
labeled streptavidin. Fluorescence was quantitated with an
Odyssey infrared imaging system using the Odyssey software
(V2.1) (LI-COR Biosciences, Bad Homburg, Germany).
For labeling of insulin, 35 nmol of the protein (105 nmol amino
functions) was reacted with 350 nmol active ester in 50 mM
phosphate buffer, pH 7.2, containing no more than 2% (v/v)
DMSO. Labeling reactions were terminated by addition of 1 mmol
of glycine. The molecular mass and heterogeneity of the samples
was determined by MALDI-TOF-MS, and the mean degree of
labeling was calculated as previously described.23 Serially diluted
labeled protein was applied onto preactivated polyvinylidene
difluoride (PVDF)-membrane-bottomed polystyrene filterplates
(1000-0.12 ng conjugate/well) and incubated for 30 min. After
washing with D-PBS containing 0.1% (v/v) Tween 20 and D-PBS
followed by blocking with 2% (w/v) casein hydrolysate in D-PBS,
the labeled protein was detected as above except that horseradish
peroxidase (HRP)-labeled detection reagents in combination with
a tetramethylbenzidine-based substrate24 were used for visualization.
Glycoproteins were labeled in situ on membranes. Serially
diluted porcine mucin was applied to nitrocellulose (2000-1 pg
glycoprotein/dot) and the carbohydrate moieties were oxidized
with sodium meta-periodate. After washing with D-PBS, mem-
branes were incubated in 2.5 µM hydrazide (7), (10), (11)
(Figure 2) or the respective hydrazide of digoxigenin or biotin,
washed again, blocked, and processed with the monoclonal
antibodies and the AlexaFluor680-labeled detection reagents as
described above (for details see the Supplementary Methods in
the Supporting Information).
EXPERIMENTAL SECTION
Additional Methods. General methods, materials, syntheses,
and analyses of the 2,4-D-based labeling derivatives are described
in the Supporting Information (Supplementary Methods and
Figures S-1-S-4).
Determination of Hydrolysis Half-Lives of Active Esters.
The deuterolysis of active esters in phosphate-buffered deuterium
1
oxide (D2O) was determined by H NMR spectroscopy. Stock
solutions of active esters (2), (5), (6), digoxigenin (digoxi-
genin-C6-N-hydroxysuccinimide (DIG-C6-NHS)) and biotin (bi-
otin-N-hydroxy-sulfosuccinimide (biotin-NHSS), biotin-C6-
NHSS, and biotin-C11-NHS) (each 50 mM) were prepared in
anhydrous DMSO-d6. Deuterolysis was initiated by adding 10
µL of these solutions to 490 µL of 50 mM deuterated phosphate-
buffer, pD 7.2, containing 0.1 mg/mL 3-(trimethylsilyl)-1-
propanesulfonic acid-d6 sodium salt (TMSPS), and monitored
with an Avance DRX-600 system (Bruker BioSpin, Rheinstetten,
1
Germany) at 600 MHz. H-spectra were recorded after 6-12,
20, 40, 60, 120, 240, 360, and 1000-1600 min (overnight). Data
were acquired and processed using the X-WIN-NMR software
(V 2.5) (Bruker BioSpin). Discrete peaks obtained at the
various points in time and originating from the (sulfo-)succin-
imidyl moiety and from the aliphatic spacer were integrated
and standardized in relation to TMSPS. From the peak
integrals, the percentage of nonhydrolyzed active ester deriva-
tive could be calculated (for details see the Supplementary
Methods in the Supporting Information).
Labeling of Biomolecules with Reactive Derivatives of 2,4-
D, Biotin, and DIG and Detection of Labeled Compounds.
Stock solutions of 2,4-D active esters (2), (5), (6) or respective
active esters of digoxigenin (DIG-C6-NHS) or biotin (biotin-NHSS,
biotin-C6-NHSS or biotin-C11-NHS) were prepared freshly in
anhydrous DMSO for each labeling experiment.
Labeling of Peptides with 2,4-D Derivatives and Detection
Thereof. Fmoc-protected lysine derivatives (19), (20), and (21)
were synthesized by reacting the active esters (2), (8), or (9)
with Fmoc-L-lysine-OH. Peptide ASQLDYKMTDAGE (N- to C-
terminus) was solid-phase-synthesized by standard Fmoc-chem-
istry. Lysine-N-ε-(biotin) was incorporated during peptide synthe-
sis. Peptides were labeled at their amino terminus with 2,4-D by
reacting 2,4-D compound (1), (3), (4), (12), (13), or (14) with
the completed peptide chain after removal of the aminoterminal
Fmoc protection group. A carboxyterminal 2,4-D label was
introduced by the use of 2,4-D-lysine derivative (19), (20), or
(21) as first amino acid in the peptide synthesis process.
To determine the limit of detection (LOD), 96 well microplates
were coated with 18.75 ng/well anti-2,4-D antibody (clone F6/
C10, E2/G2 or 4B7), washed with D-PBS, blocked with casein-
PBS, and washed again. Peptides serially diluted in casein-PBS
were applied (25.0 pmol-11.9 amol conjugate/well) and incubated
for 2.5 h at RT, and the plates were washed again. To follow the
association reaction, coated plates were incubated for 5, 10, 20,
40, or 80 min with 167 nM peptide and washed. For the
dissociation reaction, plates were incubated for 2.5 h with 167 nM
For labeling of ovalbumin, 100 nmol of the protein (2 µmol
amino functions) were reacted with 1.5 µmol of active ester in
100 mM sodium tetraborate, pH 8.2, containing no more than 5%
(v/v) DMSO. Labeling reactions were terminated by adding 1.5
mmol glycine. Serially diluted labeled protein was applied onto
nitrocellulose membranes (16000-3.12 pg conjugate/dot). After
(16) Gerdes, M.; Meusel, M.; Spener, F. J. Immunol. Methods 1999, 223, 217–
226
.
(23) Olivier, V.; Meisen, I.; Meckelein, B.; Hirst, T. R.; Peter-Katalinic, J.; Schmidt,
(17) Bauer, C. G.; Eremenko, A. V.; Ehrentreich-Forster, E.; Bier, F. F.; Makower,
M. A.; Frey, A. Bioconjugate Chem. 2003, 14, 1203–1208
(24) Frey, A.; Meckelein, B.; Externest, D.; Schmidt, M. A. J. Immunol. Methods
2000, 233, 47–56
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A.; Halsall, H. B.; Heineman, W. R.; Scheller, F. W. Anal. Chem. 1996, 68,
2453–2458
.
.
9696 Analytical Chemistry, Vol. 81, No. 23, December 1, 2009

peptide, washed, refilled with casein-PBS, incubated for 5, 10, 20,
or 80 min, and washed. In all experiments, bound peptide was
then quantitated with HRP-streptavidin as described above (for
details see the Supplementary Methods in the Supporting Infor-
mation).
Data Analysis and Statistics. Limits of detection (LOD) were
determined by t-statistics (95% confidence interval).25 For statistical
analyses, LOD data were transformed logarithmically, and an
outlier analysis (extreme studentized deviate method) and one-
way ANOVA with Bonferroni post hoc test were performed. LOD
data were then back-transformed from the logarithmic state and
are presented as geometric mean and 95% confidence interval.
Hydrolysis half-lives of active esters were calculated by nonlinear
regression (one-phase exponential decay logistic function). Maxi-
mum optical density at 450 nm (MaxOD) and steady-state
dissociation constant KD (mean
SD) were determined by
nonlinear regression analysis (one-site binding logistic func-
tion). Variation in and differences of hydrolysis half-lives,
MaxOD or KD were analyzed by an outlier analysis (extreme
studentized deviate method) and one-way ANOVA with Bon-
ferroni post hoc test. Statistical and regression analyses were
done using GraphPad Prism version 4.03 for Windows (Graph-
Pad Software, San Diego, CA). For all statistical analyses, a
probability of P < 0.05 was considered significant.
RESULTS
To achieve a broad versatility of the new labeling system, active
esters, hydrazides, and amino acid- and nucleotide-derivatives of
2,4-D were devised and synthesized (detailed synthesis schemes
and structural formulas in Figures S-1-S-4 in the Supporting
Information). These compounds, along with equivalent DIG or
biotin derivatives, were then used for labeling of proteins,
glycoproteins, peptides, and DNA, and the detection performance
of the three systems was compared in various experimental setups.
Labeling of Proteins via Primary Amines. Labeling groups
are commonly introduced in proteins by reacting active ester
derivatives of the label with the protein’s surface-exposed primary
amino functions. We, therefore, conceived appropriate active esters
of a variety of 2,4-D derivatives carrying either no aliphatic spacer
moieties or aliphatic spacer moieties of different length (Figure
1A and Figure S-1 in the Supporting Information). 2,4-D (1) was
converted into its N-hydroxysuccinimide (NHS)-ester (2) by
reacting (1) with N,N′-dicyclohexylcarbodiimide (DCC) and NHS.
Spacered 2,4-D derivatives were synthesized using aminohexanoic
(C6) and aminoundecanoic (C11) acid. Since these alkyl amino
acids were hardly soluble in organic solvents that are suited for
coupling, they were first converted into their tetrabutyl ammonium
salts before being reacted with (2) to yield the respective 2,4-D
derivatives (3) and (4). 2,4-D has low solubility in water (0.7 g/L),
and the aliphatic C6- or C11-spacers further decrease its solubility.
Hence, we replaced NHS by N-hydroxy-sulfosuccinimide (NHSS)
to introduce the amine reactive functional group along with a
hydrophilic sulfonic acid residue. NHSS-esters (5) and (6) were
synthesized by reacting carboxylic acids (3) or (4) with DCC and
NHSS. The charged NHSS-moiety renders the respective 2,4-D
derivatives suitable for protein labeling under physiological
conditions.
Figure 1. Stability of 2,4-D active ester derivatives in aqueous
solutions. (A) N-hydroxysuccinimidyl-(NHS)-ester (2) or N-hydroxy-
sulfosuccinimidyl-(NHSS)-ester derivatives (5) and (6) of 2,4-dichlo-
rophenoxyacetic acid were synthesized. Derivatives (5) and (6) had
been equipped with aliphatic 6-aminohexanoyl (C6) or 11-aminoun-
decanoyl (C11) spacers, respectively. (B, C) The deuterolysis of (2),
(5), and (6) was measured via 1H NMR spectroscopy in 50 mM
phosphate buffer, pD 7.2, containing 2% (v/v) DMSO-d6, and was
compared with deuterolyses of active ester derivatives of digoxigenin
(DIG) or biotin. (B) Exemplarily, three sections of 1H NMR spectra of
(5) after 10, 120, and 1350 min at room temperature are shown.
Signals which were altered in intensity or chemical shift due to
deuterolysis and which were not superimposed by signals of other
nuclei were integrated and put in relation to the internal standard
3-(trimethylsilyl)-1-propanesulfonic acid-d6 sodium salt. The deu-
terolysis of each active ester derivative was measured in 2 to 3
independent experiments. (C) The concentration of the nondeutero-
lyzed active ester derivatives was calculated from the change of peak
integrals and was plotted versus time (1, (2); 9, (5); ], DIG-C6-
NHS; •, biotin-NHSS; 4, biotin-C6-NHSS). Deuterolysis half-lives as
well as the 95% confidence intervals were determined by nonlinear
regression analyses (see Table S-1 in the Supporting Information).
Deuterolysis half-lives of (5) (231 min; mean) and DIG-C6-NHS (248
min, mean) are significantly above deuterolysis half-lives of all other
active esters (one-way ANOVA, Bonferroni post hoc test, P < 0.001)
and do not differ from each other (P > 0.05).
(25) Frey, A.; Di Canzio, J.; Zurakowski, D. J. Immunol. Methods 1998, 221,
35–41
.
Analytical Chemistry, Vol. 81, No. 23, December 1, 2009 9697

Table 1. Limits of Detection (LOD) of Labeled Protein Conjugatesa
limit of detection [pg] of labeled protein
label
2,4-D
primary antibody (clone)
E2/G2
spacer
ovalbumin
insulin
mucin
-
C6
C11
-
C6
C11
-
C6
C11
-
C6
C11
-
171.0b
322.8b
582.1b
171.0b
80.7b
179215.9b
1139.2c
111.9e
125.0
27.9d
42.8f
152.4
14.2d
19.4d
111.4
3.3g
E4/C2
F6/C10
B7
8277.5b
44.9c
292.4b
182.0b
192.8b
250.0b
278.6b
292.4b
479.7b
n.a.
11.7e
170215.9b
1139.2c
111.9e
15.9h
80.7i
9.8h
157398.3b
1550.2c
122.1e
15.6h
n.a.
DIG
1.71.256
n.a.
C6
C11
-
C6
C11
120.5b
n.a.
19678.9j
n.a.
76.2i
n.a.
biotin
174.6b
179.5b
35318.3
57809.6b
28906.8b
1078946.7
220.8
46.5f
125.0
a Ovalbumin and insulin were labeled with active ester derivatives (2), (5), or (6) of 2,4-D (Figure 1) or with comparable derivatives of DIG or
biotin and were immobilized on nitrocellulose or PVDF. Oxidized mucin that had been immobilized on nitrocellulose was labeled with hydrazide
derivatives (7), (10), or (11) of 2,4-D (Figure 2) or with comparable derivatives of DIG or biotin. 2,4-D and DIG labels were detected by specific
primary antibodies and AlexaFluor680-labeled (ovalbumin, mucin conjugates) or horseradish peroxidase-(HRP)-labeled (insulin conjugates) secondary
antibodies; biotin labels were visualized with streptavidin-AlexaFluor680 (ovalbumin, mucin conjugates) (numbers of experiments (Nexp): (7-8) or
HRP-streptavidin (insulin conjugates) (Nexp: 8-12). n.a.: no corresponding labeling reagent available. (95% confidence intervals of LOD are shown
in Table S-2 in the Supporting Information.) b A significantly lower (one-way ANOVA with Bonferroni post hoc test) LOD as compared to protein
labeled with C11-spacered biotin (P < 0.001). c A significantly lower (one-way ANOVA with Bonferroni post hoc test) LOD as compared to protein
labeled with C6-spacered DIG, biotin, C6-spacered biotin, C11-spacered biotin, and 2,4-D (P < 0.001). d A significantly lower (one-way ANOVA with
Bonferroni post hoc test) LOD as compared to protein labeled with C6-spacered DIG, biotin, C11-spacered biotin, and 2,4-D (P < 0.05). e A significantly
lower (one-way ANOVA with Bonferroni post hoc test) LOD as compared to protein labeled with C6-spacered DIG, biotin, C6-spacered biotin,
C11-spacered biotin, 2,4-D, and C6-spacered 2,4-D (P < 0.01). f A significantly lower (one-way ANOVA with Bonferroni post hoc test) LOD as
compared to protein labeled with biotin and C11-spacered biotin (P < 0.01). g A significantly lower (one-way ANOVA with Bonferroni post hoc test)
LOD as compared to protein labeled with all other conjugates (P < 0.001). h A significantly lower (one-way ANOVA with Bonferroni post hoc test)
LOD as compared to protein labeled with C6-spacered DIG, biotin, C6-spacered biotin, C11-spacered biotin, and 2,4-D (P < 0.05). i A significantly
lower (one-way ANOVA with Bonferroni post hoc test) LOD as compared to protein labeled with biotin (P < 0.01). j A significantly lower (one-way
ANOVA with Bonferroni post hoc test) LOD as compared to protein labeled with biotin and C11-spacered biotin (P < 0.01).
For labeling of model proteins, 2,4-D-active esters (2), (5), and
(6) as well as comparable active esters of biotin and DIG were
used. Since not all of these labeling reagents were available as
water-soluble NHSS-esters, the labeling reactions were performed
at neutral to basic pH in the presence of <5% organic solvent. To
find out whether hydrolysis of the active esters under such
conditions might diminish the labeling efficiency, their stability
was measured with a nuclear magnetic resonance (NMR)-based
method that was developed for this task and enabled direct
surveillance of the deuterolysis reaction in deuterated buffer
(Figure 1B,C). The shortest half-life of approximately 25 min was
determined for the nonspacered 2,4-D derivative (2), all other
active esters withstood deuterolysis considerably longer with half-
lives ranging from 1 h up to 4 h (see Table S-1 in the Supporting
Information for exact deuterolysis half-lives). The deuterolysis of
the C11-spacered compounds could not be determined as these
substances precipitated under the experimental conditions. Al-
though the various active esters differ in their stability, which may
influence their labeling efficacy, all measured half-lives appear to
be sufficiently high to provide enough reactive agent for labeling
in aqueous solutions.
Ovalbumin was conjugated to 2,4-D, biotin, or DIG using the
respective active esters described above. The conjugates were
immobilized on nitrocellulose membranes and detected with
fluorophore-labeled detection reagents (see Figure S-5 in the
Supporting Information). During the labeling reaction, the C11-
spacered biotin, but not the C11-spacered 2,4-D derivative (6),
precipitated, indicating that the labeling may not have been as
effective as for the other compounds used. Indeed, the limit of
detection (LOD) of all other conjugates was significantly lower
than that of the biotin-C11-ovalbumin conjugate (ANOVA, Bon-
ferroni post hoc test, P < 0.001) (Table 1). All other conjugates
showed similar LOD, the lowest value, approximately 2 fmol
labeled ovalbumin conjugate, was obtained with derivative (5) and
anti-2,4-D antibody clone E4/C2. The various aliphatic spacers in
the 2,4-D labeling compounds had no discernible effect on the
detectability of ovalbumin. A determination of the degree of
labeling of the ovalbumin conjugates by mass spectrometry was
not possible due to the high molecular mass and the heterogeneity
of the ovalbumin preparation.
Insulin was labeled analogously, and here, the mean degree
of labeling (MDOL) could be determined to be approximately 1.5
mol label/mol insulin. Only (2), (6), and biotin-C11-NHS gener-
ated lower MDOL in the range of 0.6-0.1 mol label/mol insulin
(see Figure S-6 in the Supporting Information). In variation of the
experimental setup, insulin conjugates were immobilized on PVDF
filterplates and visualized with a peroxidase-based detection
To investigate the protein labeling characteristics of the 2,4-
D-active esters and their competitors, we chose ovalbumin and
insulin as model substrates. These proteins have similar isoelectric
points (5.01 vs 5.28) but differ in molecular size (45 vs 5.7 kDa)
and number of lysines per molecule (20 vs 1).
9698 Analytical Chemistry, Vol. 81, No. 23, December 1, 2009

glycoprotein on nitrocellulose membranes. The cis-diol functions
of the carbohydrate moieties of the immobilized mucin were
oxidized, thereby generating aldehyde functions which then were
reacted with hydrazides (7), (10), or (11) or with corresponding
hydrazides of biotin or DIG. The conjugates were detected with
appropriate fluorophore-labeled detection reagents (Figure 2B,C).
The sensitivity of detection was increased up to 35-fold by the
use of alkyl-spacered 2,4-D hydrazides (10) or (11), as compared
to nonspacered 2,4-D hydrazide (7). Applying 2,4-D hydrazide
(10), the LOD was 15-23-fold lower than after biotin or DIG
labeling and allowed the detection of 3 pg (∼15 amol) of labeled
mucin (Table 1).
Effect of Polyethyleneglycol Substructures in Labeling
Compounds. In an attempt to further improve the performance
of the 2,4-D derivatives by increasing their solubility, polyethylene
glycol (PEG11) moieties were introduced in 2,4-D compounds
(1), (3), and (4) in a solid-phase synthesis such that the
aliphatic moiety remained adjacent to 2,4-D. The 2,4-D-PEG11
compounds (12), (13), and (14) (see Figure S-3 in the
Supporting Information) were converted into their corresponding
NHS-esters (15), (16), and (17) which then were used to label
and detect insulin as described above. Albeit the PEG11 moieties
brought about an excellent solubility in aqueous solutions and
improved the sensitivity of detection obtained after labeling with
(2), the sensitivity of detection obtained with the spacered
NHSS-esters (5) and (6) could not be improved further by
introducing PEG11 (see Figure S-7 in the Supporting Informa-
tion).
Double Labeling of Peptides and Evaluation of Binding
between 2,4-D and Anti-2,4-D Antibodies. A double-labeled
13 mer peptide was used to determine the binding parameters
between various 2,4-D derivatives and anti-2,4-D antibodies in situ.
A biotin label was introduced during solid-phase peptide synthesis
via incorporation of lysine-N-ε-(biotin). For the 2,4-D label, peptides
were either derivatized at their amino terminus with 2,4-D
compound (1), (3), (4), (12), (13), or (14) (see Figures S-1-S-4
in the Supporting Information) or at their carboxy terminus with
2,4-D-lysine derivative (19), (20), or (21) (Figure 3A). In an
ELISA-type setup using anti-2,4-D antibodies to capture the
peptides and HRP-labeled streptavidin for detection, the LOD for
2,4-D-labeled peptides without aliphatic spacer ranged from 0.7
to 25 fmol. The aliphatic spacers, but not the PEG-moieties,
lowered the LOD 50-65-fold and allowed the detection of about
100 amol of 2,4-D-labeled peptide (Table 2). In the same system,
the dissociation constants for the complex between 2,4-D deriva-
tives and anti-2,4-D antibodies were determined (Table 2). Here
again, a beneficial effect of the aliphatic spacers on the affinity
was observed, the respective 2,4-D derivatives showing a KD in
the range of 100 pM, while nonspacered variants showed an
approximately 10-fold higher dissociation constant. This effect
was mainly due to a decreased dissociation rate of the spacered
compared to the nonspacered 2,4-D derivatives (Figure 3B) and
gave rise to an up to 8-fold increase of the MaxOD under steady-
state conditions (Table 2).
Figure 2. Labeling of glycoprotein with hydrazide derivatives. (A)
Hydrazide derivatives of 2,4-dichlorophenoxyacetic acid (2,4-D), which
contained either no spacer, (7), an aminohexanoyl (C6) spacer, (10),
or an aminoundecanoyl (C11) spacer, (11), were synthesized and
used for the labeling of oxidized carbohydrates of the glycoprotein
mucin under physiological conditions. (B) Mucin was immobilized on
nitrocellulose, oxidized with NaIO4, and labeled with hydrazide
derivatives (7), (10), or (11), or comparable hydrazide-derivatives
of digoxigenin (DIG) or biotin. 2,4-D and DIG labels were detected
by specific primary antibodies and AlexaFluor680-labeled secondary
antibody whereas biotin labels were visualized with streptavidin-
AlexaFluor680 (Nexp: 7). (C) A representative dot blot with anti-2,4-D
antibody clone E4/C2 and mucin labeled with (10) is shown.
system (see Figure S-5 in the Supporting Information). In contrast
to ovalbumin, the use of spacered 2,4-D derivatives increased the
sensitivity of insulin detection by a factor of up to 1500 compared
to the unspacered 2,4-D derivative (Table 1). Thus, as little as 2
fmol of insulin labeled with 2,4-D derivative (6) could be detected
with anti-2,4-D antibody clone E4/C2. Thereby, the 2,4-D detection
system by far outperformed its competitors DIG and biotin which
required at least 3 pmol of labeled protein to generate a signal.
Labeling of Glycoproteins via Oligosaccharides. For tag-
ging of carbohydrate structures, we devised a specific labeling
strategy where 2,4-D hydrazides are reacted with the oxidized
carbohydrate moieties of a glycoprotein. 2,4-D hydrazide (7)
(Figure 2A) was synthesized by reacting the acid chloride of (1)
with hydrazine hydrate. The alkyl-spacered 2,4-D hydrazides (10)
and (11) (Figure 2A) were generated by first converting 2,4-D
compounds (3) and (4) into their corresponding NHS-esters (8)
and (9) which were subsequently reacted with hydrazine hydrate
(see Figure S-2 in the Supporting Information).
Reversible Labeling of Primary Amines. The possibility to
reversibly label a biomolecule is of particular interest for affinity
purification purposes. To include this option in the 2,4-D toolbox,
2,4-D dimedone (18) was synthesized which can be attached to
The highly glycosylated protein mucin (>200 kDa, 85% carbo-
hydrates) was chosen as substrate molecule for the labeling
reaction, which was carried out in situ after immobilizing the
Analytical Chemistry, Vol. 81, No. 23, December 1, 2009 9699

Figure 3. 2,4-D labeling of peptides. (A) Fmoc-protected lysines were synthesized equipped with 2,4-dichlorophenoxyacetyl (2,4-D) moieties
on their side chains. The 2,4-D moieties either carried no spacer (19), an aminohexanoyl (C6) (20) spacer, or an aminoundecanoyl (C11) (21)
spacer. (B) Peptides (sequence (N- to C-terminus): ASQLDYKMTDAGE) were synthesized in double-tagged form with 2,4-D derivatives (1),
(3), (4), (12), (13), (14), (19), (20), or (21) (see Figures S-1-S-4 in the Supporting Information) and lysine-N-ε-(biotin), respectively (Table
2). The association and dissociation behaviors of the peptides were determined by ELISA (Nexp: 3). Representative association and dissociation
profiles of peptides to anti-2,4-D antibody clone F6/C10 are shown (peptide labeled N-terminally with 2,4-D derivatives (1) (9), (3) (2), or (4)
(1)). (C, D) For reversible 2,4-D labeling, 2,4-D dimedone (18) was synthesized. Membrane anchored peptides (sequence (N- to C-terminus):
GSIGAASMEFCFDCF) derived from ovalbumin were N-terminally labeled with (18) and the bound 2,4-D label was detected with biotinylated
anti-2,4-D-antibody clone E4/C2 and AlexaFluor680-labeled streptavidin. The fluorescence signals were quantitated ((D), inset a) and (18) was
cleaved off by incubation of the membrane with 5% (v/v) hydrazine hydrate. (D) The efficacy of deprotection of (18) was monitored via
measurement of the remaining fluorescence (mean ( SD) at various points in time ((D), inset b). When the 2,4-D tag was completely removed
from the peptides, the peptides were N-terminally acetylated and detected with monoclonal anti-ovalbumin antibody and AlexaFluor680-labeled
secondary antibody ((D), inset c) (Nexp: 6).
Table 2. Binding Characteristics of 2,4-D-Labeled Peptidesa
LOD of labeled peptide [amol]
2,4-D derivative
used for labeling
MaxOD
KD
geometric
mean
95% confidence
interval
labeled peptide
mean SD
[pM] mean SD
2,4-D-Peptide-Lys(biotin)-CONH2
(1)
0.2 0.1
0.7 0.4c
1.5 0.3d
0.2 0.1
0.4 0.2
1.2 0.4d
0.4 0.1
0.6 0.2
1.1 0.3d
1339.0 865.1b
201.4 20.8
163.0 44.6
997.3 589.9e
255.5 91.9
86.7 27.9
160.0 36.8
105.9 28.5
73.6 17.5
24547.1b
1698.2
380.2
12882.5-46773.5
1000.0-2951.2
239.9-602.6
3162.3-23442.3
354.8-3235.9
49.0-588.8
144.5-3235.9
74.1-977.2
24.0-467.7
2,4-D-C6-Peptide-Lys(biotin)-CONH2
(3)
2,4-D-C11-Peptide-Lys(biotin)-CONH2
(4)
2,4-D-PEG11-Peptide-Lys(biotin)-CONH2
2,4-D-C6-PEG11-Peptide-Lys(biotin)-CONH2
2,4-D-C11-PEG11-Peptide-Lys(biotin)-CONH2
Ac-HN-Lys(biotin)-Peptide-Lys(2,4-D)-CONH2
Ac-HN-Lys(biotin)-Peptide-Lys(C6-2,4-D)-CONH2
Ac-HN-Lys(biotin)-Peptide-Lys(C11-2,4-D)-CONH2
(12)
(13)
(14)
(19)
(20)
(21)
8709.6f
1071.5
169.8
676.1
269.2
107.2
a Peptides (sequence (N- to C-terminus): ASQLDYKMTDAGE) were double labeled with 2,4-D derivatives (1), (3), (4), (12), (13), (14), (19),
(20), or (21) (see Figures S-1-S-4 in the Supporting Information) and lysine-N-ε-(biotin) (Lys(biotin)) during synthesis. The peptides were captured
by anti-2,4-D antibody clone F6/C10 on polystyrene microplates and were detected via horseradish peroxidase-labeled streptavidin (Nexp: 6). Maximum
optical density at 450 nm (MaxOD), steady-state dissociation constant KD and limit of detection (LOD) were determined as described. Statistical
analyses were performed by applying a one-way ANOVA and Bonferroni post hoc test. Results of other anti-2,4-D antibody clones tested were
comparable.ThebestaffinityofKD)2.8×10-11 MwasobtainedwithantibodycloneE2/G2incombinationwithAc-HN-Lys(Biotin)-Peptide-Lys(C11-2,4-D)-CONH2.
b Higher KD and LOD than obtained with all other 2,4-D derivatives (P < 0.05) except with derivative (4) (P > 0.05). c Higher MaxOD than obtained
with 2,4-D derivatives without spacer (P < 0.05). d Higher MaxOD than obtained with 2,4-D derivatives without and with C6-spacer (P < 0.05).
e Higher LOD than obtained with all other 2,4-D derivatives (P < 0.05) except with derivatives (1) and (2) (P > 0.05). f Higher KD than obtained
with all other 2,4-D derivatives (P < 0.05) except with derivative (1) (P > 0.05).
and removed from proteinaceous substrates under mild condi-
tions18 (Figure 3C and Figure S-4 in the Supporting Information).
2,4-D dimedone (18) was reacted with the free amino terminus
of cellulose-bound peptides after completion of the solid-phase
9700 Analytical Chemistry, Vol. 81, No. 23, December 1, 2009

peptide synthesis. The labeled peptides were detected with
biotinylated anti-2,4-D antibody and AlexaFluor680-labeled strepta-
vidin. (Figure 3D). The 2,4-D tag was cleaved off with aqueous
hydrazine which resulted in an almost complete removal of the
2,4-D tag after approximately 100 min. The integrity of the peptide
was not compromised by the reversible labeling procedure as
verified by subsequent reaction with peptide-specific antibodies.
Labeling of DNA with 2′-Deoxyuridine-5′-triphosphate
(dUTP) Derivatives. The applicability of the 2,4-D tag was further
expanded by establishing a DNA-labeling system based on dUTP
derivatives of 2,4-D. 2,4-D-11-dUTP (22) (Figure 4A) was synthe-
sized by reacting 5-(3-aminoallyl)-dUTP with the 2,4-D-C6-NHSS-
ester (5) (see Figure S-4 in the Supporting Information) and
incorporated into a DNA fragment via polymerase chain reaction.
For comparison, biotin-11-dUTP and DIG-11-dUTP were used
analogously. A ratio of 80:20 of 2,4-D-11-dUTP to dTTP was
tolerated by Taq-DNA polymerase which was higher than for
biotin-11-dUTP or DIG-11-dUTP (Figure 4B). In dot blot experi-
ments on nylon membranes, approximately 9 amol of a 629 bp
DNA fragment was detectable with the 2,4-D and the DIG systems,
while the LOD for biotin-labeled DNA was 325 amol (Figure 4C).
DISCUSSION
We present a new indirect labeling system which is based on
2,4-dichlorophenoxyacetic acid (2,4-D) derivatives and 2,4-D-
specific antibodies and which is suited for the specific labeling
and sensitive detection of biomolecules in a variety of applications.
In comparison with commonly used labeling reagents, the 2,4-D
system excels in the achievement of particularly low limits of
detection and, in general, compares favorably with the biotin/
streptavidin and the DIG/anti-DIG systems. We attribute this high
quality of performance to a consequent enhancement of the affinity
between the 2,4-D tag and the monoclonal 2,4-D-specific antibodies
which was achieved by linking aliphatic spacer moieties to the
2,4-D labeling molecules. This increase in affinity may be ascribed
to a “spacer-binding” effect, which up to now has mainly been
alleged as an undesirable interference in case of competition-type
hapten immunoassays for the detection of low molecular weight
analytes in environmental probes.19,20 We decided to take advan-
tage of this effect which is presumably due to the fact that
antibodies induced by immunization with hapten-protein conju-
gates also recognize, to a certain extent, the linker structure
between hapten and protein. Such linker structures are often made
up of the carrier protein’s lysine side chains. Yet, 2,4-D coupling
to its target molecule merely via a lysine, as achieved by derivative
(19), did not result in the optimal binding partner for the 2,4-D-
specific antibodies. The appendage of an additional aliphatic chain
to the 2,4-D residue greatly improves the detectability by the
antibodies when combining the spacered 2,4-D-labeling derivatives
with the proper anti-2,4-D antibody. Not only peptides but also
insulin, where the single lysine present is not reactive and the
amino-termini are utilized in the labeling reaction,21 and mucin
Figure 4. 2,4-D labeling of DNA. (A) A 2′-deoxyuridine-5′-
triphosphate (dUTP) derivative equipped with a C6-spacered 2,4-
dichlorophenoxyacetyl (2,4-D) moiety, 2,4-D-11-dUTP (22), was
prepared. (B) A 629 base pair (bp) DNA fragment of murine prion-
DNA was amplified via polymerase chain reaction and Taq-DNA
polymerase in the presence of different ratios of either unlabeled
dUTP/dTTP (upper panel; 0-100% dUTP) or 2,4-D-, biotin-, or
digoxigenin-(DIG)-11-dUTP/dTTP (0-100% labeled dUTP). The
incorporation of the different dUTPs could be visualized by the
band shifts of the PCR fragment toward higher molecular weights
in the ethidium bromide stained agarose gel (dashed red line: level
of unlabeled PCR fragment). (C) The PCR products labeled with
(22), biotin-11-dUTP, or DIG-11-dUTP were purified, and their
concentration was determined after electrophoretic separation by
densitometric scanning. The labeled DNA fragments were serially
diluted and immobilized on nylon membrane. 2,4-D and DIG labels
were detected by specific primary and AlexaFluor680-labeled
secondary antibodies whereas biotin labels were visualized with
streptavidin-AlexaFluor680 (Nexp: 2-4). Limits of detection (LOD;
amol labeled DNA) (mean ( SD) were determined as described
and plotted versus the respective dUTP/dTTP ratios used for
amplification of the DNA fragments (9, (22); 1, DIG-11-dUTP; 2,
biotin-11-dUTP). The respective minimum LOD was determined
by nonlinear regression analyses (one-phase exponential curve
fit). The minimum LOD (lower plateau, 95% confidence interval)
of 2,4-D-labeled DNA (9.2 amol, 2.7-15.7 amol) and DIG-labeled
DNA (7.9 amol, -7.6 to +23.4 amol) was significantly lower than
the minimum LOD of biotin-labeled DNA (324.5 amol, -21.4 to
+670.4 amol) (one-way ANOVA, Bonferroni post hoc test, P <
0.001), whereas the minimum LOD of 2,4-D- and DIG-labeled DNA
did not differ significantly (P > 0.05).
(18) Kellam, B.; Chan, W. C.; Chhabra, S. R.; Bycroft, B. W. Tetrahedron Lett.
1997, 38, 5391–5394
(19) Eremin, S. A.; Lunskaya, I. M.; Egorov, A. M. Bioorg. Khim. (Russ.) 1993,
19, 836–843
(20) Abuknesha, R. A.; Luk, C. Analyst 2005, 130, 956–963
(21) Nakaya, K.; Horinishi, H.; Shibata, K. J. Biochem. (Tokyo) 1967, 61, 337–
344
.
.
.
.
Analytical Chemistry, Vol. 81, No. 23, December 1, 2009 9701

labeled via the carbohydrate residues greatly benefit from the
aliphatic spacers. In a situation, however, where the labeled target
apparently mimics the situation of the hapten-carrier protein
conjugate used for immunization very closely, the positive effect
of the aliphatic spacers may be negligible. This is exemplified by
our labeling of ovalbumin which has a blocked, acetylated
N-terminus and can only be derivatized via its lysines.22 The use
of nonspacered 2,4-D (2) for labeling already establishes a C4-
bridge between 2,4-D and the protein backbone. In this case, the
sensitivity of detection cannot be improved by the use of deriva-
tives (5) or (6) that comprise longer aliphatic spacers.
CONCLUSION
We have developed a new nonradioactive labeling system for
the sensitive detection of biomolecules. The 2,4-D tag is highly
versatile and robust and can be used with excellent results under
all conditions generally involved in the labeling and detection of
nucleic acids, peptides, proteins, or carbohydrates. The new
labeling system is specifically suited for analytical applications
which focus on small target molecules, such as peptides, and, as
a novel member of the bioanalytical toolbox, greatly improves the
possibilities for multilabel setups.
The 2,4-D system demonstrates its power best when used on
substrate molecules that carry only one label, such as the synthetic
peptides, or where numerous labels are rather distant from each
other, as on mucin labeled via its carbohydrate residues. In
connection with substrate molecules that can carry a high number
of labels in relatively close proximity to each other, such as
ovalbumin or the DNA fragment tested, the biotin and DIG/anti-
DIG systems catch up to some extent. This may be due to space
constraints on the labeled substrate molecule which allow binding
of only a limited number of anti-2,4-D antibodies until the target
molecule’s surface is covered completely. By use of smaller
detection reagents such as single-chain antibodies, the 2,4-D
system can probably be pushed to even higher sensitivities.
ACKNOWLEDGMENT
This work was supported by grants from the German Federal
Ministry of Education and Research (BMBF, Grants 01KO0113
and 13N8473) and in part by the German Research Council (DFG,
program project 1089 “Novel vaccination strategies“, Grant Fr958/
4-1).
SUPPORTING INFORMATION AVAILABLE
Additional information as noted in text. This material is
available free of charge via the Internet at http://pubs.acs.org.
Received for review August 23, 2009. Accepted October
14, 2009.
(22) Narita, K.; Ishii, J. J. Biochem. (Tokyo) 1962, 52, 367–373
.
AC901900N
9702 Analytical Chemistry, Vol. 81, No. 23, December 1, 2009