Site-specific conjugation of metal carbonyl dendrimer to antibody and its use as detection reagent in immunoassay

Article abstract of DOI:10.1016/j.ab.2010.08.027

We describe here the conjugation of polyclonal goat anti-rabbit antibody to generation 4 polyamidoamine (G4-PAMAM) dendrimers carrying (i) (η⁵-cyclopentadienyl) iron dicarbonyl succinimidato complexes as infrared (IR) probes, (ii) nitroaniline

Full text of DOI:10.1016/j.ab.2010.08.027

Analytical Biochemistry 407 (2010) 211–219
Contents lists available at ScienceDirect
Analytical Biochemistry
journal homepage: www.elsevier.com/locate/yabio
Site-specific conjugation of metal carbonyl dendrimer to antibody and its use
as detection reagent in immunoassay
Nathalie Fischer-Durand a, Michèle Salmain a, Bogna Rudolf b, Lili Dai a, Lauriane Jugé c, Vincent Guérineau d,
Olivier Laprévote d, Anne Vessières a, , Gérard Jaouen a
⇑
a Chimie ParisTech (Ecole Nationale Supérieure de Chimie de Paris), Laboratoire Charles Friedel, CNRS UMR 7223, 75005 Paris, France
b Department of Organic Chemistry, University of Lodz, 91-403 Lodz, Poland
c Chimie ParisTech, Service de RMN, 75231 Paris Cedex 05, France
d Centre de recherche de Gif, Institut de Chimie des Substances Naturelles, CNRS, 91198 Gif sur Yvette Cedex, France
a r t i c l e i n f o
a b s t r a c t
Article history:
Received 9 June 2010
Received in revised form 28 July 2010
Accepted 20 August 2010
Available online 15 September 2010
We describe here the conjugation of polyclonal goat anti-rabbit antibody to generation 4 polyamido-
amine (G4–PAMAM) dendrimers carrying (i) (g
5-cyclopentadienyl) iron dicarbonyl succinimidato com-
plexes as infrared (IR) probes, (ii) nitroaniline entities as nuclear magnetic resonance (NMR) probes,
(iii) acetamide groups for surface neutralization, and (iv) hydrazide-terminated spacer arms for the reac-
tion with aldehyde. To preserve a high binding affinity, the conjugation was performed on the carbohy-
drate moieties located on the Fc fragment. The resulting conjugates were characterized by Fourier
transform–IR, ultraviolet (UV), and high-mass matrix-assisted laser desorption/ionization time-of-flight
(MALDI–TOF) mass spectrometry. On the basis of relative concentration ratios of IR probes and antibody,
an average labeling of 30 IR probes per antibody was reached (i.e., more than twice the value obtained
with our previous strategy that generated no spacer arm). Immunoassays revealed that the antibody–
dendrimer conjugates retained 55.1% of immunoreactivity on average with respect to underivatized
antibody. Finally, the conjugates were used to quantify their antigen by solid-phase carbonyl metallo
immunoassay (CMIA). Results showed a significant enhancement of the IR signal, demonstrating the effi-
ciency of the new conjugation strategy and the potential of the new antibody–dendrimer conjugates as
universal immunoanalytical reagents.
Keywords:
Antibody labeling
Dendrimers
Carbonyl ligands
Immunochemistry
IR spectroscopy
Ó 2010 Elsevier Inc. All rights reserved.
Dendrimers, and particularly polyaminoamine (PAMAM)1 den-
drimers [1–4], are playing an increasing role in the field of bioconju-
gation. This growing interest is attested by the number of articles
and reviews devoted to this subject and is illustrated by a specific
chapter titled ‘‘Dendrimers and Dendrons,” including their mode of
conjugation, in the 2008 second edition of the well-known book Bio-
conjugate Techniques [5]. Their popularity in the field is due to their
water solubility, their lack of immunogenicity, their availability in a
variety of sizes (termed generations), and their versatile surface
functionalities. These properties make dendrimers ideal platforms
for the construction of complex protein conjugates for a wide range
of applications, including drug delivery [6–18] diagnosis [19–23],
biosensing interfaces for the design of biosensors [24–29], and signal
enhancement in immunoassays [30–32]. Some of these applications
involved the conjugation between engineered dendrimers and anti-
bodies so as to introduce a high number of active compounds on the
antibodymolecule or immunoglobulin G (IgG) so as to improve a given
effect (e.g., fluorescence signal, delivery of therapeutics). Most of the
previously reported conjugation strategies involved the use of hetero-
bifunctional cross-linkers such as sulfosuccinimidyl-4-(N-maleimi-
domethyl)cyclohexane-1-carboxylate (sulfo-SMCC) and N-succini-
midyl-3-(2-pyridyldithio)propionate (SPDP) to introduce maleimide
[23,33–38] and thiol [39,40] groups on the IgG molecule. These conju-
gation procedures involve amine groups that are located uniformly on
the surface of the immunoglobulin, thereby leading to random addi-
tion of the cross-linker and to potential blockingof the antigen binding
site. On the contrary, one of the possible site-selective conjugation
strategies takes advantage of the carbohydrate chains located in the
Fc region, thereby preserving the remote antigen binding sites. Mild
oxidation of the carbohydrate moieties generates aldehyde groups
allowing conjugation reactions with amines or hydrazides. Only a
⇑
Corresponding author. Fax: +33 1 43 26 00 61.
E-mail address: a-vessieres@chimie-paristech.fr (A. Vessières).
Abbreviations used: PAMAM, polyaminoamine; IgG, immunoglobulin G; sulfo-
1
SMCC, sulfosuccinimidyl-4-(N-maleimidomethyl) cyclohexane-1-carboxylate; SPDP,
N-succinimidyl-3-(2-pyridyldithio) propionate; CMIA, carbonyl metallo immunoas-
say; IR, infrared; G4, generation 4; NMR, nuclear magnetic resonance; FT, Fourier
transform; UV, ultraviolet; MALDI–TOF, matrix-assisted laser desorption/ionization
time-of-flight; DIPEA, N,N-diisopropylethylamine; TSTU, N,N,N’,N’-tetramethyl-O-(N-
succinimidyl) uronium tetrafluoroborate; Vis, visible; NaPB, sodium phosphate
buffer; MeOH, methanol; SA, sinapic acid; DMF, dimethylformamide; PBS, phos-
phate-buffered saline; HRP, horseradish peroxidase; b-LG, b-lactoglobulin.
0003-2697/$ - see front matter Ó 2010 Elsevier Inc. All rights reserved.
doi:10.1016/j.ab.2010.08.027

212
Conjugation of metal carbonyl dendrimer to antibody / N. Fischer-Durand et al. / Anal. Biochem. 407 (2010) 211–219
few authors reported the linkage of dendrimers to antibodies using
this site-directed chemical reaction [41–44]. Whatever the conjuga-
tion strategy, coupling ratios of approximately 1 dendrimer per IgG
were reported.
We are currently involved in the labeling of antibodies with tran-
sition metal carbonyl complexes using amine-terminated PAMAM
dendrimers as label carriers. These antibody–dendrimer metal car-
bonyl complexes are intended to be used as new universal detection
reagents in a modified solid-phase format of the carbonyl metallo
immunoassay (CMIA) that we introduced during the early 1990s
[45–49]. CMIA takes advantage of the unique signals generated by
transition metal carbonyl complexes that display intense vibration
PEA), acetic anhydride, 1,1,1-trifluoroethanol, and N,N,N’,N’-
tetramethyl-O-(N-succinimidyl)uronium tetrafluoroborate (TSTU)
were purchased from Sigma–Aldrich. Fp–maleimide was synthe-
sized as described previously [51]. Dialysis was performed with
Spectra/Por 1 membranes with a molecular weight cutoff of 6000
to 8000 Da (Spectrum Laboratories). Nitrocellulose membranes
(pore size 0.45
162-0117). Flash chromatography was performed on silica gel 60
(40–63 m, Merck). Gel filtration column Superdex 200 HiLoad
lm) were purchased from Bio-Rad (product no.
l
16/60 and 5-ml HiTrap desalting column were purchased from
GE Healthcare. UV/visible (Vis) spectra were recorded on a UV/
mc2 spectrometer (Safas, Monaco). NMR spectra were recorded
on Bruker Avance 300 and Avance 400 spectrometers. Sodium
phosphate buffer (NaPB) was a 10 mM NaPB (pH 7.2) containing
0.2 M NaH2PO4 (11 ml) and 0.2 M Na2HPO4 (39 ml) diluted to 1 L
of deionized water with 0.15 M NaCl. Citrate buffer (pH 6.0) was
10 mM citrate buffer containing 130 mM NaCl.
bands
2200 cmÀ1) where few other molecules absorb, thereby allowing
their quantification by measuring the height of the CO bands, which
(mCO) in the mid-infrared (IR) spectral range (1800–
m
is proportional to the quantity of complex. As a consequence, the
more metal carbonyl complexes (IR probes) are linked to the anti-
body, the higher the resulting IR signal is. In this regard, PAMAM
dendrimers are ideal carriers to covalently couple multiple IR probes
to antibodies. We previously described thesite-directed conjugation
of PAMAM–(NH2)64 or generation 4 (G4)–PAMAM carrying 10 to 25
FT–IR spectroscopy
FT–IR spectra were recorded on a benchtop Bruker Tensor 27 IR
spectrometer equipped with a DTGS (deuterated triglycine sulfate)
detector and a 6-mm-diameter membrane holder perpendicularly
positioned with respect to the IR beam. FT–IR data were recorded
and manipulated on a Windows-operating PC using OPUS 6.5 soft-
ware. Routinely, 44 scans were co-added in 40 s, and the resulting
interferogram was apodized using a Blackman–Harris three-term
function and then Fourier-transformed to yield a 4-cmÀ1 resolution
spectrum.
(g
5-cyclopentadienyl) iron dicarbonyl succinimidato (Fp) units to
oxidized goat anti-rabbit IgG through reductive amination using
the remaining surface amines of the dendrimer [44]. This strategy
afforded immunoconjugates with a loading of metal carbonyl den-
drimer highly dependent on the steric hindrance around the free
amino groups on the surface of the dendrimer. We showed that
the dendrimer carrying 10 Fp complexes led to the attachment of
1.4 dendrimers per IgG, whereas the dendrimer carrying 25 Fp com-
plexes led to the attachment of only 0.5 dendrimer per IgG. In other
words, we were able to bind 13 Fp units per IgG on average. To fur-
ther enhance the labeling of antibodies with Fp complexes, thereby
affording a larger amplification of the IR signal, and to fully exploit
the 64 potential conjugation sites of G4–PAMAM, we then designed
a more advanced generation of PAMAM dendrimers carrying 20 to
30 Fp complexes and a functionalized spacer arm [50] intended to
move the dendrimer away from the IgG so as to decrease the steric
hindrance between the two macromolecules. The spacer encom-
passed a nitroaromatic entity whose aromatic protons served as nu-
clear magnetic resonance (NMR) probe to estimate the average
number of substituents conjugated at each functionalization step
(as a consequence, all of the coupling ratios reported here corre-
spond to average, and not absolute, values). It was terminated by a
hydrazide function known to react with aldehydes at slightly acidic
pH, thereby avoiding self-condensation of oxidized IgG with amine
residues that may occur at neutral or basic pH.
The calibration curve used for the quantification of the metallo-
dendrimers and the immunoconjugates was established as follows.
Standard solutions of Fp–maleimide in the range of 50 to
660 nmol/ml in 10 mM NaPB (pH 7.2) were spotted (4
punched nitrocellulose membranes (diameter = 6 mm). A blank
was made by spotting 4 l of NaPB. Membranes were dried for at
ll) onto
l
least 2 h at room temperature before IR recording in the range of
1800 to 2200 cmÀ1. The calibration curve was constructed by plot-
ting the absorbance at 2052 cmÀ1 versus the concentration of the
standard solutions of Fp–maleimide. This experiment was repeated
several times and gave reproducible standard straight lines.
Quantification of Fp entities in the metallodendrimer solutions
was carried out as follows. The conjugate was dissolved in 2 ml of
methanol (MeOH) and then diluted appropriately with 10 mM NaPB
(pH 7.2). Then 4
membranes and dried. A blank membrane was prepared by spotting
l of NaPB. In the case of antibody–dendrimer conjugate samples,
ll in duplicate was spotted onto nitrocellulose
4
l
In the current article, we describe the conjugation of oxidized
goat anti-rabbit IgG to our new hydrazide-based Fp-labeled G4–PA-
MAM dendrimers whose improved synthetic route is also reported.
The new immunoconjugates were analyzed by Fourier transform
(FT)–IR, ultraviolet (UV), and high-mass matrix-assisted laser
desorption/ionization time-of-flight (MALDI–TOF) mass spectrome-
try to evaluate the efficiency of the new dendrimer design on the
coupling ratio. Immunoreactivity studies were then conducted for
the first time on these antibody–metallodendrimer conjugates to as-
sess the influence of the dendrimer loading and the coupling ratio on
their affinity for rabbit IgG. Finally, they were involved in the quan-
tification of rabbit IgG by solid-phase CMIA to assess the efficiency of
the new bioconjugation strategy on the intensity of the IR signal.
the final solution after purification and concentration was diluted 4
times in 10 mM NaPB (pH 7.2). FT–IR spectra were then recorded
between 1800 and 2200 cmÀ1. Absorbances at 2052 cmÀ1 were used
to calculate the average concentration of Fp entities using the cali-
bration curve established above.
MALDI–TOF mass spectrometry
A Voyager DE-STR MALDI–TOF mass spectrometer (Perseptive
Biosystems, Framingham, MA, USA), equipped with a 337-nm
pulsed nitrogen laser (20 Hz) and an Acqiris 2-GHz digitizer board,
was used for all experiments.
The detection was done with a dedicated high-mass detection
system (HM1, CovalX, Zurich, Switzerland). This system, which is
based on unique conversion dynode technology, enhances sensitiv-
ity in the high-mass range up to 100 kDa and avoids detector sat-
uration due to intense low-mass (e.g., matrix) species [52]. The low
saturation is critical to ensure detection over a broad dynamic
range.
Materials and methods
Materials
Amine-terminated G4–PAMAM dendrimer ethylenediamine
core, 4-fluoro-3-nitrobenzoic acid, N,N-diisopropylethylamine (DI-

Conjugation of metal carbonyl dendrimer to antibody / N. Fischer-Durand et al. / Anal. Biochem. 407 (2010) 211–219
213
Mass spectra were obtained in linear positive ion mode with the
following settings: accelerating voltage of 25 kV, grid voltage 93%
of accelerating voltage, and extraction delay time of 700 ns. The la-
ser intensity was set just above the ion generation threshold to ob-
tain peaks with the highest possible signal-to-noise ratio without
significant peak broadening.
The mass spectrometer was externally calibrated using the pro-
tonated monomer and dimer of IgG. All data were processed using
the Data Explorer software package (Applied Biosystems).
Sinapic acid (SA) was used as the matrix. The SA matrix solution
8.0, or 7.17 ppm versus the 744 protons of G4–PAMAM at 2.40,
2.61, and 2.82 ppm. The dendrimer was then allowed to react with
Fp–maleimide (40 equiv) in MeOH at room temperature in the
dark for 3 to 4 days and was purified by dialysis in MeOH. The
resulting conjugate was analyzed (i) by 1H NMR to assess the aver-
age number of Fp labels attached per dendrimer, (ii) by FT–IR spec-
troscopy to quantify the concentration of Fp entities according to
the procedure described in the ‘‘FT–IR spectroscopy” section above,
leading to the concentration of the dendrimer based on the cou-
pling ratio estimated by 1H NMR spectroscopy (80% yield from
commercial G4–PAMAM).
was prepared by dissolving 10 mg in 500
acetonitrile. Antibody–dendrimer conjugates were extensively dia-
lyzed in 20 mM ammonium acetate before analysis. Then 1 l of
the analyte and 9 l of the matrix solution were mixed together,
and 1 l of the resulting solution was spotted on the MALDI plate
ll of water and 500 ll of
l
Synthesis of PAMAM–G4(ArHydrNHBoc)xFpyNHAcz
This procedure was described in our previous report [50].
Removal of the t-Boc protective group
l
l
and air-dried (‘‘dried droplet” method).
Precautions for handling dendrimers
The reaction was performed as described previously [50], but a
different workup was applied; at the end of the reaction time, the
The functionalized dendrimers described in this article must be
kept in solution at all times otherwise it is not possible to dissolve
them again. Consequently, compounds cannot be thoroughly dried
before NMR recording. Accordingly, to eliminate MeOH traces,
each sample is evaporated to dryness for no longer than 1 min
and then is dissolved two times with 0.8 ml of MeOH-d4 and evap-
orated. Because functionalized dendrimers were not unimolecular,
their characterization by NMR represented the average value of
their polymeric distribution.
reaction mixture was diluted with H2O (280
adjusted to approximately 6.0 with DIPEA (130
l
l) and the pH was
ll) to yield a clear or-
ange solution. This solution was dialyzed in 50 mM AcONa +
150 mM NaCl (pH 5.0) for 18 h and purified on a gel filtration column
(Superdex 200 HiLoad 16/60) [50]. A calibration curve at 280 nm
was established by plotting A280 versus standard concentrations of
this dendrimer solution in the range of 0.312 to 20 nmol/ml for fur-
ther characterization of the antibody–dendrimer conjugates.
Synthesis of hydrazine carboxylic acid, 2-[6-(4-fluoro-3-
nitrobenzoyl)amino-1-oxohexyl]-1-tert-butyl ester (1)
Preparation of antibody–dendrimer conjugates
NaIO4 (0.1 M) in citric acid solution (10 mM, 24.7
to a solution of goat anti-rabbit IgG (2 mg) in NaPB (142.4
the pH was adjusted to 3.7 with citric acid solution (10 mM, 80
This solution was incubated for 1 h at room temperature in the
dark and then quickly purified on a 5-ml HiTrap fast desalting col-
umn (50 mM AcONa + 150 mM NaCl, pH 5.0). Oxidized IgG solu-
tion was concentrated and transferred into 0.1 M AcONa (pH 5.1)
and allowed to react with the PAMAM–G4(ArHydrNH2)xFpyNHAcz
conjugate (8 mol equiv) at room temperature in the dark for
l
l) was added
l), and
l).
DIPEA (98.5
l
l, 0.56 mmol) and TSTU (136 mg, 0.45 mmol) were
l
added to a solution of 4-fluoro-3-nitrobenzoic acid (69.8 mg,
0.377 mmol) in dimethylformamide (DMF, 2 ml). The solution
was stirred at room temperature for 45 min, and then hydrazine
carboxylic acid [2-(6-amino-1-oxohexyl)-1-tert-butyl ester] pre-
pared previously [50] (92.4 mg, 0.377 mmol) in DMF (1 ml) was
added dropwise and the reaction mixture was stirred at room tem-
perature for 7 h. EtOAc (15 ml) and a saturated aqueous solution of
NaHCO3 (15 ml) were added, and the aqueous layer was extracted
with EtOAc (3 Â 15 ml). Organic layers were combined and washed
with water (2 Â 20 ml) and then dried (Na2SO4). The crude com-
pound was purified by flash chromatography on silica gel (EtOAc,
Rf = 0.35) to afford compound 1 as a pale yellow oil (50 mg, 30%
yield): 1H NMR (300 MHz, CDCl3) d 1.33 (s, 9 H), 1.38 (m, 2 H),
1.53–1.68 (m, 4 H), 2.20 (t, J = 7.20 Hz, 2 H), 3.38 (q, J = 6.0 Hz, 2
H), 6.79 (s, NH), 7.28 (dd, JHF = 10.3 Hz, JHH = 8.8 Hz, H5Ar), 8.16
(ddd, JHH = 2.2 Hz, JHF = 4.2 Hz, JHH = 8.6 Hz, H6Ar), 8.55 (dd,
JHF = 7.0 Hz, JHH = 2.2 Hz, H2Ar); 13C NMR (75.5 MHz, CDCl3) d
24.21, 25.95, 28.05, 28.41, 33.42, 39.87, 81.82, 118.65 (d,
JCF = 21.1 Hz), 125.10, 131.51, 134.90 (d, JCF = 9.1 Hz), 136.96 (d,
JCF = 8.0 Hz), 155.75, 156.95 (d, JCF = 269.3 Hz), m/z (CI/NH3) 430
(40%, [M+NH4]+), 413 (25%, [M+H]+), 374 (10), 357 (15), 330 (20),
313 (100); HRMS (CI/CH4) m/z calculated for C18H26O6N4F (M+H)+
413.1836, observed 413.1853.
l
18 h. NaBH3CN (2.5 M in H2O, 32
was added, and the incubation was pursued for 2.5 h before etha-
nolamine–HCl (1 M in H2O, pH 10.3, 32 l) was added and the
ll, final concentration = 100 mM)
l
incubation was continued for 2 h. The solution was dialyzed in
50 mM AcONa + 150 mM NaCl (pH 5.0) at 4 °C for 24 h and then
purified on a gel filtration column (Superdex 200 HiLoad 16/60)
using the BioLogic DuoFlow chromatography system (Bio-Rad)
equipped with an analytical UV flow cell. The purification was
done in 50 mM AcONa + 150 mM NaCl (pH 5.0) at a flow rate of
1 ml/min (fraction size = 0.5 ml from 56 to 80 min). The course of
the purification was monitored by UV at 280 nm, and fractions be-
tween 59 and 70 ml (see chromatogram in Fig. 1) were combined,
concentrated, dialyzed in phosphate-buffered saline (PBS, pH 7.4),
and analyzed by FT–IR to quantify the concentration of Fp entities,
thereby leading to the dendrimer concentration. Absorbance mea-
surement at 280 nm afforded the IgG concentration (A280 = 1.4 for
1 mg/ml solution) after subtraction of the contribution of the den-
drimer obtained from the previously established calibration curve
A280 = f[dendrimer].
Synthesis of PAMAM–G4(ArHydrNHBoc)xFpy
Compound 1 (5 equiv) and DIPEA (10 equiv) were added to a
solution of G4–PAMAM in MeOH and stirred at room temperature
for 48 h. The resulting conjugate was purified by dialysis in MeOH
and analyzed by 1H NMR (see ‘‘Precautions for handling dendri-
mers” section above) to estimate the average number of aromatic
residues covalently linked to the dendrimer amino groups through
comparative peak integration of one of the aromatic protons at 8.7,
Immunoreactivity of the antibody–dendrimer conjugates
Binding of antibody–dendrimer conjugates to rabbit IgG-coated
microplates was compared with binding of unmodified goat anti-
rabbit IgG in the following manner. A rabbit IgG solution (100
ll/
well, 5 g/ml, in PBS, pH 7.4) was pipetted in a 96-well microtiter
l

214
Conjugation of metal carbonyl dendrimer to antibody / N. Fischer-Durand et al. / Anal. Biochem. 407 (2010) 211–219
spacer arm (N-Boc protected) and the nitroaromatic NMR probe.
For this purpose, compound 1 was first prepared independently
by acylation of the aminohydrazide spacer with the activated ester
of 4-fluoro-3-nitrobenzoic acid (Scheme 1, Eq. A) and was allowed
to react with the dendrimer (Scheme 1, Eq. B). Labeling with Fp–
maleimide, acetylation of the remaining accessible primary amino
groups of the dendrimer and cleavage of the N-Boc protective
group were then carried out according to established procedures
[50]. This new synthetic route brought several advantages com-
pared with the previous one [50]: (i) it reduced the number of
steps from five to four; (ii) it prevented polymerization or aggrega-
tion observed when the acylation of the aminohydrazide spacer
was carried out on the dendrimer, leading to an overall yield twice
as high as that first reported; and (iii) it brought functional homo-
geneity on the dendrimer surface because all of the nitroaromatic
residues now carry the hydrazide-terminated spacer brought by
compound 1.
Fig.1. Chromatographic profile obtained for the purification of IgG–dendrimer
conjugate from excess dendrimer with a Superdex 200 HiLoad column at a flow rate
of 1 ml/min.
Coupling of the hydrazide-based Fp-labeled G4–PAMAM dendrimer to
goat anti-rabbit IgG and characterization of IgG–dendrimer conjugates
plate (F-bottom, Greiner) and left overnight at 4 °C. Nonspecific
binding was further blocked by PBS + 4% goat serum (200
Goat anti-rabbit IgG or antibody–dendrimer conjugate dilutions
ranging from 0.23 to 120 g/ml were prepared in 50 mM cit-
rate + 130 mM NaCl (pH 5.0, citrate buffer) with 2% goat serum,
applied to the wells in duplicate (100 l/well), and incubated for
ll/well).
The conjugation procedure was based on the orthogonal liga-
tion between aldehyde groups of goat anti-rabbit IgG generated
by mild oxidation of the carbohydrate moieties [44,53] and hydra-
zide residues of the dendrimers (Scheme 2). After stabilization of
the hydrazone linkage by reduction with NaBH3CN and the addi-
tion of ethanolamine to block the remaining free aldehyde groups
[5], the immunoconjugate was isolated from excess dendrimer
using size exclusion gel chromatography (Fig. 1).
l
l
2 h at room temperature in the dark. Wells were washed four times
with citrate buffer + 0.05% Tween 20, and then goat anti-rabbit
IgG–horseradish peroxidase (HRP) conjugate diluted 1:8000 in cit-
rate buffer + 2% goat serum was applied to the wells (100 ll/well)
for 2 h at room temperature in the dark. Wells were washed as
above, and a 0.7-mg/ml solution of o-phenylenediamine in cit-
rate–phosphate buffer (pH 5.0) + 0.012% (v/v) H2O2 was applied
To evaluate the average number of dendrimers conjugated per
IgG, concentrations of both IgG and dendrimer in the conjugate
solutions were then estimated by combining UV and FT–IR
spectroscopies. First, the concentration of Fp labels was quantified
by FT–IR. Solutions in PBS were spotted on nitrocellulose mem-
branes, and then dried membranes were analyzed (Fig. 2A). The
2052-cmÀ1 peak height was measured and then related to the
concentration of Fp units using the calibration curve established
from standard solutions of Fp–maleimide in the range of 50 to
660 nmol/ml (Fig. 2B). This concentration gave access to the esti-
mated concentration of the dendrimer knowing the average num-
ber of Fp units attached per dendrimer determined independently
by NMR measurements [50]. On the other hand, for each dendri-
mer solution ready for antibody coupling, a UV calibration curve
was established by plotting the absorbance at 280 nm (A280) versus
dendrimer concentration in the range of 0.625 to 20 nmol/ml. This
calibration curve allowed us to determine the contribution of the
dendrimer absorption at 280 nm in the conjugate solution know-
ing its concentration from FT–IR quantification. Then A280 of the
IgG in the conjugate solution was easily determined as the differ-
ence between total A280 of the conjugate solution and A280 of the
to the wells (100
and the enzymatic reaction was stopped by the addition of 2.5 M
H2SO4 (50 l/well). The absorbance was read at 490 nm with a
ll/well). The color was developed for 10 min,
l
microtiter plate reader (model 550, Bio-Rad).
Quantification of rabbit IgG on nitrocellulose membrane by CMIA
Rabbit IgG solutions (0.2–40 lg IgG) were spotted onto punched
nitrocellulose disks (6 mm diameter) with the amount of b-lacto-
globulin (b-LG) solution (prepared at 10 mg/ml) necessary to coat
each membrane with the same total amount of protein (40
lg).
Two other membranes were coated with b-LG (40 g): one to pro-
l
vide a reference IR spectrum (reference) and the other to measure
nonspecific interactions (blank). Membranes were air-dried for 1 h
and then incubated with blocking buffer (NaPB + 4% skimmed milk)
for 90 min at room temperature. Membranes were washed two
times with citrate buffer + 1.25% skimmed milk (0.8 ml/membrane
in microtube with stirring). Each membrane was then incubated
for 2 h at room temperature in the dark with Fp-labeled goat anti-
rabbit IgG (0.3 nmol) in diluted blocking buffer (citrate buffer [pH
dendrimer (e
IgG = 210,000 molÀ1 cmÀ1).
Two conjugates were also analyzed by MALDI–TOF mass spec-
trometry using a dedicated high-mass detection system (HM1)
allowing enhanced sensitivity in the high-mass range and avoiding
detector saturation due to intense low-mass species (e.g., matrix)
[52,54]. Fig. 3 shows the mass spectra of the underivatized IgG (pa-
nel A) and two antibody–dendrimer conjugates with conjugation
rates of 0.5 for conjugate 1 (panel B) and 1.2 for conjugate 2 (panel
C) as estimated by UV and FT–IR measurements. Figs. 3B and 3C
display broad peaks around m/z 168,000, corresponding to the
antibody labeled with 1 dendrimer. Peaks are broadened, as is usu-
ally observed with PAMAM dendrimers higher than generation 3
[50,55–57], and this feature is retained even when conjugated to
a protein. Underivatized IgG was present in a large amount in con-
jugate 1 (Fig. 3B), consistent with the estimation of 0.5 dendrimer
per IgG determined by combining UV and FT–IR quantification.
6.0] + 2% skimmed milk, 150 ll) except for the reference membrane,
which was incubated without antibody. Membranes were washed
five times with citrate buffer + 1.25% skimmed milk (0.8 ml/mem-
brane in microtube with stirring) and air-dried for at least 2 h before
IR analysis. Absorbances at 2052 cmÀ1 were corrected by measure-
ments taken from the blank.
Results and discussion
A new synthetic route to homogeneous hydrazide-based Fp-labeled
G4–PAMAM dendrimer
The first functionalization step of G4–PAMAM dendrimer con-
sisted in the combined introduction of the hydrazide-terminated

Conjugation of metal carbonyl dendrimer to antibody / N. Fischer-Durand et al. / Anal. Biochem. 407 (2010) 211–219
215
Scheme 1. Improved synthetic route to the hydrazide-based trifunctional G4–PAMAM dendrimer.
Conversely, nearly all of the IgG molecules appear to be derivatized
in conjugate 2, also consistent with the estimation of 1.2 dendrimers
per IgG determined by UV and FT–IR quantification. Moreover, in
this latter conjugate, a peak around m/z 183,000 can be assigned
to the antibody labeled with two dendrimers. Our method combin-
ing UV and FT–IR measurements to estimate the average number of
dendrimers attached per antibody is in agreement with the distribu-
tion patterns obtained by MALDI–TOF mass spectrometry.
To optimize the multilabeling of the antibody, we studied the
influence of pH, temperature, and reaction time on the conjugation
reaction. First, Table 1 shows the number of dendrimers linked per
antibody when the conjugation was carried out in acetate buffer at
pH 6.5 or 5.1. Results clearly show that lowering the pH of the reac-
tion buffer has a positive effect on the coupling rate regardless of
the average number of Fp units attached to the dendrimer. Second,
with the conjugation reaction being fixed at pH 5.1, the effect of
temperature was studied. With a dendrimer carrying 28 Fp units,
an average conjugation ratio of 0.6 dendrimer per antibody was
obtained when the macromolecules were reacted for 4 h at room
temperature, and then for 14 h at 4 °C, versus 1.0 dendrimer per
antibody when the reaction mixture was left for 18 h at room tem-
perature, indicating that the reaction was slow.
Experimental conditions being fixed at pH 5.1 and room tem-
perature for 18 h, Table 2 shows the number of dendrimers linked
per antibody starting from two dendrimer samples carrying 28 and
22 Fp units (i.e., an average of 25 Fp units). Average coupling ratios
of 1.0 and 1.5, respectively, were obtained, indicating a positive ef-
fect of the spacer given that we previously reported a conjugation
ratio of 0.5 with a dendrimer without spacer carrying 25 Fp units
[44]. Regarding the average number of Fp probes linked per anti-
body, we were able to prepare immunoconjugates with averages
of 33 and 28 Fp probes with G4(ArHydr)4Fp22NHAc21 and G4(Ar-
Hydr)5Fp28NHAc18, respectively. An average of 30 Fp probes per
antibody represents more than twice the number of 13 previously
reported without the spacer. This result clearly shows that the
newly engineered dendrimer significantly improved the loading
in IR probes of the immunoconjugates.
Immunoreactivity
The immunoreactivity of the antibody–dendrimer conjugates
was then determined by a two-step immunoassay with micro-
plates coated with rabbit IgG. The first incubation step was per-
formed by the addition of standard solutions of commercial

216
Conjugation of metal carbonyl dendrimer to antibody / N. Fischer-Durand et al. / Anal. Biochem. 407 (2010) 211–219
Scheme 2. Synthetic route to IgG–dendrimer conjugates.
(underivatized) or dendrimer-conjugated goat anti-rabbit IgG. Per-
oxidase-conjugated goat anti-rabbit IgG was incubated in a second
step, and the immunoreactivities relative to the commercial IgG
were calculated from the corresponding IC50 values. An assay
was performed with the dendrimer alone to assess the level of
nonspecific interaction between the dendrimer and the sensitized
plate and/or between the dendrimer and the secondary antibody.
The working concentrations in the assay were equal to those of
the dendrimer in the conjugate samples taking into account the
conjugation rate. It was shown that running the assay in classical
PBS buffer at pH 7.4 led to nonspecific interactions (the engineered
dendrimer tends to stick to the protein coated to the plate, leading
to the formation of a sandwich coated protein/dendrimer/second-
ary antibody, presumably owing to electrostatic interactions),
whereas running the assay at pH 5.0 in citrate buffer eliminated
nonspecific interactions at the working concentrations used. Con-
sequently, all of the assays were performed at pH 5.0 in citrate buf-
fer. The immunoreactivity of the antibody–dendrimer conjugates
was found to be between 41.4 and 66.2% of that of the underiv-
atized antibody depending on the degree of antibody derivatiza-
Fig.3. High-mass MALDI–TOF mass spectrometry for underivatized IgG (A), IgG–
[G4(ArHydr)4Fp29NHAc14 (B), and IgG–[G4(ArHydr)4Fp22NHAc21 (C).
]
0.5
]
1.2
tion (Table 3), with an average immunoreactivity of 55.1%. A high
labeling decreased the immunoreactivity, as evidenced by entry 5
in Table 3, although the conjugation strategy was site selective to
Fig.2. Quantification of Fp labels by FT–IR spectroscopy on nitrocellulose membranes. (A) Here 4
l
l of the following solutions was deposited and left to dry: IgG–
[G4(ArHydr)4Fp29NHAc14 5.4 nmol/ml (a); IgG–[G4(ArHydr)4Fp22NHAc21 11.8 nmol/ml (b); IgG–[G4(ArHydr)3Fp26NHAc9]1.3,
]
0.6
,
]
1.3
,
5.7 nmol/ml (c). The 2052 cmÀ1
‘‘analytical peak” height is proportional to the concentration of the Fp complex. a.u., arbitrary units. (B) Calibration curve of Fp–maleimide (Fpm) in the range of 50 to
660 nmol/ml.

Conjugation of metal carbonyl dendrimer to antibody / N. Fischer-Durand et al. / Anal. Biochem. 407 (2010) 211–219
217
Table 1
Number of dendrimers linked per antibody when the conjugation was carried out at
pH 6.5 or 5.1.
Dendrimer
pH 6.5
pH 5.1
G4(ArHydr)4Fp29NHAc14
G4(ArHydr)4Fp22NHAc21
0.6 0.2
1.0 0.2
1.0 0.2
1.3 0.2
Note. Values are means standard deviations (n = 2).
Table 2
Number of dendrimers and Fp probes attached per antibody molecule depending on
the synthetic strategy followed for the preparation of the dendrimer.
Scheme 3. Schematic interaction between rabbit IgG (primary antibody) and goat
anti-rabbit IgG–dendrimer conjugate (secondary antibody) at the surface of
nitrocellulose membranes. IR probes located on the IgG–dendrimer conjugate
allow the quantification of rabbit IgG by FT–IR (see Fig. 4).
Dendrimer
Number of dendrimers
per antibody
Number of Fp
probes per antibody
G4(ArHydr)5Fp28NHAc18
G4(ArHydr)4Fp22NHAc21
G4(Fp25) [44]
1.0 0.2
1.5 0.2
0.5 0.1
28
33
13
7
5
2
Note. Values are means standard deviations (n = 3).
Table 3
Immunoreactivity of the antibody–dendrimer conjugates relative to the immunore-
activity of the underivatized antibody.
Entry
Antibody–dendrimer conjugate
Immunoreactivity retained (%)
1
2
3
4
5
IgG–[G4(ArHydr)5Fp28NHAc18
IgG–[G4(ArHydr)5Fp28NHAc18
IgG–[G4(ArHydr)4Fp22NHAc21
IgG–[G4(ArHydr)5Fp29NHAc14
IgG–[G4(ArHydr)4Fp22NHAc21
]
]
]
]
]
66.2
60.0
52.9
55.8
41.4
0.6
0.8
1
1
1.8
Fig.4. Quantification of rabbit IgG on nitrocellulose membranes by using an excess
of Fp-labeled goat anti-rabbit IgG with 0.3 nmol per assay of Fp-labeled antibody–
the Fc region of the IgG and thereby remote from the antibody–
antigen binding site. On the other hand, the number of bulky metal
carbonyl complexes carried by the dendrimer did not influence the
immunoreactivity, as exemplified by entries 3 and 4 in Table 3.
Antibody–dendrimer conjugates preserved good immunoreactivity
provided that the conjugation ratio was not too high, which seems
to favor a high dendrimer loading over a high antibody–dendrimer
conjugation ratio.
dendrimer conjugate with spacer arm (sample IgG–[G4(ArHydr)4Fp22NHAc21
]
)
1.3
(a) and with 0.8 nmol per assay of Fp-labeled antibody–dendrimer conjugate
without spacer arm (sample IgG–[G4(Fp10 1.4]) (b) [44].
)
sured for the membrane coated with 10 lg (66.7 pmol) of rabbit
IgG instead of 3.3 x 10–3 using the first conjugate generation. More-
over, this result was obtained with a lower amount of labeled anti-
body in the assay (0.3 vs. 0.8 nmol). This experiment clearly shows
the efficiency of the new multilabeling procedure given that the IR
signal was more than twice as high, although 2.7 times less labeled
antibody was employed in the assay.
Quantification of rabbit IgG by CMIA
In a second experiment, we then studied the ability of these
new IgG–dendrimer conjugates to quantify their related antigen,
namely rabbit IgG, by CMIA on nitrocellulose membrane. In fact,
this test corresponds to the second step in classical immunoassays,
the interaction between primary and secondary antibodies, with
secondary antibodies commonly labeled with enzyme, fluorescent
compound, or biotin as detectable probes. Here secondary antibod-
ies were labeled with IR probes (Scheme 3). Rabbit IgG in the range
of 0.2 to 40 lg (1.3–266.7 pmol) was spotted onto membranes
with concomitant quantities of b-LG, which is not recognized by
goat anti-rabbit IgG, so that the total amount of protein adsorbed
per membrane remained the same (40 lg). Membranes were air-
Conclusion
We have described the site-directed conjugation of hydrazide-
based G4–PAMAM dendrimers carrying 22 to 29 organometallic
Fp labels on average to the oxidized glycosylated part of an anti-
body. First an improved synthetic route to the labeled dendrimers
was reported, and then the bioconjugation conditions were opti-
mized so as to reach an average coupling ratio of 1 dendrimer
per IgG with a dendrimer carrying 28 Fp labels or 1.5 dendrimers
per IgG with a dendrimer carrying 22 Fp labels. At the organome-
tallic probe level, we were able to prepare immunoconjugates with
30 Fp units on average, which is more than twice the value previ-
ously reported without the spacer. Thus, the newly designed Fp-la-
beled dendrimer proved to be more efficient for the multilabeling
of aldehyde-containing molecules with metal carbonyl probes.
These antibody–dendrimer conjugates were characterized by com-
bining UV and FT–IR measurements. High-mass MALDI–TOF mass
spectrometry experiments confirmed the covalent coupling as well
as the estimated conjugation ratio. For the first time, immunoreac-
tivity studies were conducted and revealed that our antibody–
dried and blocked with 4% dried skimmed milk. Each membrane
was then incubated with 0.3 nmol of goat anti-rabbit IgG–dendri-
mer conjugate (IgG–[G4(ArHydr)4Fp22NHAc21 1.3) in citrate buffer
]
(pH 6.0), washed, dried, and analyzed by IR in the transmission
mode. Curve a in Fig. 4 shows the absorbance of the 2052-cm–1
band versus the amount of rabbit IgG on the membrane. For a gi-
ven amount of antigen on the membrane, a higher absorbance
was measured compared with results obtained with the first gen-
eration of Fp-labeled IgG–dendrimer conjugate (IgG–[G4(Fp
10)1.4])
(Fig. 4, curve b). For instance, an absorbance of 7.8 x 10–3 was mea-

218
Conjugation of metal carbonyl dendrimer to antibody / N. Fischer-Durand et al. / Anal. Biochem. 407 (2010) 211–219
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