Estrogen conjugation and antibody binding interactions in surface plasmon resonance biosensing

Article abstract of DOI:10.1016/j.steroids.2006.03.004

Thioether-linked 3-mercaptopropionic acid derivatives of 17β-estradiol and estrone were formed at the A-ring 4-position of the steroids by substitution of their 4-bromo analogues. The carboxylic acid terminal was used to link to an oligoethylene glycol (OEG) chain of 15-atoms in length. The OEG derivative of 17β-estradiol was then in situ immobilized on a carboxymethylated dextran-coated gold sensor surface used to detect refractive index changes upon protein binding to the surface by surface plasmon propagation in a BIAcore surface plasmon resonance (SPR) instrument. Two other estradiol-OEG derivatives with Mannich reaction linkage at the 2-position and hemisuccinate linkage at the 3-position were also immobilized on the sensor surfaces for comparison. Binding performance between these immobilized different positional conjugates and monoclonal anti-estradiol antibody, raised from a 6-position conjugate, clearly demonstrated that both 2- and 4-conjugates, not conjugated through existing functional groups, gave strong antibody bindings, whereas the 3-conjugate through an existing functional group (3-OH) gave very little binding (2% compared to the 2-conjugate). Both 2- and 4-position conjugates were then applied in a highly sensitive estradiol SPR immunoassay with secondary antibody mediated signal enhancement that gave up to a 9.5-fold signal enhancement of primary antibody binding, and a detection limit of 25 pg/mL was achieved for a rapid and convenient flow-through immunoassay of estradiol.

Full text of DOI:10.1016/j.steroids.2006.03.004

steroids 7 1 ( 2 0 0 6 ) 618–631
available at www.sciencedirect.com
journal homepage: www.elsevier.com/locate/steroids
Estrogen conjugation and antibody binding interactions in
surface plasmon resonance biosensing
John S. Mitchella,b, Yinqiu Wua,∗, Christian J. Cooka, Lyndsay Mainb
a
Bioengineering Sector, HortResearch, East Street, Private Bag 3123, Hamilton, New Zealand
Department of Chemistry, University of Waikato, Private Bag 3105, Hamilton, New Zealand
b
a r t i c l e i n f o
a b s t r a c t
Article history:
Thioether-linked 3-mercaptopropionic acid derivatives of 17␤-estradiol and estrone were
formed at the A-ring 4-position of the steroids by substitution of their 4-bromo analogues.
The carboxylic acid terminal was used to link to an oligoethylene glycol (OEG) chain of
15-atoms in length. The OEG derivative of 17␤-estradiol was then in situ immobilized on
a carboxymethylated dextran-coated gold sensor surface used to detect refractive index
changes upon protein binding to the surface by surface plasmon propagation in a BIAcore
surface plasmon resonance (SPR) instrument. Two other estradiol—OEG derivatives with
Mannich reaction linkage at the 2-position and hemisuccinate linkage at the 3-position
were also immobilized on the sensor surfaces for comparison. Binding performance between
these immobilized different positional conjugates and monoclonal anti-estradiol antibody,
raised from a 6-position conjugate, clearly demonstrated that both 2- and 4-conjugates,
not conjugated through existing functional groups, gave strong antibody bindings, whereas
the 3-conjugate through an existing functional group (3-OH) gave very little binding (2%
compared to the 2-conjugate). Both 2- and 4-position conjugates were then applied in
a highly sensitive estradiol SPR immunoassay with secondary antibody mediated signal
enhancement that gave up to a 9.5-fold signal enhancement of primary antibody binding,
and a detection limit of 25 pg/mL was achieved for a rapid and convenient flow-through
immunoassay of estradiol.
Received 16 November 2005
Received in revised form 23 March
2006
Accepted 23 March 2006
Published on line 15 May 2006
Keywords:
17␤-Estradiol
Surface plasmon resonance
Anti-estradiol mAb
Immunoassay
© 2006 Elsevier Inc. All rights reserved.
1.
Introduction
change in the chemical environment of the noble metal sens-
ing surface causes a shift in the angle or wavelength required
The production of coating steroid antigens for use in
immunoassay is of great importance in the development of
biosensors for steroid analysis. Biosensors are instruments
that detect changes in chemical concentration by the use
of a biochemical interaction, such as antibody/target bind-
ing, and convert the interaction into an electrical signal. One
such transduction technique is surface plasmon resonance
(SPR). SPR is an optical–electrical phenomenon whereby pho-
tons incident on a noble metal surface cause electrons in the
metal to move as a plasmon and generate an electrical field. A
to induce surface plasmon resonance, providing a means of
detecting small mass changes on the sensing surface [1]. The
presence of a small molecule, such as a steroid, can compete
with labeled or surface bound steroid for binding to the anti-
body and thus can change the level of antibody/target binding
detected. The use of biosensor technology has been greatly
hindered by poor sensor surface stability resulting in a lack of
repeatability in binding results and limiting the lifetime of the
biosensor systems. Biosensors, especially those based on sur-
face plasmon resonance also suffer from poor signal strength
∗
Corresponding author. Tel.: +64 7 858 4751; fax: +64 7 858 4705.
E-mail address: ywu@hortresearch.co.nz (Y. Wu).
0039-128X/$ – see front matter © 2006 Elsevier Inc. All rights reserved.
doi:10.1016/j.steroids.2006.03.004

steroids 7 1 ( 2 0 0 6 ) 618–631
619
so maximizing signal from specific binding is very important.
There is a clear need to develop functionalized biosensor sur-
faces that are both stable and allow for maximum specific
antibody binding through conjugation at points on the anti-
gen that do not bear existing functional groups. By conjugat-
ing through existing functional groups, the antigenicity of the
bound antigen is compromised, potentially reducing binding
to antibodies that specifically recognize all functional groups
on the analyte. Intuitively, greatest inhibition immunoassay
sensitivity should be obtained by using conjugates that attach
without modifying existing functional groups on the antigen
both for production of the coating antigen and raising of the
antibody, because with such conjugations the antibody should
bind both the free analyte and the coating antigen strongly so
that free analyte will significantly inhibit antibody binding to
the sensor surface and at the same time there will be maxi-
mum surface binding signal in the immunobiosensor.
Most conjugation of the steroid hormones has involved
attachment through an existing functional group, such as the
formation of hemisuccinates of alcohols and carboxymethy-
loximes of carbonyl groups. This has certainly been the case
for the estrogens 17␤-estradiol and estrone [2,3]. Conjugations
to estrogens have also been performed by introducing new
functional groups such as amines and conjugating by such
methods as diazo formation and glutaraldehyde linkage [4,5].
These methods however often involve many steps, with the
diazo method often leading to self-conjugation of the anti-
gen or intra-molecular coupling of the carrier protein, and the
glutaraldehyde method produces unstable Schiff bases that
need to be reduced, and can lead to self-conjugation. Man-
nich condensation is another popular method but, when done
in its conventional one-step conjugation, a mixture of 2- and
4-position conjugated products results.
The use of thioethers to attach linkers or other substituents
directly to the steroid without altering existing functional
groups has been demonstrated for attachment at the 7-
position of the estrogens [6] whilst carboxymethyloxime con-
jugation has been used for attachment at the 6-position [7,8].
Thioether attachment to the 4-position of the A-ring of the
steroid has been achieved only through an epoxide-mediated
route for estradiol [9,10] or by formation of the o-quinone of
the 3-hydroxyl derivative, which leaves the final product still
containing an unwanted 3-hydroxyl group [11–13]. Production
of 4-position thioether bridged steroids with a non-aromatic
A-ring has been achieved through reaction from the 6-bromo-
derivative [14–16].
It has previously been shown for progesterone, that conju-
gation through the 4-position using a thioether linkage gives
superior antibody binding over 7-position conjugation in flow-
through biosensing formats [17]. It is therefore of interest to
further extend the approach to produce the analogous con-
jugation for the estrogens through the 4-position on their
aromatic A-rings. As for progesterone, the 4-position of the
estrogens has the advantage that no epimeric mixtures are
formed at point of attachment, and it can direct linkers away
from functional groups. The position is also on the same side
of the steroid as most of the conjugates commonly used to
raise antibodies. Thioether linkages have also been shown in
the past to have the necessary stability to function as coating
antigens in SPR [18]. The carboxylic acid group then allows
easy attachment of a range of linkers including oligoethy-
lene glycol (OEG) chains. Such chains are water-soluble, have
low immunogenicity [19,20] and their length can be easily
incremented up or down as desired. Recent studies with pro-
gesterone immunoassay have shown that covalent immobi-
lization of the amine terminal of OEG-derivatives of proges-
terone onto carboxymethylated dextran-coated gold biosen-
sor surfaces is possible in situ in a controlled format, thus
producing coating antigens that can be applied in surface plas-
mon resonance (SPR)-based assays using BIAcore instruments
[18].
There is a dearth of reports from studies on the effects
of changing conjugation position of steroids on the antibody
binding and assay performance of small molecule conju-
gates either with carrier proteins or as coating antigens on
solid surfaces. To rationally design immunobiosensors with
maximum specific antibody-binding capacity, various link-
ing positions and conjugation methods need to be exam-
ined. SPR biosensors provide a unique and convenient way
of assessing antibody bindings in real-time for comparing
different conjugations in a flow-through format. SPR has
been applied to good effect to probe estrogen/receptor lig-
and interactions [21,22] and antibody binding of chemilumi-
nescent estradiol conjugates [23]. It is also of great interest
to develop new analytical techniques for sensitive detection
of estrogens for reproductive and environmental monitoring.
A consideration of the effects of the position of conjuga-
tion on antibody binding and assay performance in a flow-
through SPR immunobiosensor is crucial to maximizing assay
sensitivity.
In this study, we report the production of 17␤-estradiol
and estrone mercaptopropionate derivatives at the 4-position
and the use of such derivatives to attach an OEG chain
of 15-atoms in length. Use of the estradiol derivative as a
surface coating antigen in SPR binding studies, in compari-
son to more conventional attachment methods, is discussed
with respect to position of conjugation. Construction of a
sensitive and convenient estradiol SPR immunoassay is also
reported.
2.
Experimental
2.1.
Materials
All chemicals were reagent grade and used without fur-
ther purification. All solvents were analytical grade for reac-
tions and HPLC grade for chromatography. All dried sol-
vents were dried over molecular sieves. 17␤-Estradiol and N-
hydroxysuccinimide (NHS) were purchased from ICN (Aurora,
OH, USA), estrone from Acros Organics (Geel, Belgium), di-tert-
butyl dicarbonate and 4,7,10-trioxa-1,13-tridecanediamine
were purchased from Fluka Chemie (Buchs, Germany). All
other chemicals were purchased from Sigma–Aldrich (Milwau-
kee, WI, USA). Silica column chromatography was carried out
˚
using silica gel, Merck 60 A, grade 9385, 230–400 mesh. TLC was
run on silica gel 60 F254 plates, aluminum-backed.
Monoclonal anti-17␤-estradiol (E3550-29, US Biologicals,
Swampscott, MA, USA) was raised to estradiol-6-17␤-6-
carboxymethyloxime-bovine serum albumin conjugate as

620
steroids 7 1 ( 2 0 0 6 ) 618–631
white crystalline solid. Yield: 572 mg (55%). mp 203–205 ◦C
(lit. [25] 207–208 ◦C). Analytical RP-HPLC [MeOH/H2O (90:10,
v/v), 283 nm]: Rt = 2.62 min 96%. IR: 1053, 1437, 2926, 3242,
mouse anti-human. It had a calculated affinity constant
of approximately 1 × 1010 L/M. The anti-mouse IgG (whole
molecule) secondary antibody (M7023, Sigma, Milwaukee,
WI, USA) was developed in rabbit as the IgG fraction of
the antiserum. The SPR coupling kit was supplied by BIA-
core (BR-1000-50, Uppsala, Sweden), consisting of 1-ethyl-3-(3-
dimethylaminopropyl)-carbodiimide hydrochloride (EDC) and
NHS solutions.
Melting point determinations were done using a Reichert
Thermopan instrument and were uncorrected. 1H, 13C, and
DEPT 135 NMR spectra were obtained on Bruker AC300
300 MHz and Bruker Avance 400 MHz spectrometers. Chem-
ical shifts (ppm) were referenced to tetramethylsilane. Ana-
lytical reversed-phase HPLC was performed on a Shimadzu
(Kyoto, Japan) Class VP instrument including an SPD-M10A VP
UV–vis detector. Data was collected using Class VP software.
Analyses were performed on a Sphereclone 3 ␮m ODS (2) col-
umn (150 mm × 4.6 mm internal diameter), at a flow rate of
1 mL/min at 35 ◦C.
3504 cm−1 1H NMR (CDCl3): ı 0.80 (3H, s, 18-CH3), 1.98 (2H,
.
dd, J = 2.7 Hz, J = 12 Hz, 12-H), 2.16 (2H, m, 11-H), 2.72 (1H, m,
9-H), 2.93 (1H, dd, J = 5.6, J = 11.9 Hz, 17-H), 3.75 (2H, q, J = 7.0 Hz,
6-H), 5.59 (1H, s, OH), 6.88 (1H, d, J = 8.5 Hz, 2-H), 7.21 (1H, d,
J = 8.3 Hz, 1-H). 13C NMR (CDCl3): ı 11.1, 23.2, 26.7, 27.4, 27.6,
30.0, 31.3, 36.8, 38.2, 43.3, 44.3, 81.6, 113.1, 113.5, 125.2, 134.1,
137.2. ESI-MS m/z (MeOH, −45 V) 349.1, 351.1 (M − H)−.
2.3.
4-Bromoestrone (2)
Estrone (400 mg, 1.48 mmol) was dissolved in dry EtOH
(10 mL) and acetone (10 mL). N-Bromosuccinimide (263 mg,
1.48 mmol) was added to the vigorously stirring solution
and the solution was stirred at room temperature for 24 h.
The white solid formed was filtered off and washed with
ethanol to yield 2. The filtrate solvent was removed and the
resultant solid recrystalised to increase yield. Yield: 221 mg
(43%). mp 264–265 ◦C (lit. [26] 264–265 ◦C). Analytical RP-HPLC
[MeOH/H2O (90:10, v/v), 281 nm]: Rt = 2.55 min, 96%. IR: 1472,
Low-resolution mass spectra were obtained on
a VG
Platform II electrospray mass spectrometer (ESI-MS). High-
resolution mass spectra were obtained on a VG-70SE mass
spectrometer with MASPEC 2 data analysis software. BIA-
1731, 3420 cm−1 1H NMR (CDCl3): ı 0.90 (3H, s, 18-CH3), 5.37
.
core SPR analysis was done on
a BIAcore 3000 instru-
(1H, s, OH), 6.86 (1H, d, J = 8.6 Hz, 2-H), 7.18 (1H, d, J = 8.6 Hz, 1-
H). 13C NMR (CDCl3): ı 14.0, 21.8, 26.3, 26.9, 31.2, 31.7, 36.2, 37.8,
44.4, 49.1, 50.5, 113.2, 118.9, 125.3, 133.3, 137.0, 151.4, 222.6.
ESI-MS m/z (MeOH, −40 V) 346.7 and 348.7 (M − H)−. HRESI-MS:
348.0727 and 350.0708 (M+) (calcd. for C18H21O2Br, 348.0725 and
350.0705).
ment (BIAcore Inc., Piscataway, NJ, USA) using CM5 research
grade chips and 10 mM (N-[2-hydroxyethyl]piperazine-Nꢀ-
[2-ethanesulfonic acid]), 150 mM NaCl with 0.11% (w/v)
ethylenediaminetetra-acetic acid and 0.05% (v/v) P-20 surfac-
tant, pH 7.4 as running buffer. All BIAcore experiments were
performed at 25 ◦C.
For statistical analysis, all error bars shown on graphs rep-
resent one standard deviation of the mean. All assay stan-
dard curves have been fitted to a four-parameter logistic plot
using Sigma Plot 8.02 (SPSS, Chicago, IL, USA). All limits of
detection (LOD) have been computed as the concentration
corresponding to the blank less two standard deviations of
the blank determination. The inhibitory concentration at 50%
bound (IC50) values have been computed as a parameter of
the curve fitting. Standard deviations (S.D.) in the LOD and
IC50 have been computed by inputting the standard devia-
tion of a neighboring standard point into the rearranged four-
parameter logistic equation, calculating from the S.D. in the
response. Sensitivities represent the slope of the linear por-
tion of the assay curve. The enhancement ratios are calculated
by dividing the slope of the enhanced antibody-binding plot
by the slope of the primary antibody plot. Errors quoted in
the text are the standard error of the determination. All dif-
ferences between association and dissociation constants and
antibody binding values are statistically significant by t-test
(P < 0.02).
2.4.
3-(3,17ˇ-Dihydroxyestra-1,3,5(10)-trien-4-yl)
thiopropanoic acid (3)
Compound 1 (200 mg, 0.57 mmol) was dissolved in dry MeOH
(20 mL). Methanolic potassium hydroxide (20 mL, 7.8 mg/mL)
was added followed by 3-mercaptopropionic acid (550 ␮L). The
solution was refluxed using oven-dried glassware and a dry-
ing tube for 24 h in the dark. The solvent was removed and the
sample reconstituted in distilled water (50 mL). The aqueous
phase was washed with ethyl acetate (2× 25 mL, 1× 50 mL).
The ethyl acetate phase was then washed with distilled water
and the product was reacted directly to the next stage. 1H NMR
(CDCl3): ı 0.81 (3H, s, 18-CH3), 1.38–2.3 (m, steroidal ring struc-
ture), 2.75 (3H, t, J = 4.6 Hz, 17-CH), 2.81 (2H, t, J = 4.5 Hz, S-CH2),
6.89 (1H, d, J = 6.3 Hz, 2-H), 7.22 (1H, d, J = 6.7 Hz, 1-H) (spectrum
was overlaid with 3-mercaptopropionic acid peaks). 13C NMR
(CDCl3): ı 14.2, 21.2, 21.4, 22.8, 23.1, 24.0, 25.4, 26.8, 29.1, 29.8,
30.2, 31.0, 33.8, 34.2, 37.1, 50.9, 74.6, 90.5, 171.5, 194.0. ESI-MS
(MeOH): 399.1 [M + Na]+, 406.8 (M + OMe)−.
2.5.
3-(3-Hydroxyestra-1,3,5(10)-trien-17-on-4-yl)
2.2.
4-Bromo-17ˇ-estradiol (1)
thiopropanoic acid (4)
The method was adapted from previous reports [24,25]. 17␤-
Estradiol (800 mg, 2.94 mmol) was dissolved in dry EtOH
(40 mL) and N-bromosuccinimide (522 mg, 2.94 mmol in 15 mL
of dry EtOH) was added dropwise to the vigorously stir-
ring solution. The solution was stirred for 24 h and a white
solid formed within 1 h of reaction. The solid was collected
by filtration and recrystalized from CHCl3 to yield 1 as a
Compound 2 (150 mg, 0.43 mmol) was dissolved in dry
MeOH (20 mL) and potassium hydroxide (15 mL, 23 mg/mL
in dry MeOH) was added whilst stirring, followed by 3-
mercaptopropionic acid (425 ␮L) and the solution heated
under reflux for 24 h using oven-dried glassware and a dry-
ing tube. The sample was then cooled and solvent removed.
The sample was reconstituted in distilled water (25 mL) and

steroids 7 1 ( 2 0 0 6 ) 618–631
621
with aqueous sodium bicarbonate solution. Yield: 1.925 g
(69%). Analytical RP-HPLC [MeOH/H2O (60:40, v/v), 201 nm]:
washed with ethyl acetate (2× 12.5 mL, 1× 25 mL). The water
was removed in vacuo and the sample separated by silica
column chromatography using CHCl3/MeOH (1:1) eluant to
yield compound 4 as a white solid. Yield: 43 mg (27%). mp:
Rt = 1.34 min, 97%. IR: 1113, 1555, 1694, 2874, 2931 cm−1
.
1H
+
NMR (CDCl3, compound 7 in R-NH3 form): ı 1.43 (s, 9H,
t-butoxy carbonyl (Boc) methyl), 1.77 (2H, quin, J = 6.3 Hz,
CH2–CH2–NH3+), 1.94 (2H, quin, J = 5.8 Hz, CH2–CH2–NH–CO),
3.09 (2H, t, 6.1 Hz, CH2–NH3+), 3.19 (2H, d of t, Jd = 5.9 Hz,
CH2–NH–CO), 3.53 (2H, t, J = 6.0 Hz, CH2–O), 3.58 (2H, t, J = 3.3 Hz,
CH2–O), 3.62 (6H, m, CH2–O), 3.66 (2H, t, J = 5.6 Hz CH2–O). 13C
NMR (CDCl3): ı 28.4, 29.7, 33.4, 38.4, 39.6, 69.4, 70.19, 70.22,
70.57, 70.60, 156.1. ESI-MS m/z (MeOH): 321.1 (M + H)+.
108–112 ◦C. IR: 1241, 1400, 1698, 1735, 3429 cm−1 1H NMR
.
(CDCl3 + d3-MeOH): ı 0.93 (3H, s, 18-CH3), 2.54 (2H, t, J = 9.4 Hz,
CH2–COOH), 2.88 (2H, t, J = 8.8 Hz, CH2-S), 6.81 (1H, d, J = 11 Hz,
H-2), 7.22 (1H, d, J = 11 Hz, H-1). 13C NMR (CDCl3 + d3-MeOH): ı
13.8, 21.6, 26.2, 26.7, 29.5, 31.0, 31.5, 35.9, 37.4, 38.5, 44.0, 48.0,
50.3, 113.0, 125.4, 133.6, 136.5, 150.6, 156.2, 175.4, 223.0. ESI-MS
m/z (MeOH): 397.4 (M + Na)+, 373.4 (M − H)−.
2.6.
2,5-Dioxo-1-pyrrolidinyl 3-(3,17ˇ-dihydroxyestra-
2.9.
N-(13-(t-Butoxycarbonylamino)-4,7,10-
1,3,5(10)-trien-4-yl)thiopropionate (5)
trioxatridecanyl)-3-(3,17ˇ-dihydroxyestra-1,3,5(10)-trien-
4-yl)thiopropanamide (8)
The entire semi-solid from the ethyl acetate phase of
the compound
3
reaction was dissolved in dry N,Nꢀ-
Compound 5 (50 mg, 0.075 mmol) was dissolved in dry DMF
(1 mL). 7 (Na2CO3 washed, 21 mg, 0.064 mmol in 0.5 mL of
CHCl3) was added dropwise with vigorous stirring followed by
triethylamine (100 ␮L, dried over molecular sieves). The solu-
tion was stirred in the dark at room temperature for 60 h. Sol-
vent was then removed and the product separated by silica col-
umn chromatography using successively CHCl3/MeOH (15:1),
CHCl3/MeOH (10:1), CHCl3/MeOH (5:1), CHCl3/MeOH (1:1) and
then MeOH as eluant to yield compound 8 as a clear, color-
less oil. Yield: 44 mg (86%). Analytical RP-HPLC [MeOH/H2O
(70:30, v/v), 207 nm]: Rt = 12.03 min, 95%. IR: 1542, 1655, 1695,
2919, 3410. 1H NMR (CDCl3): ı 0.77 (3H, s, 18-CH3), 1.44 (9H, s,
Boc CH3), 2.56 (2H, t, J = 7.1 Hz, CH2COO–), 2.99 (2H, t, J = 7.1 Hz
Ar–S–CH2), 3.60 (12H, m, OEG CH2–O), 3.73 (1H, t, J = 8.5 Hz,
H-17), 6.85 (1H, d, J = 8.5 Hz, H-2), 7.17 (1H, d, J = 8.5Hz, H-
1). 13C NMR (CDCl3): ı 11.0, 23.2, 27.2, 28.1, 28.4, 28.5, 28.9,
29.2, 29.5, 29.7, 31.1, 34.2, 35.9, 36.5, 37.9, 38.1, 43.2, 44.2,
49.9, 69.5, 69.9, 70.1, 70.2, 70.5, 81.8, 82.3, 112.8, 113.7, 125.4,
134.2, 136.5, 150.3, 156.1, 170.9. ESI-MS m/z (H2O, 60 V): 718.9
(M + H2O + Na)+.
dimethylformamide (DMF) (1 mL), and dicyclohexylcarbod-
imide (DCC) (176 mg in 1 mL of dry DMF) was added dropwise
followed by NHS (98 mg in 1 mL of dry DMF). A white solid pre-
cipitated within 1 h and the reaction was left stirring in the
dark overnight. The solvent was removed in vacuo and the
sample separated by silica column chromatography using in
succession, CHCl3, CHCl3/MeOH (15:1), CHCl3/MeOH (10:1) and
CHCl3/MeOH (2:1) as eluant to yield compound 5 as a white
solid. Yield: 141 mg (52%) from compound 1. mp 194–196 ◦C.
IR: 1242, 1576, 1626, 1736, 1783, 2851, 2929, 3327 cm−1 1H NMR
.
(CDCl3 + d3-MeOH): ı 0.79 (3H, s, 18-CH3), 2.97 (4H, s, NHS),
3.05 (2H, t, J = 6.7 Hz, CH2S, overlaid with 1H, s, H-9), 3.75 (1H,
t, J = 10.9 Hz, H-17), 6.87 (1H, d, J = 11.3 Hz, H-2), 7.20 (1H, d,
J = 11.4 Hz, H-1). 13C NMR (CDCl3 + d3-MeOH): ı 11.1, 23.1, 24.7,
25.0, 25.5, 26.6, 27.4, 30.7, 31.2, 32.4, 32.9, 38.1, 43.2, 44.2, 52.9,
81.9, 112.8, 113.8, 125.6, 134.4, 136.5, 150.2, 162.6, 179.3. ESI-MS
m/z (MeCN, 40 V): 515.5 (M + MeCN + H)+.
2.7.
2,5-Dioxo-1-pyrrolidinyl-3-(3-hydroxyestra-
1,3,5(10)-trien-3-ol-17-on-4-yl)thiopropionate (6)
2.10. N-(13-(t-Butoxycarbonylamino)-4,7,10-
trioxatridecanyl)-3-(3-hydroxyestra-1,3,5(10)-trien-17-
on-4-yl)thiopropanamide (9)
Compound 4 (100 mg, 0.268 mmol) was dissolved in dry DMF
(1 mL), and DCC (72 mg, 0.348 mmol, in 0.5 mL of dry DMF) was
added to the rapidly stirring solution dropwise. This was fol-
lowed by NHS (40 mg, 0.348 mmol, in 0.5 mL of dry DMF). The
solution was left stirring at room temperature overnight in
the dark. The solvent was removed in vacuo and the result-
ing semi-solid purified by silica column chromatography using
CHCl3/MeOH (15:1) eluant to yield compound 6 as a white
solid. Yield: 40 mg (32%). mp 193–197 ◦C. IR: 1576, 1627, 1736,
Compound 6 (37 mg, 0.079 mmol) was dissolved in dry DMF
(1 mL). A solution of 7 (51 mg, 0.159 mmol, in 750 ␮L of dry
CHCl3) was added dropwise to the stirring steroid solution.
Triethylamine (dry, 250 ␮L) was then added and the reac-
tion stirred in the dark at room temperature for 60 h. After
48 h another 250 ␮L of dry CHCl3 was added to aid solubil-
ity. The solvent was removed in vacuo and the sample sep-
arated by silica column chromatography using successively
CHCl3 and CHCl3/MeOH (15:1) eluant to yield compound 9 as
a waxy white solid. Yield: 27 mg (50%). Analytical RP-HPLC
[MeOH/H2O (65:35, v/v), 203 nm]: Rt = 12.76 min, 96%. IR: 1626,
1780, 2851, 2929, 3328 cm−1 1H NMR (CDCl3 + d3-MeOH): ı 0.90
.
(3H, s, 18-CH3), 2.89 (4H, s, NHS), 4.31 (1H, d, J = 9.4 Hz), 6.85
(1H, d, J = 11.4 Hz, H-2), 7.16 (1H, d, J = 11.4 Hz, H-1). 13C NMR
(CDCl3 + d3-MeOH): ı 13.9, 21.6, 24.8, 25.0, 25.5, 25.7, 26.2, 26.7,
31.1, 32.9, 34.0, 36.6, 37.6, 44.2, 48.0, 52.8, 113.0, 125.5, 133.6,
136.4, 150.6, 157.1, 162.7, 179.3, 220.9. ESI-MS m/z (MeOH 40 V):
471.6 (M + H)+.
1655, 1702, 1736, 2850, 2928, 3327 cm−1 1H NMR (CDCl3): ı
.
0.91 (3H, s, 18-CH3), 1.44 (9H, s, Boc CH3), 3.63 (13H, m, OEG),
4.62 (1H, d, J = 10.0 Hz), 6.83 (1H, d, J = 11.3 Hz, 2-H), 7.15 (1H,
d, J = 11.7 Hz, 1-H). 13C NMR (CDCl3): ı 13.8, 21.6, 25.0, 25.6,
26.2, 28.4, 29.6, 31.1, 31.5, 33.8, 36.0, 37.6, 38.2, 39.3, 44.1, 48.9,
49.0, 50.3, 69.4, 69.9, 70.2, 70.5, 79.2, 113.0, 117.5, 125.3, 133.3,
136.6, 157.5, 161.9, 178.7, 221.8. ESI-MS m/z (MeOH, 40 V): 695.6
(M + H2O + H)+.
2.8.
N-(t-Butoxycarbonyl)-4,7,10-trioxa-1,13-
tridecanediamine (7)
Performed according to the method of [18] adapted from
[27]. To return to free amine form, compound 7 was washed

622
steroids 7 1 ( 2 0 0 6 ) 618–631
NMR (CDCl3 + d3-MeOH): ı 11.1, 23.2, 25.7, 26.5, 27.4, 28.5,
28.9, 29.5, 29.6, 29.8, 30.1, 30.8, 33.9, 36.8, 37.5, 39.1, 44.1, 48.8,
48.9, 50.2, 69.6, 70.2, 70.6, 81.7, 83.3, 118.6, 121.5, 126.4, 131.8,
138.0, 148.5, 158.0, 172.1, 173.8. ESI-MS m/z (MeOH, 40 V):
697.5 (M + Na)+. HRFAB-MS: m/z 675.4202 (MH+) (calcd. for
2.11. N-(13-Amino-4,7,10-trioxatridecanyl)-(3,17ˇ-
dihydroxyestra-1,3,5(10)-trien-4-yl)thiopropanamide (10)
Compound 8 (28 mg, 0.041 mmol) was dissolved in formic acid
(4 mL) and stirred at room temperature for 4 h before removal
of acids in vacuo. Yield: 24 mg (100%). Analytical RP-HPLC
[MeOH/H2O (60:40, v/v), 212 nm] Rt = 1.73 min, 98%. IR: 1653,
C37H58O9N2, 675.4202).
1718, 1734, 2847, 2929 cm−1 1H NMR (CDCl3): ı 0.85 (3H, s, 18-
.
2.14. (13-Amino-4,7,10-trioxatridecanyl)-3-(17ˇ-
hydroxyestra-1,3,5(10)-trien-3-
yl)oxycarbonylpropanamide (13)
CH3), 3.62 (13H, OEG CH2-O), 4.80 (1H, t, J = 8.4 Hz), 6.86 (1H,
d, J = 8.4 Hz, 2-H), 7.15 (1H, d, J = 8.4 Hz, 1-H). 13C NMR (CDCl3):
ı 12.1, 23.3, 25.7, 26.4, 27.4, 27.6, 29.2, 31.1, 34.0, 35.9, 36.7,
36.9, 37.8, 43.0, 44.0, 49.7, 69.0, 69.7, 69.8, 69.9, 70.1, 70.4, 82.5,
113.0, 113.6, 125.4, 133.8, 136.5, 150.7, 161.2, 171.7. ESI-MS m/z
(MeOH): 615.2 (M + 2H2O + H)+, 637.3 (M + 2H2O + Na)+.
Compound 12 (48 mg, 0.071 mmol) was dissolved in formic
acid (4 mL) and stirred at room temperature for 2.5 h before
adding chloroform (1 mL) to improve solubility and stirred for
an additional 1.5 h. Solvent was removed in vacuo and the
product column separated using MeOH/AcOH (10:1) eluant.
AcOH was removed in vacuo. Yield: 36 mg (81%). Analytical
RP-HPLC [MeOH, 203 nm]: Rt = 1.64 min, 95%. IR: 1414, 1561,
2.12. 17ˇ-Hydroxyestra-1,3,5(10)-trien-3-yl
hemisuccinate (11)
1638, 3434 cm−1 1H NMR (D2O): ı 1.14 (3H, s, 18-CH3), 2.52 (2H,
.
Method adapted from [28]. 17␤-Estradiol (100 mg, 0.367 mmol)
was dissolved in pyridine (9 mL). Succinic anhydride (37 mg,
0.367 mmol in 1 mL of pyridine) was added dropwise to the
rapidly stirring solution. The reaction was then stirred at 45 ◦C
for 24 h. The pyridine was then removed in vacuo and the
resultant sample purified by silica column chromatography
using successively, CHCl3, CHCl3/MeOH (15:1), CHCl3/MeOH
(10:1) as eluant to yield compound 11 as a semi-solid. Yield:
t, J = 6.0 Hz, CH2-amide), 2.65 (2H, t, J = 7.2 Hz, CH2-ester), 2.81
(2H, d, J = 12.0 Hz), 3.09 (2H, t, J = 7.4 Hz), 3.23 (2H, m), 3.55 (3H,
m, OEG), 3.66 (11H, m, OEG CH2–O), 6.68 (m, 2-H), 6.88 (m, 4-
H), 7.23 (m, 1-H). 13C NMR (d3-MeOH): ı 21.1, 23.3, 29.8, 38.8,
70.1, 70.4, 83.4, 127.0, 174.8. ESI-MS m/z (MeOH, 40 V): 575.2
(M + H)+.
33 mg (24%). IR: 1588, 1739, 3584 cm−1 1H NMR (CDCl3 + d3-
.
2.15. 2-(N-(13-(t-Butoxycarbonylamino)-4,7,10-trioxa-
1,13-tridecanyl)-(aminomethyl)-3,17ˇ-dihydroxyestra-
1,3,5(10)-triene) (14)
MeOH): ı 0.77 (3H, s, 18-CH3), 2.74 (2H, t, J = 8.9 Hz, CH2-ester),
2.84 (2H, t, J = 7.3 Hz, CH2–COOH), 6.55 (1H, m, 4-H), 6.84 (1H,
m, 1-H), 7.28 (1H, m, 2-H). 13C NMR (CDCl3 + d3-MeOH): ı 11.1,
23.1, 26.2, 27.1, 27.3, 29.1, 29.5, 36.7, 38.6, 43.2, 44.2, 49.8, 50.1,
81.6, 118.5, 121.4, 126.4, 131.7, 138.0, 148.4, 171.6, 174.9. ESI-MS
m/z (MeOH, 40 V): 395.3 (M + Na)+.
Estradiol (100 mg, 0.367 mmol) was dissolved in absolute EtOH
(10 mL). Compound 7 (236 mg, 0.734 mmol in EtOH/water (4:1)
(5 mL)) was added gradually to the solution whilst stirring.
This was followed by 37% (v/v) formaldehyde solution (222 ␮L,
8.06 mmol) and the solution refluxed with stirring for 6 h. The
solvent was removed under vacuum and the sample separated
by silica column chromatography using CHCl3, CHCl3/MeOH
(15:1), CHCl3/MeOH (10:1) as eluant to yield compound 14 as a
2.13. N-(13-(t-Butoxycarbonylamino)-4,7,10-
trioxatridecanyl)-3-(17ˇ-hydroxyestra-1,3,5(10)-trien-3-
yl)oxycarbonylpropanamide (12)
Compound 11 (72 mg, 0.194 mmol) was dissolved in
DMF/CHCl3 (1:1) (2.5 mL, dry). This solution was stirred
whilst DCC (60 mg, 0.292 mmol, in 500 ␮L of dry DMF) was
added dropwise followed by NHS (34 mg, 0.292 mmol, in
500 ␮L of dry DMF), also dropwise. The solution was stirred
in the dark overnight. White solid was filtered from the
solution and washed with CHCl3 and DMF before removing
all solvent in the filtrate under vacuum. The resultant white
semi-solid was reconstituted in DMF/CHCl3 (1:1) (2 mL, dry)
and 7 was added (94 mg in 3.25 mL of dry DMF/chloroform
(2:1)) dropwise to the stirring solution. Triethylamine (1 mL,
dried over molecular sieves) was then added and the reac-
tion stirred in the dark overnight. The solvent was then
removed and the sample dried under vacuum. The sample
was then separated by silica column chromatography using
successively CHCl3, CHCl3/MeOH (15:1), and CHCl3/MeOH
(10:1) as eluant to yield compound 12 as a white semi-solid.
Yield: 64 mg (49%). IR: 1627, 1654, 1702, 1736, 2850, 2926,
clear colorless oil. Yield: 30 mg (13%). IR: 1692, 3584 cm−1 1H
.
NMR (CDCl3 + d3-MeOH): ı 0.77 (3H, s, 18-CH3), 1.43 (9H, s, Boc
methyl), 2.80 (2H, m, CH2–Ar), 3.60 (14H, m, OEG CH2–O), 6.51
(1H, s, 4-H), 6.88 (1H, s, 1-H). 13C NMR (CDCl3 + d3-MeOH): ı 11.1,
23.2, 26.5, 27.3, 28.2, 28.4, 28.5, 29.4, 29.4, 29.6, 30.1, 36.8, 38.2,
39.0, 43.3, 44.1, 48.3, 48.9, 49.5, 69.1, 69.3, 69.5, 70.2, 70.6, 72.1,
81.6, 82.4, 116.0, 117.3, 124.4, 133.0, 136.5, 151.8, 155.1. COSY
(CDCl3 + d3-MeOH): no aromatic coupling cross-peaks. ESI-MS
m/z (MeOH, 40 V): 605.5 (M + H)+.
2.16. 2-(13-Amino-4,7,10-trioxa-1,13-tridecanyl)-
(aminomethyl)-3,17ˇ-dihydroxyestra-1,3,5(10)-
triene) (15)
Compound 14 (21 mg, 0.034 mmol) was dissolved in formic
acid (3 mL) and stirred for 4 h at room temperature before
removal of acid in vacuo. The product was column purified
using MeOH/AcOH (2:1) to elute the product. Yield: 15 mg
(86%). Analytical RP-HPLC [MeOH, 203 nm]: Rt = 1.63 min, 97%.
.
IR: 1134, 1421, 2981, 3438 cm−1 1H NMR (d3-MeOH) ı: 0.89 (3H,
18-CH3), 2.83 (2H, m, 7-CH2), 3.64 (m, OEG CH2–O), 6.65 (4-H),
6.99 (1-H). ESI-MS m/z (MeOH): 505.3 (M + H)+.
3326 cm−1 1H NMR (CDCl3 + d3-MeOH): ı 0.77 (3H, s, 18-CH3),
.
1.44 (9H, s, Boc methyls), 2.47 (2H, t, J = 9.3 Hz, hemisuccinate
CH2-amide), 2.65 (2H, t, J = 9.4 Hz, hemisuccinate CH2-ester),
6.62 (1H, m, 2-H), 6.79 (1H, m, 4-H), 7.12 (1H, m, 1-H). 13C

steroids 7 1 ( 2 0 0 6 ) 618–631
623
mary antibody (50 ng/mL, 70 ␮L) with 17␤-estradiol solutions
(70 ␮L; 0, 0.5, 1, 5, 10, 50, 100, and 500 pg/mL, 1 and 5 ng/mL,
five replicates each) in a microwell plate, mixing, incubat-
ing and injecting as above. This injection was then imme-
diately followed by injection of secondary antibody (60 ␮L,
200 ␮g/mL, 10 ␮L/min), a wait of 120 s and then regeneration
as above.
2.17. SPR chip surface immobilization
A CM5 sensor chip was docked in a BIAcore 3000 instru-
ment and primed twice with running buffer. The surface was
activated with BIAcore coupling solutions of EDC/NHS (1:1)
(50 ␮L, 5 ␮L/min). A solution of the compound to be immo-
bilized (13, 15 or 10, 1 mg/mL in 1% (v/v) DMF in 10 mM
phosphate buffered saline with 0.05% (w/v) Tween 20 (PBS/T)
buffer, pH 9.7) was centrifuged to remove any insoluble mate-
rial and then injected (1× 20 ␮L, 1× 80 ␮L, 1× 100 ␮L, 1×
200 ␮L, 5 ␮L/min). This was immediately followed by further
injections of estrogen derivative at higher pH (1 mg/mL, 1%
(v/v) DMF in PBS/T buffer, pH 11.7, 1× 200 ␮L, 2× 100 ␮L,
5 ␮L/min) and then deactivation with 1.0 M ethanolamine
(50 ␮L, 5 ␮L/min). Compound 13 was immobilized in flow cell
2, compound 15 in flow cell 3 and compound 10 in flow cell 4.
Flow cell 1 was activated as for the others and then immedi-
ately deactivated with ethanolamine (50 ␮L, 5 ␮L/min) without
immobilization of estrogens to serve as a reference flow cell.
The immobilization was repeated on another chip but at pH
4.2 to ensure alkaline degradation was not occurring to the
surface coatings and antibody binding was checked as below.
3.
Results and discussion
3.1.
Synthesis of 4-position mercaptopropionate
conjugates
To attach linkers by thioether bridging at the A-ring 4-position
of non-aromatic steroids such as progesterone, it has been
shown that formation of the 6-bromo-derivative and subse-
quent alkaline reflux with the thiol will produce the desired
derivative. This method cannot be replicated when the A-ring
of the steroid is aromatic, such as in the case of the estro-
gens. Whilst previous studies have used reaction via a 4,5-
epoxide [9], a potentially simpler route is to form the 4-bromo-
derivative and to follow this with an aromatic substitution of
the bromo-derivative with the thiol, thus producing the link-
age in two simple steps (Scheme 1).
2.18. SPR antibody binding studies
4-Bromo-17␤-estradiol was produced by adapting an exist-
ing generalized method whereby the steroid is simply mixed
with N-bromosuccinimide in dry ethanol and the product
precipitates [24,25] (Scheme 1). The existing methods [24,25]
however provided no experimental details of the synthesis
and so this paper is the first to provide clear details of a
one-step synthesis of 4-bromoestradiol. Attempts have been
made to synthesize 4-bromoestrone in one step but with lim-
ited success [25]. Instead, a two-step method whereby estrone
is brominated with N-bromosuccinimide to form the 2,4-
dibromoestrone followed by regioselective reductive dehalo-
genation using palladium on carbon has been previously
adopted as the best method [29,30]. In this study it has been
found that the two-step method resulted in a low yield of
the product and so a method was developed whereby the 4-
bromoestrone could be produced in one step by simply adjust-
ing the solvent combination used in the reaction to precipitate
the desired product (Scheme 1).
Production of derivatives with thioether linkages was then
achieved by refluxing the corresponding bromo-derivatives
in methanolic potassium hydroxide under dry conditions
(Scheme 1). By simply extracting the product into ethyl acetate
and washing with water it could be mostly purified, however
in the case of the 17␤-estradiol derivative there was still some
residual 3-mercaptopropionic acid contamination. 4-Position
attachment could be confirmed through the 1H NMR signals,
which clearly showed two doublets with chemical shifts con-
sistent with those expected for 4-substituted estrogens.
This is a new example of replacement of an aryl bromo-
derivative with an alkyl thiol group and speculatively may
occur through a radical SRN1 mechanism, as the 4-position
is not activated toward nucleophilic aromatic substitution.
This reaction attached a carboxylic acid group to the steroid
A-ring without compromising existing functional groups
and attached solely at the 4-position, therefore allowing
Plots of response versus primary antibody concentration with
no secondary antibody enhancement were prepared using
a BIAcore wizard program by injecting primary monoclonal
antibody (60 ␮L, at 0, 0.25, 0.5, 1, 2, 5, 10, and 25 ␮g/mL, five
replicates of each, 20 ␮L/min) and waiting 120 s before regen-
eration with two pulses of 50 mM NaOH, 10% (v/v) MeCN (20 ␮L
each, 20 ␮L/min). These binding sensorgrams were then fitted
using BIAevaluation 3.1 software and the affinity constants
(KA) and dissociation constants (KD) for the interactions were
calculated taking IgG mass at 150 kDa.
Plots of response versus secondary antibody concen-
tration for secondary antibody signal enhancement were
prepared by injecting monoclonal primary antibody (60 ␮L,
20 ␮L/min, 0.5 ␮g/mL) followed immediately by secondary
antibody (60 ␮L; 0, 10, 20, 50, 100, 200, 300 ␮g/mL; 10 ␮L/min)
and then a 120 s wait before regeneration as above.
Plots of response versus monoclonal antibody concentra-
tion with secondary antibody enhancement were prepared by
injecting monoclonal primary antibody (60 ␮L; 0, 10, 25, 50,
100, 200, 350, 500 ng/mL; 20 ␮L/min) immediately followed by
secondary antibody (60 ␮L, 200 ␮g/mL), a 120 s wait and then
regeneration as above.
2.19. SPR 17ˇ-estradiol assays
Assay curves for 17␤-estradiol assay without secondary anti-
body enhancement were prepared by mixing monoclonal pri-
mary antibody (70 ␮L, 1 ␮g/mL) with 17␤-estradiol solutions
(70 ␮L; 0, 1, 5, 10, 50, 100, and 500 pg/mL, 1, 5, and 10 ng/mL; five
replicates each) in a microwell plate, mixing using the auto-
matic mixing function, incubating for 5 min at 25 ◦C and then
injecting (60 ␮L, 20 ␮L/min), and then after a 120 s wait regen-
erating as above.
Assay curves for 17␤-estradiol assay with secondary anti-
body enhancement were prepared by mixing monoclonal pri-

624
steroids 7 1 ( 2 0 0 6 ) 618–631
Scheme 1 – Synthesis of 4-position thiopropionate derivatives of 17␤-estradiol and estrone and the attachment of
oligoethylene glycol (OEG) chains.
attachment of a large range of different linkers tailored
to individual requirements for various conjugations of the
estrogens.
estrogen derivatives could also project the estrogen antigens
into the aqueous phase effectively when tethered to a solid
surface as a coating antigen. To attach such linkers to these
derivatives, the carboxylic acid was first activated using NHS,
with DCC used as a dehydrating agent (Scheme 1). This worked
well for both steroids.
3.2.
Attachment of oligoethylene glycol linkers
Oligoethylene glycol linkers are water-soluble, and as such, in
aqueous media would be envisaged to improve the solubility
of the estrogen derivatives for convenient in situ immobiliza-
tion of these estrogens onto a solid surface such as a BIAcore
biosensor chip in a flow-through format. Such water-soluble
An OEG linker was attached to the estrogens by reaction
with the corresponding NHS active ester (Scheme 1). The chain
had an amine terminal at both ends and so one end was pro-
tected with a Boc protecting group to prevent dimerization.
The reaction conditions were simple mixing and addition of

steroids 7 1 ( 2 0 0 6 ) 618–631
625
Scheme 2 – Synthesis of 3-hemisuccinate derivative of estradiol and attachment of an oligoethylene glycol (OEG) chain.
the protein being present, but rather using mono-protected
OEG with one free amine terminal exposed (Scheme 3). This
reaction then enabled chromatographic purification of the
main product. The yield of this reaction was quite low (13%);
variable yields having been reported as a disadvantage with
Mannich conjugations [31]. The product isolated was analyzed
by 1H NMR and 2-D COSY, both of which showed clearly that
there was no coupling between the two proton singlet signals
and the aromatic chemical shifts matched those expected for
2-position conjugation.
a mild base (triethylamine) and room temperature stirring in
dry solvent.
3.3.
Attachment at 3- and 2-positions
To compare the newly synthesized 4-estrogen OEG deriva-
tive (4-E2) as a coating antigen in a flow-through BIAcore
SPR biosensor, it was necessary to synthesize reference com-
pounds attached at other positions on the aromatic ring of
the steroid. 3-Position conjugation through a hemisuccinate
linkage via the 3-OH (3-E2), and the 2-position via Mannich
reaction (2-E2) were selected. Estradiol-3-hemisuccinate was
produced as reported previously [28], and a 15-atom OEG chain
was then attached in the same way as for the 4-thioether
derivatives (Scheme 2).
Mannich conjugations are normally done in one step with
both the estrogen and the carrier protein present. This pro-
duces a mixture of 2- and 4-conjugated estrogen–protein con-
jugates. To compare the effect of position of attachment on the
ability to bind primary antibody, it was necessary to isolate one
of the positions by performing the Mannich reaction without
3.4.
Immobilization of the sensor surface
To expose the amine terminal of the linker chain, all the
estradiol-oligoethylene glycol derivatives were treated with
formic acid to remove the Boc protecting group (Schemes 1–3).
The estradiol derivatives could then be in situ immobilized
on the sensor surface through their primary amine terminals.
The carboxymethylated dextran surface of a BIAcore CM5 chip
is functionalized with carboxylic acid groups that can be eas-
ily activated with a combination of NHS and EDC coupling

626
steroids 7 1 ( 2 0 0 6 ) 618–631
Scheme 3 – Synthesis of 2-E2.
reagents. As for previous reports for progesterone [18], the sur-
face was immobilized in situ by flowing high concentrations of
the primary amines over the surface under basic conditions.
To ensure surface saturation in each case, the concentrations
were high (1 mg/mL) and a large volume of solution was flowed
over the surfaces (800 ␮L per surface in total) giving a high
excess of immobilizing agent. A very slow flow rate was used
(5 ␮L/min) to maximize immobilization. Two alkaline pH were
used in each case to help ensure that the maximum amount
of steroid derivative was bound with the pH above the pKa of
the amine in each case. Each surface was immobilized under
identical conditions with the first flow cell being simply acti-
vated and then deactivated with ethanolamine to act as the
blank.
In the first instance, the binding of monoclonal antibody to
the surfaces was examined (Fig. 1) and a plot of the binding
response, in response units (RU), versus primary monoclonal
antibody concentration was prepared (Fig. 2). This showed
clearly very little antibody binding to the 3-E2 conjugated sur-
face and quite strong binding to the 4-E2 conjugated surface
but even stronger binding to the 2-E2 conjugated surface. Anti-
body binding to the surface of the 4-E2 conjugate was 84% that
of the 2-E2 conjugate at saturation (25 ␮g/mL primary anti-
body), whilst its binding to the 3-E2 conjugated surface was
only 2% that of the corresponding 2-E2 (Fig. 2). This suggests
that the formation of the conjugate through the 3-position of
the steroid is greatly reducing the antibody binding capacity of
the antigen, most likely through modification of the 3-hydroxy
group of the steroid reducing its antigenicity when an antibody
raised to the 6-position is employed. As 6-position conjuga-
tion involves attachment that does not compromise existing
functional groups, the antibody would be expected to have a
relatively high specificity to free estradiol.
3.5.
Antibody binding studies using SPR
It was necessary to use monoclonal antibody as the binding
antibody because the use of commercial polyclonal antibody
(E2885, Sigma, St. Louis, MO, USA) resulted in no detectable
binding to any of the flow cells in this flow-through format.
Clearly, compromising one functional group of the estra-
diol has greatly reduced binding. This result has important

steroids 7 1 ( 2 0 0 6 ) 618–631
627
Fig. 1 – Schematic representation of primary antibody binding to 17␤-estradiol-linker modified SPR sensor surface,
subsequent binding of secondary antibody to enhance sensor signal and regeneration of the sensor surface. Signal
responses are shown for each step as a plot of response vs. time, relating signal to binding event.
implications for biosensor design as the 3-position is widely
used in enzyme and chemiluminescent label conjugations
of estradiol and yet is of no use in flow-through biosensors
using antibody raised to the 6-poistion conjugate. This result
is different from that previously reported for enzyme-linked
immunosorbent assay (ELISA) of progesterone, where signif-
icant antibody binding was obtained when the antibody was
raised to the 6-position and the coating antigen was conju-
gated through the 3-position [32] indicating the importance of
assay format in antibody binding performance. An antibody
raised to the 3-position could be used to gain better biosen-
sor signal but such an antibody would likely bind the 3-E2
conjugated coating antigen more strongly than the free ana-
lyte and so immunoassay sensitivity would likely be reduced.
Hemisuccinate linkages have been reported to be somewhat
sensitive to alkaline degradation but this has not occurred in
this case as immobilization at non-alkaline pH demonstrated
comparable binding results for the hemisuccinate.
It is also interesting to note that the 2-E2 system performs
slightly better than that of the 4-E2 (Table 1). As the anti-
body has been raised to the 6-position conjugate, it would
be expected that any changes to the steroid structure on the
opposite side of the antigen would greatly diminish the anti-
body binding. The 2-position however, appears to be near
enough to the same side of the steroid as the conjugate
used to raise the antibody for it to have no major effect on

628
steroids 7 1 ( 2 0 0 6 ) 618–631
Table 1 – Summary of 17␤-estradiol immunoassay parameters of LOD, IC50, sensitivity, and signal enhancement with
changes in primary antibody concentration and signal enhancement labelinga
Assay format
Conjugation mAb concentration LOD (pg/mL) IC50 (pg/mL) Sensitivity Enhancement
position
(ng/mL)
(RU mL/ng)
ratio
mAb only
mAb only
mAb only
mAb only
Secondary antibody enhanced
Secondary antibody enhanced
2
4
2
4
2
4
500
500
25
25
25
96 32
111 33
66 27
95 32
25 22
61 39
1457 9.6
1432 14
414.9 5.9
351 14
324 21
396 28
22
16
6.9
4.1
65
41
n/a
n/a
9.5
8.9
9.5
8.9
25
a
Errors quoted are standard errors.
the ability of the antibody to recognize the antigen; indeed
the 2-E2 gave the strongest antibody binding (Fig. 2). This
result differs from that seen with fluoresecent conjugates of
estradiol where a 2-position conjugation via a carboxypenty-
loxime linkage gave no binding to an antibody raised to the
6-carboxymethyloxime conjugate, whereas thioether conju-
gation through the 4-position did give binding in an ELISA
format [9]. This difference is due either to the different format
used (flow-through or ELISA) or the method of linkage at the
2-position. Speculatively, in a static ELISA format, 2-position
of attachment close to the opposite side of the antigen from
that used in raising the antibody may result in the linker and
fluorescent label obstructing approach of the antibody to this
side of the antigen whereas in flow-through formats the linker
tether is suspended in the fluid stream away from the antigen
allowing sterically favorable approach of the antibody. These
results show how important the binding format and conjuga-
tion method is in determining levels of antibody binding.
The differences in antibody binding responses are also
reflected in the calculated affinity and dissociation constants
for the antibody binding to the sensor surfaces, Table 2. The
affinity constants increase significantly when changing conju-
gation from 3-E2 to 4-E2 and then to 2-E2, showing increasing
antibody binding affinity. Correspondingly, the dissociation
constants decrease as the affinity constants increase.
Saturation of all binding sites occurred at close to 25 ␮g/mL
of primary antibody under the flow-through conditions, with
rapid decline in binding signal at concentrations less than
2 ␮g/mL. A primary antibody concentration of 0.5 ␮g/mL was
adequate to achieve sufficient response for conducting an SPR
immunoassay.
To produce sensitive immunoassays for small molecules
where the competing antigen is immobilized on the surface,
it is desirable to reduce the primary antibody concentration
as much as possible so that smaller amounts of analyte can
inhibit the antibody binding to the surface. The problem with
this approach in SPR is that the lower the primary antibody
concentration, the lower the signal and so the poorer the
signal: noise ratio. One technique that has proved very suc-
cessful with progesterone is to enhance the signal with sec-
ondary antibody that recognizes the bound primary antibody
[18] (Fig. 1). Enhancements of signal up to eight-fold have been
seen for progesterone using this method [18].
To determine the best secondary antibody concentration
to use as a label for the binding, a plot of response ver-
sus secondary antibody concentration at fixed primary anti-
body was prepared (Fig. 3). This showed that primary anti-
body at 0.5 ␮g/mL had maximum binding with secondary
antibody label at concentrations of about 200 ␮g/mL. Use of
300 ␮g/mL produced only approximately 4% additional bind-
ing but could potentially significantly increase non-specific
binding. To determine just how much signal enhancement had
been obtained and thus how low the primary antibody concen-
tration could be reduced, a plot of response versus primary
antibody concentration at fixed secondary antibody concen-
tration was prepared (Fig. 4). By comparing the slopes of the
plots in the linear region, this gave signal enhancements of
Table 2 – Affinity constants (KA) and dissociation
constants (KD) for the binding of estradiol monoclonal
antibody to SPR sensor surfaces with conjugation at the
2-, 3- and 4-positionsb
Compound
Position of
KA (107 M−1
)
KD (nM)
attachment
15
13
10
2
3
4
148 13
2.02 0.23
95.6 3.7
0.70 0.06
44.8 7.7
1.05 0.04
Fig. 2 – Plot of response (RU) vs. primary antibody
concentration (␮g/mL) for 3-E2 (ꢀ), 4-E2 (ꢁ) and 2-E2 (ꢂ)
conjugates (error bars too small to see).
b
Errors quoted are standard errors, values are the average of five
replicates.

steroids 7 1 ( 2 0 0 6 ) 618–631
629
Fig. 5 – Estradiol immunoassay curve plot for secondary
antibody enhancement and 2-E2 conjugation showing the
logistic-log fitted assay curve.
Fig. 3 – Plot of response (RU) vs. secondary antibody
concentration (␮g/mL) for 4-E2 (ꢁ) and 2-E2 (ꢂ) linked
conjugates (error bars too small to see).
3.6.
Sensitive SPR assay of 17ˇ-estradiol
9.5-fold for the 2-E2 surface and 8.9-fold for the 4-E2 surface.
These enhancements were slightly better than those seen pre-
viously with progesterone [18] and support the previous obser-
vation that secondary antibody can enhance primary antibody
binding signal more than would be expected from simple one-
to-one binding [18]. These plot also show that the primary
antibody concentration can be reduced to 25 ng/mL and still
maintain about 100 RU of specific binding after secondary anti-
body enhancement (Fig. 4).
Having optimized the antibody concentrations that can be
used, assays could be performed for 17␤-estradiol. The first
assay was constructed without secondary antibody signal
enhancement and so used primary antibody at 0.5 ␮g/mL. The
assay gave a limit of detection (LOD) of 96 pg/mL using the
2-E2 surface (Table 1). It was not possible to conduct assays
using the 3-E2 surface because the binding signals were too
small.
The assay was then repeated but adding the secondary
antibody signal enhancement (Fig. 1) and using a final pri-
mary antibody concentration of 25 ng/mL (Fig. 5). This assay
gave a LOD of 25 pg/mL with the 2-E2 surface (Table 1). The
LOD for the unenhanced primary antibody at the same con-
centration was 66 pg/mL (Table 1). The IC50 for the assay
had dropped by approximately 75% upon secondary antibody
enhancement whilst the sensitivity had increased three-fold
(Table 1). These assays have the lowest LOD of reported SPR
assays of estradiol and show how LOD for this steroid can
be obtained that are comparable to certain ELISA tests [33,34]
but much more rapid and simple. The only previous study of
estradiol SPR immunoassay demonstrated eight-fold higher
LOD, used manual mixing and injecting after prolonged incu-
bation at 4 ◦C and used a much less stable protein conju-
gate format with conjugation through an existing functional
group [35].
The in situ covalently immobilized sensor surface, like the
previously reported progesterone surface [18], has proved to
be quite stable over a large number of cycles (in excess of 200
cycles), with no noticeable decline in antibody binding capac-
ity, a major factor in a practical biosensor.
Production of stable biosensor surfaces that project small
molecule antigens with conjugation at positions not bear-
ing functional groups is essential to retaining high specific
binding signal in flow-through biosensors. A novel route
has been demonstrated for the attachment of carboxylic
acid groups to the 4-position of 17␤-estradiol and estrone
Fig. 4 – Plot of response (RU) vs. primary antibody
concentration (ng/mL) using 2-E2 conjugate and showing
mAb binding only (ꢀ) and secondary antibody-enhanced
binding (ꢁ). The linear portions of the curves are from 0 to
100 ng/mL and the line equations are y = 0.574x + 3.98
(R2 = 1.00) for mAb only and y = 5.43x + 92.7 (R2 = 0.99) for
secondary antibody enhancement (error bars too small to
see.

630
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Acknowledgement
We thank the New Zealand Foundation for Research Science
and Technology for funding this work and providing a Top
Achiever doctoral scholarship for John Mitchell.
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