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Estrogen conjugation and antibody binding interactions in
surface plasmon resonance biosensing
John S. Mitchell , Yinqiu Wu , Christian J. Cook , Lyndsay Main
a Bioengineering Sector, HortResearch, East Street, Private Bag 3123, Hamilton, New Zealandb Department of Chemistry, University of Waikato, Private Bag 3105, Hamilton, New Zealand Article history: Thioether-linked 3-mercaptopropionic acid derivatives of 17␤-estradiol and estrone were Received 16 November 2005 formed at the A-ring 4-position of the steroids by substitution of their 4-bromo analogues.
Received in revised form 23 March 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 Accepted 23 March 2006 a carboxymethylated dextran-coated gold sensor surface used to detect refractive index Published on line 15 May 2006 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 Surface plasmon resonance these immobilized different positional conjugates and monoclonal anti-estradiol antibody, Anti-estradiol mAb 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.
2006 Elsevier Inc. All rights reserved.
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 to induce surface plasmon resonance, providing a means of immunoassay is of great importance in the development of detecting small mass changes on the sensing surface biosensors for steroid analysis. Biosensors are instruments presence of a small molecule, such as a steroid, can compete that detect changes in chemical concentration by the use with labeled or surface bound steroid for binding to the anti- of a biochemical interaction, such as antibody/target bind- body and thus can change the level of antibody/target binding ing, and convert the interaction into an electrical signal. One detected. The use of biosensor technology has been greatly such transduction technique is surface plasmon resonance hindered by poor sensor surface stability resulting in a lack of (SPR). SPR is an optical–electrical phenomenon whereby pho- repeatability in binding results and limiting the lifetime of the tons incident on a noble metal surface cause electrons in the biosensor systems. Biosensors, especially those based on sur- metal to move as a plasmon and generate an electrical ﬁeld. A face plasmon resonance also suffer from poor signal strength ∗ Corresponding author. Tel.: +64 7 858 4751; fax: +64 7 858 4705.
E-mail address: (Y. Wu).
0039-128X/$ – see front matter 2006 Elsevier Inc. All rights reserved.
so maximizing signal from speciﬁc binding is very important.
easy attachment of a range of linkers including oligoethy- There is a clear need to develop functionalized biosensor sur- lene glycol (OEG) chains. Such chains are water-soluble, have faces that are both stable and allow for maximum speciﬁc low immunogenicity their length can be easily antibody binding through conjugation at points on the anti- incremented up or down as desired. Recent studies with pro- gen that do not bear existing functional groups. By conjugat- gesterone immunoassay have shown that covalent immobi- ing through existing functional groups, the antigenicity of the lization of the amine terminal of OEG-derivatives of proges- bound antigen is compromised, potentially reducing binding terone onto carboxymethylated dextran-coated gold biosen- to antibodies that speciﬁcally recognize all functional groups sor surfaces is possible in situ in a controlled format, thus on the analyte. Intuitively, greatest inhibition immunoassay producing coating antigens that can be applied in surface plas- sensitivity should be obtained by using conjugates that attach mon resonance (SPR)-based assays using BIAcore instruments without modifying existing functional groups on the antigen both for production of the coating antigen and raising of the There is a dearth of reports from studies on the effects antibody, because with such conjugations the antibody should of changing conjugation position of steroids on the antibody bind both the free analyte and the coating antigen strongly so binding and assay performance of small molecule conju- that free analyte will signiﬁcantly inhibit antibody binding to gates either with carrier proteins or as coating antigens on the sensor surface and at the same time there will be maxi- solid surfaces. To rationally design immunobiosensors with mum surface binding signal in the immunobiosensor.
maximum speciﬁc antibody-binding capacity, various link- Most conjugation of the steroid hormones has involved ing positions and conjugation methods need to be exam- attachment through an existing functional group, such as the ined. SPR biosensors provide a unique and convenient way formation of hemisuccinates of alcohols and carboxymethy- of assessing antibody bindings in real-time for comparing loximes of carbonyl groups. This has certainly been the case different conjugations in a ﬂow-through format. SPR has for the estrogens 17␤-estradiol and estrone ations been applied to good effect to probe estrogen/receptor lig- to estrogens have also been performed by introducing new and interactions antibody binding of chemilumi- functional groups such as amines and conjugating by such nescent estradiol conjugates It is also of great interest methods as diazo formation and glutaraldehyde linkage to develop new analytical techniques for sensitive detection These methods however often involve many steps, with the of estrogens for reproductive and environmental monitoring.
diazo method often leading to self-conjugation of the anti- A consideration of the effects of the position of conjuga- gen or intra-molecular coupling of the carrier protein, and the tion on antibody binding and assay performance in a ﬂow- glutaraldehyde method produces unstable Schiff bases that through SPR immunobiosensor is crucial to maximizing assay need to be reduced, and can lead to self-conjugation. Man- nich condensation is another popular method but, when done In this study, we report the production of 17␤-estradiol in its conventional one-step conjugation, a mixture of 2- and and estrone mercaptopropionate derivatives at the 4-position 4-position conjugated products results.
and the use of such derivatives to attach an OEG chain The use of thioethers to attach linkers or other substituents of 15-atoms in length. Use of the estradiol derivative as a directly to the steroid without altering existing functional surface coating antigen in SPR binding studies, in compari- groups has been demonstrated for attachment at the 7- son to more conventional attachment methods, is discussed position of the estrogens carboxymethyloxime con- with respect to position of conjugation. Construction of a jugation has been used for attachment at the 6-position sensitive and convenient estradiol SPR immunoassay is also Thioether attachment to the 4-position of the A-ring of the steroid has been achieved only through an epoxide-mediatedroute for estradiol by formation of the o-quinone ofthe 3-hydroxyl derivative, which leaves the ﬁnal product still containing an unwanted 3-hydroxyl group roductionof 4-position thioether bridged steroids with a non-aromatic A-ring has been achieved through reaction from the 6-bromo-derivative All chemicals were reagent grade and used without fur- It has previously been shown for progesterone, that conju- ther puriﬁcation. All solvents were analytical grade for reac- gation through the 4-position using a thioether linkage gives tions and HPLC grade for chromatography. All dried sol- superior antibody binding over 7-position conjugation in ﬂow- vents were dried over molecular sieves. 17␤-Estradiol and N- through biosensing formats t is therefore of interest to hydroxysuccinimide (NHS) were purchased from ICN (Aurora, further extend the approach to produce the analogous con- OH, USA), estrone from Acros Organics (Geel, Belgium), di-tert- jugation for the estrogens through the 4-position on their butyl dicarbonate and 4,7,10-trioxa-1,13-tridecanediamine aromatic A-rings. As for progesterone, the 4-position of the were purchased from Fluka Chemie (Buchs, Germany). All estrogens has the advantage that no epimeric mixtures are other chemicals were purchased from Sigma–Aldrich (Milwau- formed at point of attachment, and it can direct linkers away kee, WI, USA). Silica column chromatography was carried out from functional groups. The position is also on the same side using silica gel, Merck 60 ˚ A, grade 9385, 230–400 mesh. TLC was of the steroid as most of the conjugates commonly used to run on silica gel 60 F254 plates, aluminum-backed.
raise antibodies. Thioether linkages have also been shown in Monoclonal anti-17␤-estradiol (E3550-29, US Biologicals, the past to have the necessary stability to function as coating Swampscott, MA, USA) was raised to estradiol-6-17␤-6- antigens in SPR The carboxylic acid group then allows carboxymethyloxime-bovine serum albumin conjugate as mouse anti-human. It had a calculated afﬁnity constant white crystalline solid. Yield: 572 mg (55%). mp 203–205 ◦C of approximately 1 × 1010 L/M. The anti-mouse IgG (whole (lit. ◦C). Analytical RP-HPLC [MeOH/H2O (90:10, molecule) secondary antibody (M7023, Sigma, Milwaukee, v/v), 283 nm]: Rt = 2.62 min 96%. IR: 1053, 1437, 2926, 3242, WI, USA) was developed in rabbit as the IgG fraction of 3504 cm−1. 1H NMR (CDCl3): ı 0.80 (3H, s, 18-CH3), 1.98 (2H, the antiserum. The SPR coupling kit was supplied by BIA- dd, J = 2.7 Hz, J = 12 Hz, 12-H), 2.16 (2H, m, 11-H), 2.72 (1H, m, core (BR-1000-50, Uppsala, Sweden), consisting of 1-ethyl-3-(3- 9-H), 2.93 (1H, dd, J = 5.6, J = 11.9 Hz, 17-H), 3.75 (2H, q, J = 7.0 Hz, dimethylaminopropyl)-carbodiimide hydrochloride (EDC) and 6-H), 5.59 (1H, s, OH), 6.88 (1H, d, J = 8.5 Hz, 2-H), 7.21 (1H, d, NHS solutions.
J = 8.3 Hz, 1-H). 13C NMR (CDCl3): ı 11.1, 23.2, 26.7, 27.4, 27.6, Melting point determinations were done using a Reichert 30.0, 31.3, 36.8, 38.2, 43.3, 44.3, 81.6, 113.1, 113.5, 125.2, 134.1, Thermopan instrument and were uncorrected. 1H, 13C, and 137.2. ESI-MS m/z (MeOH, −45 V) 349.1, 351.1 (M − H)−.
DEPT 135 NMR spectra were obtained on Bruker AC300300 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 Estrone (400 mg, 1.48 mmol) was dissolved in dry EtOH (Kyoto, Japan) Class VP instrument including an SPD-M10A VP (10 mL) and acetone (10 mL). N-Bromosuccinimide (263 mg, UV–vis detector. Data was collected using Class VP software.
1.48 mmol) was added to the vigorously stirring solution Analyses were performed on a Sphereclone 3 m ODS (2) col- and the solution was stirred at room temperature for 24 h.
umn (150 mm × 4.6 mm internal diameter), at a ﬂow rate of The white solid formed was ﬁltered off and washed with 1 mL/min at 35 ◦C.
ethanol to yield 2. The ﬁltrate solvent was removed and the
Low-resolution mass spectra were obtained on a VG resultant solid recrystalised to increase yield. Yield: 221 mg Platform II electrospray mass spectrometer (ESI-MS). High- (43%). mp 264–265 ◦C (lit. ◦C). Analytical RP-HPLC resolution mass spectra were obtained on a VG-70SE mass [MeOH/H2O (90:10, v/v), 281 nm]: Rt = 2.55 min, 96%. IR: 1472, 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- ment (BIAcore Inc., Piscataway, NJ, USA) using CM5 research H). 13C NMR (CDCl3): ı 14.0, 21.8, 26.3, 26.9, 31.2, 31.7, 36.2, 37.8, grade chips and 10 mM (N-[2-hydroxyethyl]piperazine-N- 44.4, 49.1, 50.5, 113.2, 118.9, 125.3, 133.3, 137.0, 151.4, 222.6.
[2-ethanesulfonic acid]), 150 mM NaCl with 0.11% (w/v) ESI-MS m/z (MeOH, −40 V) 346.7 and 348.7 (M − H)−. HRESI-MS: ethylenediaminetetra-acetic acid and 0.05% (v/v) P-20 surfac- 348.0727 and 350.0708 (M+) (calcd. for C18H21O2Br, 348.0725 and 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- thiopropanoic acid (3)
dard curves have been ﬁtted to a four-parameter logistic plotusing Sigma Plot 8.02 (SPSS, Chicago, IL, USA). All limits of Compound 1 (200 mg, 0.57 mmol) was dissolved in dry MeOH
detection (LOD) have been computed as the concentration (20 mL). Methanolic potassium hydroxide (20 mL, 7.8 mg/mL) corresponding to the blank less two standard deviations of was added followed by 3-mercaptopropionic acid (550 L). The the blank determination. The inhibitory concentration at 50% solution was reﬂuxed using oven-dried glassware and a dry- bound (IC50) values have been computed as a parameter of ing tube for 24 h in the dark. The solvent was removed and the the curve ﬁtting. Standard deviations (S.D.) in the LOD and sample reconstituted in distilled water (50 mL). The aqueous IC50 have been computed by inputting the standard devia- phase was washed with ethyl acetate (2× 25 mL, 1× 50 mL).
tion of a neighboring standard point into the rearranged four- The ethyl acetate phase was then washed with distilled water parameter logistic equation, calculating from the S.D. in the and the product was reacted directly to the next stage. 1H NMR response. Sensitivities represent the slope of the linear por- (CDCl3): ı 0.81 (3H, s, 18-CH3), 1.38–2.3 (m, steroidal ring struc- tion of the assay curve. The enhancement ratios are calculated ture), 2.75 (3H, t, J = 4.6 Hz, 17-CH), 2.81 (2H, t, J = 4.5 Hz, S-CH2), by dividing the slope of the enhanced antibody-binding plot 6.89 (1H, d, J = 6.3 Hz, 2-H), 7.22 (1H, d, J = 6.7 Hz, 1-H) (spectrum by the slope of the primary antibody plot. Errors quoted in was overlaid with 3-mercaptopropionic acid peaks). 13C NMR the text are the standard error of the determination. All dif- (CDCl3): ı 14.2, 21.2, 21.4, 22.8, 23.1, 24.0, 25.4, 26.8, 29.1, 29.8, ferences between association and dissociation constants and 30.2, 31.0, 33.8, 34.2, 37.1, 50.9, 74.6, 90.5, 171.5, 194.0. ESI-MS antibody binding values are statistically signiﬁcant by t-test (MeOH): 399.1 [M + Na]+, 406.8 (M + OMe)−.
(P < 0.02).
thiopropanoic acid (4)
The method was adapted from previous reports 17␤- Compound 2 (150 mg, 0.43 mmol) was dissolved in dry
Estradiol (800 mg, 2.94 mmol) was dissolved in dry EtOH MeOH (20 mL) and potassium hydroxide (15 mL, 23 mg/mL (40 mL) and N-bromosuccinimide (522 mg, 2.94 mmol in 15 mL in dry MeOH) was added whilst stirring, followed by 3- of dry EtOH) was added dropwise to the vigorously stir- mercaptopropionic acid (425 L) and the solution heated ring solution. The solution was stirred for 24 h and a white under reﬂux for 24 h using oven-dried glassware and a dry- solid formed within 1 h of reaction. The solid was collected ing tube. The sample was then cooled and solvent removed.
by ﬁltration and recrystalized from CHCl3 to yield 1 as a
The sample was reconstituted in distilled water (25 mL) and washed with ethyl acetate (2× 12.5 mL, 1× 25 mL). The water with aqueous sodium bicarbonate solution. Yield: 1.925 g was removed in vacuo and the sample separated by silica (69%). Analytical RP-HPLC [MeOH/H2O (60:40, v/v), 201 nm]: column chromatography using CHCl3/MeOH (1:1) eluant to Rt = 1.34 min, 97%. IR: 1113, 1555, 1694, 2874, 2931 cm−1. 1H yield compound 4 as a white solid. Yield: 43 mg (27%). mp:
3, compound 7 in R-NH3
form): ı 1.43 (s, 9H, 108–112 ◦C. IR: 1241, 1400, 1698, 1735, 3429 cm−1. 1H NMR t-butoxy carbonyl (Boc) methyl), 1.77 (2H, quin, J = 6.3 Hz, 3 + d3-MeOH): ı 0.93 (3H, s, 18-CH3), 2.54 (2H, t, J = 9.4 Hz, CH2–CH2–NH3 ), 1.94 (2H, quin, J = 5.8 Hz, CH2–CH2–NH–CO), 2–COOH), 2.88 (2H, t, J = 8.8 Hz, CH2-S), 6.81 (1H, d, J = 11 Hz, 3.09 (2H, t, 6.1 Hz, CH2–NH3 ), 3.19 (2H, d of t, Jd = 5.9 Hz, H-2), 7.22 (1H, d, J = 11 Hz, H-1). 13C NMR (CDCl3 + d3-MeOH): ı CH2–NH–CO), 3.53 (2H, t, J = 6.0 Hz, CH2–O), 3.58 (2H, t, J = 3.3 Hz, 13.8, 21.6, 26.2, 26.7, 29.5, 31.0, 31.5, 35.9, 37.4, 38.5, 44.0, 48.0, CH2–O), 3.62 (6H, m, CH2–O), 3.66 (2H, t, J = 5.6 Hz CH2–O). 13C 50.3, 113.0, 125.4, 133.6, 136.5, 150.6, 156.2, 175.4, 223.0. ESI-MS NMR (CDCl3): ı 28.4, 29.7, 33.4, 38.4, 39.6, 69.4, 70.19, 70.22, m/z (MeOH): 397.4 (M + Na)+, 373.4 (M − H)−.
70.57, 70.60, 156.1. ESI-MS m/z (MeOH): 321.1 (M + H)+.
The entire semi-solid from the ethyl acetate phase of
the compound 3 reaction was dissolved in dry N,N-
Compound 5 (50 mg, 0.075 mmol) was dissolved in dry DMF
dimethylformamide (DMF) (1 mL), and dicyclohexylcarbod- (1 mL). 7 (Na2CO3 washed, 21 mg, 0.064 mmol in 0.5 mL of
imide (DCC) (176 mg in 1 mL of dry DMF) was added dropwise CHCl3) was added dropwise with vigorous stirring followed by followed by NHS (98 mg in 1 mL of dry DMF). A white solid pre- triethylamine (100 L, dried over molecular sieves). The solu- cipitated within 1 h and the reaction was left stirring in the tion was stirred in the dark at room temperature for 60 h. Sol- dark overnight. The solvent was removed in vacuo and the vent was then removed and the product separated by silica col- sample separated by silica column chromatography using in umn chromatography using successively CHCl3/MeOH (15:1), succession, CHCl3, CHCl3/MeOH (15:1), CHCl3/MeOH (10:1) and CHCl3/MeOH (10:1), CHCl3/MeOH (5:1), CHCl3/MeOH (1:1) and CHCl3/MeOH (2:1) as eluant to yield compound 5 as a white
then MeOH as eluant to yield compound 8 as a clear, color-
solid. Yield: 141 mg (52%) from compound 1. mp 194–196 ◦C.
less oil. Yield: 44 mg (86%). Analytical RP-HPLC [MeOH/H2O IR: 1242, 1576, 1626, 1736, 1783, 2851, 2929, 3327 cm−1. 1H NMR (70:30, v/v), 207 nm]: Rt = 12.03 min, 95%. IR: 1542, 1655, 1695, (CDCl3 + d3-MeOH): ı 0.79 (3H, s, 18-CH3), 2.97 (4H, s, NHS), 2919, 3410. 1H NMR (CDCl3): ı 0.77 (3H, s, 18-CH3), 1.44 (9H, s, 3.05 (2H, t, J = 6.7 Hz, CH2S, overlaid with 1H, s, H-9), 3.75 (1H, Boc CH3), 2.56 (2H, t, J = 7.1 Hz, CH2COO–), 2.99 (2H, t, J = 7.1 Hz t, J = 10.9 Hz, H-17), 6.87 (1H, d, J = 11.3 Hz, H-2), 7.20 (1H, d, Ar–S–CH2), 3.60 (12H, m, OEG CH2–O), 3.73 (1H, t, J = 8.5 Hz, J = 11.4 Hz, H-1). 13C NMR (CDCl3 + d3-MeOH): ı 11.1, 23.1, 24.7, H-17), 6.85 (1H, d, J = 8.5 Hz, H-2), 7.17 (1H, d, J = 8.5Hz, H- 25.0, 25.5, 26.6, 27.4, 30.7, 31.2, 32.4, 32.9, 38.1, 43.2, 44.2, 52.9, 1). 13C NMR (CDCl3): ı 11.0, 23.2, 27.2, 28.1, 28.4, 28.5, 28.9, 81.9, 112.8, 113.8, 125.6, 134.4, 136.5, 150.2, 162.6, 179.3. ESI-MS 29.2, 29.5, 29.7, 31.1, 34.2, 35.9, 36.5, 37.9, 38.1, 43.2, 44.2, m/z (MeCN, 40 V): 515.5 (M + MeCN + H)+.
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)+.
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 Compound 6 (37 mg, 0.079 mmol) was dissolved in dry DMF
solution was left stirring at room temperature overnight in (1 mL). A solution of 7 (51 mg, 0.159 mmol, in 750 L of dry
the dark. The solvent was removed in vacuo and the result- CHCl3) was added dropwise to the stirring steroid solution.
ing semi-solid puriﬁed by silica column chromatography using Triethylamine (dry, 250 L) was then added and the reac- CHCl3/MeOH (15:1) eluant to yield compound 6 as a white
tion stirred in the dark at room temperature for 60 h. After solid. Yield: 40 mg (32%). mp 193–197 ◦C. IR: 1576, 1627, 1736, 48 h another 250 L of dry CHCl3 was added to aid solubil- 1780, 2851, 2929, 3328 cm−1. 1H NMR (CDCl3 + d3-MeOH): ı 0.90 ity. The solvent was removed in vacuo and the sample sep- (3H, s, 18-CH3), 2.89 (4H, s, NHS), 4.31 (1H, d, J = 9.4 Hz), 6.85 arated by silica column chromatography using successively (1H, d, J = 11.4 Hz, H-2), 7.16 (1H, d, J = 11.4 Hz, H-1). 13C NMR CHCl3 and CHCl3/MeOH (15:1) eluant to yield compound 9 as
(CDCl3 + d3-MeOH): ı 13.9, 21.6, 24.8, 25.0, 25.5, 25.7, 26.2, 26.7, a waxy white solid. Yield: 27 mg (50%). Analytical RP-HPLC 31.1, 32.9, 34.0, 36.6, 37.6, 44.2, 48.0, 52.8, 113.0, 125.5, 133.6, [MeOH/H2O (65:35, v/v), 203 nm]: Rt = 12.76 min, 96%. IR: 1626, 136.4, 150.6, 157.1, 162.7, 179.3, 220.9. ESI-MS m/z (MeOH 40 V): 1655, 1702, 1736, 2850, 2928, 3327 cm−1. 1H NMR (CDCl3): ı 471.6 (M + H)+.
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, Performed according to the method of from 136.6, 157.5, 161.9, 178.7, 221.8. ESI-MS m/z (MeOH, 40 V): 695.6 To return to free amine form, compound 7 was washed
(M + H2O + H)+.
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, Compound 8 (28 mg, 0.041 mmol) was dissolved in formic acid
138.0, 148.5, 158.0, 172.1, 173.8. ESI-MS m/z (MeOH, 40 V): (4 mL) and stirred at room temperature for 4 h before removal 697.5 (M + Na)+. HRFAB-MS: m/z 675.4202 (MH+) (calcd. for 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: 1653,1718, 1734, 2847, 2929 cm−1. 1H NMR (CDCl3): ı 0.85 (3H, s, 18- 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, Compound 12 (48 mg, 0.071 mmol) was dissolved in formic
113.0, 113.6, 125.4, 133.8, 136.5, 150.7, 161.2, 171.7. ESI-MS m/z acid (4 mL) and stirred at room temperature for 2.5 h before (MeOH): 615.2 (M + 2H2O + H)+, 637.3 (M + 2H2O + Na)+.
adding chloroform (1 mL) to improve solubility and stirred foran 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%). AnalyticalRP-HPLC [MeOH, 203 nm]: Rt = 1.64 min, 95%. IR: 1414, 1561, Method adapted from 7␤-Estradiol (100 mg, 0.367 mmol) 1638, 3434 cm−1. 1H NMR (D2O): ı 1.14 (3H, s, 18-CH3), 2.52 (2H, was dissolved in pyridine (9 mL). Succinic anhydride (37 mg, t, J = 6.0 Hz, CH2-amide), 2.65 (2H, t, J = 7.2 Hz, CH2-ester), 2.81 0.367 mmol in 1 mL of pyridine) was added dropwise to the (2H, d, J = 12.0 Hz), 3.09 (2H, t, J = 7.4 Hz), 3.23 (2H, m), 3.55 (3H, rapidly stirring solution. The reaction was then stirred at 45 ◦C m, OEG), 3.66 (11H, m, OEG CH2–O), 6.68 (m, 2-H), 6.88 (m, 4- for 24 h. The pyridine was then removed in vacuo and the H), 7.23 (m, 1-H). 13C NMR (d3-MeOH): ı 21.1, 23.3, 29.8, 38.8, resultant sample puriﬁed by silica column chromatography 70.1, 70.4, 83.4, 127.0, 174.8. ESI-MS m/z (MeOH, 40 V): 575.2 using successively, CHCl3, CHCl3/MeOH (15:1), CHCl3/MeOH (M + H)+.
(10:1) as eluant to yield compound 11 as a semi-solid. Yield:
33 mg (24%). IR: 1588, 1739, 3584 cm−1. 1H NMR (CDCl3 + d3-
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, Estradiol (100 mg, 0.367 mmol) was dissolved in absolute EtOH 81.6, 118.5, 121.4, 126.4, 131.7, 138.0, 148.4, 171.6, 174.9. ESI-MS (10 mL). Compound 7 (236 mg, 0.734 mmol in EtOH/water (4:1)
m/z (MeOH, 40 V): 395.3 (M + Na)+.
(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
clear colorless oil. Yield: 30 mg (13%). IR: 1692, 3584 cm−1. 1H DMF/CHCl3 (1:1) (2.5 mL, dry). This solution was stirred NMR (CDCl3 + d3-MeOH): ı 0.77 (3H, s, 18-CH3), 1.43 (9H, s, Boc whilst DCC (60 mg, 0.292 mmol, in 500 L of dry DMF) was methyl), 2.80 (2H, m, CH2–Ar), 3.60 (14H, m, OEG CH2–O), 6.51 added dropwise followed by NHS (34 mg, 0.292 mmol, in (1H, s, 4-H), 6.88 (1H, s, 1-H). 13C NMR (CDCl3 + d3-MeOH): ı 11.1, 500 L of dry DMF), also dropwise. The solution was stirred 23.2, 26.5, 27.3, 28.2, 28.4, 28.5, 29.4, 29.4, 29.6, 30.1, 36.8, 38.2, in the dark overnight. White solid was ﬁltered from the 39.0, 43.3, 44.1, 48.3, 48.9, 49.5, 69.1, 69.3, 69.5, 70.2, 70.6, 72.1, solution and washed with CHCl3 and DMF before removing 81.6, 82.4, 116.0, 117.3, 124.4, 133.0, 136.5, 151.8, 155.1. COSY all solvent in the ﬁltrate under vacuum. The resultant white (CDCl3 + d3-MeOH): no aromatic coupling cross-peaks. ESI-MS semi-solid was reconstituted in DMF/CHCl3 (1:1) (2 mL, dry) m/z (MeOH, 40 V): 605.5 (M + H)+.
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 samplewas then separated by silica column chromatography using Compound 14 (21 mg, 0.034 mmol) was dissolved in formic
successively CHCl3, CHCl3/MeOH (15:1), and CHCl3/MeOH acid (3 mL) and stirred for 4 h at room temperature before (10:1) as eluant to yield compound 12 as a white semi-solid.
removal of acid in vacuo. The product was column puriﬁed Yield: 64 mg (49%). IR: 1627, 1654, 1702, 1736, 2850, 2926, using MeOH/AcOH (2:1) to elute the product. Yield: 15 mg 3326 cm−1. 1H NMR (CDCl3 + d3-MeOH): ı 0.77 (3H, s, 18-CH3), (86%). Analytical RP-HPLC [MeOH, 203 nm]: Rt = 1.63 min, 97%.
1.44 (9H, s, Boc methyls), 2.47 (2H, t, J = 9.3 Hz, hemisuccinate IR: 1134, 1421, 2981, 3438 cm−1. 1H NMR (d3-MeOH) ı: 0.89 (3H, CH2-amide), 2.65 (2H, t, J = 9.4 Hz, hemisuccinate CH2-ester), 18-CH3), 2.83 (2H, m, 7-CH2), 3.64 (m, OEG CH2–O), 6.65 (4-H), 6.62 (1H, m, 2-H), 6.79 (1H, m, 4-H), 7.12 (1H, m, 1-H). 13C 6.99 (1-H). ESI-MS m/z (MeOH): 505.3 (M + H)+.
SPR chip surface immobilization
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, A CM5 sensor chip was docked in a BIAcore 3000 instru- ﬁve replicates each) in a microwell plate, mixing, incubat- ment and primed twice with running buffer. The surface was ing and injecting as above. This injection was then imme- activated with BIAcore coupling solutions of EDC/NHS (1:1) diately followed by injection of secondary antibody (60 L, (50 L, 5 L/min). A solution of the compound to be immo- 200 g/mL, 10 L/min), a wait of 120 s and then regeneration 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× Results and discussion
200 L, 5 L/min). This was immediately followed by furtherinjections of estrogen derivative at higher pH (1 mg/mL, 1% Synthesis of 4-position mercaptopropionate
(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
To attach linkers by thioether bridging at the A-ring 4-position 2, compound 15 in ﬂow cell 3 and compound 10 in ﬂow cell 4.
of non-aromatic steroids such as progesterone, it has been Flow cell 1 was activated as for the others and then immedi- shown that formation of the 6-bromo-derivative and subse- ately deactivated with ethanolamine (50 L, 5 L/min) without quent alkaline reﬂux with the thiol will produce the desired immobilization of estrogens to serve as a reference ﬂow cell.
derivative. This method cannot be replicated when the A-ring The immobilization was repeated on another chip but at pH of the steroid is aromatic, such as in the case of the estro- 4.2 to ensure alkaline degradation was not occurring to the gens. Whilst previous studies have used reaction via a 4,5- surface coatings and antibody binding was checked as below.
epoxide potentially simpler route is to form the 4-bromo-derivative and to follow this with an aromatic substitution of SPR antibody binding studies
the bromo-derivative with the thiol, thus producing the link-age in two simple steps ( Plots of response versus primary antibody concentration with 4-Bromo-17␤-estradiol was produced by adapting an exist- no secondary antibody enhancement were prepared using ing generalized method whereby the steroid is simply mixed a BIAcore wizard program by injecting primary monoclonal with N-bromosuccinimide in dry ethanol and the product antibody (60 L, at 0, 0.25, 0.5, 1, 2, 5, 10, and 25 g/mL, ﬁve precipitates The existing methods replicates of each, 20 L/min) and waiting 120 s before regen- however provided no experimental details of the synthesis eration with two pulses of 50 mM NaOH, 10% (v/v) MeCN (20 L and so this paper is the ﬁrst to provide clear details of a each, 20 L/min). These binding sensorgrams were then ﬁtted one-step synthesis of 4-bromoestradiol. Attempts have been using BIAevaluation 3.1 software and the afﬁnity constants made to synthesize 4-bromoestrone in one step but with lim- (KA) and dissociation constants (KD) for the interactions were ited success a two-step method whereby estrone calculated taking IgG mass at 150 kDa.
is brominated with N-bromosuccinimide to form the 2,4- Plots of response versus secondary antibody concen- dibromoestrone followed by regioselective reductive dehalo- tration for secondary antibody signal enhancement were genation using palladium on carbon has been previously prepared by injecting monoclonal primary antibody (60 L, adopted as the best method In this study it has been 20 L/min, 0.5 g/mL) followed immediately by secondary found that the two-step method resulted in a low yield of antibody (60 L; 0, 10, 20, 50, 100, 200, 300 g/mL; 10 L/min) the product and so a method was developed whereby the 4- and then a 120 s wait before regeneration as above.
bromoestrone could be produced in one step by simply adjust- Plots of response versus monoclonal antibody concentra- ing the solvent combination used in the reaction to precipitate tion with secondary antibody enhancement were prepared by the desired product injecting monoclonal primary antibody (60 L; 0, 10, 25, 50, Production of derivatives with thioether linkages was then 100, 200, 350, 500 ng/mL; 20 L/min) immediately followed by achieved by reﬂuxing the corresponding bromo-derivatives secondary antibody (60 L, 200 g/mL), a 120 s wait and then in methanolic potassium hydroxide under dry conditions regeneration as above.
By simply extracting the product into ethyl acetateand washing with water it could be mostly puriﬁed, however in the case of the 17␤-estradiol derivative there was still someresidual 3-mercaptopropionic acid contamination. 4-Position Assay curves for 17␤-estradiol assay without secondary anti- attachment could be conﬁrmed through the 1H NMR signals, body enhancement were prepared by mixing monoclonal pri- which clearly showed two doublets with chemical shifts con- mary antibody (70 L, 1 g/mL) with 17␤-estradiol solutions sistent with those expected for 4-substituted estrogens.
(70 L; 0, 1, 5, 10, 50, 100, and 500 pg/mL, 1, 5, and 10 ng/mL; ﬁve This is a new example of replacement of an aryl bromo- replicates each) in a microwell plate, mixing using the auto- derivative with an alkyl thiol group and speculatively may matic mixing function, incubating for 5 min at 25 ◦C and then occur through a radical SRN1 mechanism, as the 4-position injecting (60 L, 20 L/min), and then after a 120 s wait regen- is not activated toward nucleophilic aromatic substitution.
erating as above.
This reaction attached a carboxylic acid group to the steroid Assay curves for 17␤-estradiol assay with secondary anti- A-ring without compromising existing functional groups body enhancement were prepared by mixing monoclonal pri- and attached solely at the 4-position, therefore allowing 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 estrogen derivatives could also project the estrogen antigens to individual requirements for various conjugations of the into the aqueous phase effectively when tethered to a solid surface as a coating antigen. To attach such linkers to thesederivatives, the carboxylic acid was ﬁrst activated using NHS, Attachment of oligoethylene glycol linkers
with DCC used as a dehydrating agent This workedwell for both steroids.
Oligoethylene glycol linkers are water-soluble, and as such, in An OEG linker was attached to the estrogens by reaction aqueous media would be envisaged to improve the solubility with the corresponding NHS active ester (The chain of the estrogen derivatives for convenient in situ immobiliza- had an amine terminal at both ends and so one end was pro- tion of these estrogens onto a solid surface such as a BIAcore tected with a Boc protecting group to prevent dimerization.
biosensor chip in a ﬂow-through format. Such water-soluble The reaction conditions were simple mixing and addition of Scheme 2 – Synthesis of 3-hemisuccinate derivative of estradiol and attachment of an oligoethylene glycol (OEG) chain.
a mild base (triethylamine) and room temperature stirring in the protein being present, but rather using mono-protected dry solvent.
OEG with one free amine terminal exposed Thisreaction then enabled chromatographic puriﬁcation of the Attachment at 3- and 2-positions
main product. The yield of this reaction was quite low (13%);variable yields having been reported as a disadvantage with To compare the newly synthesized 4-estrogen OEG deriva- Mannich conjugations product isolated was analyzed tive (4-E2) as a coating antigen in a ﬂow-through BIAcore by 1H NMR and 2-D COSY, both of which showed clearly that SPR biosensor, it was necessary to synthesize reference com- there was no coupling between the two proton singlet signals pounds attached at other positions on the aromatic ring of and the aromatic chemical shifts matched those expected for the steroid. 3-Position conjugation through a hemisuccinate linkage via the 3-OH (3-E2), and the 2-position via Mannichreaction (2-E2) were selected. Estradiol-3-hemisuccinate was Immobilization of the sensor surface
produced as reported previously a 15-atom OEG chainwas then attached in the same way as for the 4-thioether To expose the amine terminal of the linker chain, all the estradiol-oligoethylene glycol derivatives were treated with Mannich conjugations are normally done in one step with formic acid to remove the Boc protecting group ( both the estrogen and the carrier protein present. This pro- The estradiol derivatives could then be in situ immobilized duces a mixture of 2- and 4-conjugated estrogen–protein con- on the sensor surface through their primary amine terminals.
jugates. To compare the effect of position of attachment on the The carboxymethylated dextran surface of a BIAcore CM5 chip ability to bind primary antibody, it was necessary to isolate one is functionalized with carboxylic acid groups that can be eas- of the positions by performing the Mannich reaction without ily activated with a combination of NHS and EDC coupling Scheme 3 – Synthesis of 2-E2.
reagents. As for previous reports for progesterone sur- In the ﬁrst instance, the binding of monoclonal antibody to face was immobilized in situ by ﬂowing high concentrations of the surfaces was examined and a plot of the binding the primary amines over the surface under basic conditions.
response, in response units (RU), versus primary monoclonal To ensure surface saturation in each case, the concentrations antibody concentration was prepared (This showed were high (1 mg/mL) and a large volume of solution was ﬂowed clearly very little antibody binding to the 3-E2 conjugated sur- over the surfaces (800 L per surface in total) giving a high face and quite strong binding to the 4-E2 conjugated surface excess of immobilizing agent. A very slow ﬂow rate was used but even stronger binding to the 2-E2 conjugated surface. Anti- (5 L/min) to maximize immobilization. Two alkaline pH were body binding to the surface of the 4-E2 conjugate was 84% that used in each case to help ensure that the maximum amount of the 2-E2 conjugate at saturation (25 g/mL primary anti- of steroid derivative was bound with the pH above the pKa of body), whilst its binding to the 3-E2 conjugated surface was the amine in each case. Each surface was immobilized under only 2% that of the corresponding 2-E2 (This suggests identical conditions with the ﬁrst ﬂow cell being simply acti- that the formation of the conjugate through the 3-position of vated and then deactivated with ethanolamine to act as the the steroid is greatly reducing the antibody binding capacity of the antigen, most likely through modiﬁcation of the 3-hydroxygroup of the steroid reducing its antigenicity when an antibody Antibody binding studies using SPR
raised to the 6-position is employed. As 6-position conjuga-tion involves attachment that does not compromise existing It was necessary to use monoclonal antibody as the binding functional groups, the antibody would be expected to have a antibody because the use of commercial polyclonal antibody relatively high speciﬁcity to free estradiol.
(E2885, Sigma, St. Louis, MO, USA) resulted in no detectable Clearly, compromising one functional group of the estra- binding to any of the ﬂow cells in this ﬂow-through format.
diol has greatly reduced binding. This result has important
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 lyte and so immunoassay sensitivity would likely be reduced.
used in enzyme and chemiluminescent label conjugations Hemisuccinate linkages have been reported to be somewhat of estradiol and yet is of no use in ﬂow-through biosensors sensitive to alkaline degradation but this has not occurred in using antibody raised to the 6-poistion conjugate. This result this case as immobilization at non-alkaline pH demonstrated is different from that previously reported for enzyme-linked comparable binding results for the hemisuccinate.
immunosorbent assay (ELISA) of progesterone, where signif- It is also interesting to note that the 2-E2 system performs icant antibody binding was obtained when the antibody was slightly better than that of the 4-E2 As the anti- raised to the 6-position and the coating antigen was conju- body has been raised to the 6-position conjugate, it would gated through the 3-position the importance of be expected that any changes to the steroid structure on the assay format in antibody binding performance. An antibody opposite side of the antigen would greatly diminish the anti- raised to the 3-position could be used to gain better biosen- body binding. The 2-position however, appears to be near sor signal but such an antibody would likely bind the 3-E2 enough to the same side of the steroid as the conjugate conjugated coating antigen more strongly than the free ana- used to raise the antibody for it to have no major effect on
Table 1 – Summary of 17␤-estradiol immunoassay parameters of LOD, IC50, sensitivity, and signal enhancement with
changes in primary antibody concentration and signal enhancement labeling
mAb concentration Secondary antibody enhanced Secondary antibody enhanced a Errors quoted are standard errors.
the ability of the antibody to recognize the antigen; indeed Saturation of all binding sites occurred at close to 25 g/mL the 2-E2 gave the strongest antibody binding (This of primary antibody under the ﬂow-through conditions, with result differs from that seen with ﬂuoresecent conjugates of rapid decline in binding signal at concentrations less than estradiol where a 2-position conjugation via a carboxypenty- 2 g/mL. A primary antibody concentration of 0.5 g/mL was loxime linkage gave no binding to an antibody raised to the adequate to achieve sufﬁcient response for conducting an SPR 6-carboxymethyloxime conjugate, whereas thioether conju- gation through the 4-position did give binding in an ELISA To produce sensitive immunoassays for small molecules format difference is due either to the different format where the competing antigen is immobilized on the surface, used (ﬂow-through or ELISA) or the method of linkage at the it is desirable to reduce the primary antibody concentration 2-position. Speculatively, in a static ELISA format, 2-position as much as possible so that smaller amounts of analyte can of attachment close to the opposite side of the antigen from inhibit the antibody binding to the surface. The problem with that used in raising the antibody may result in the linker and this approach in SPR is that the lower the primary antibody ﬂuorescent label obstructing approach of the antibody to this concentration, the lower the signal and so the poorer the side of the antigen whereas in ﬂow-through formats the linker signal: noise ratio. One technique that has proved very suc- tether is suspended in the ﬂuid stream away from the antigen cessful with progesterone is to enhance the signal with sec- allowing sterically favorable approach of the antibody. These ondary antibody that recognizes the bound primary antibody results show how important the binding format and conjuga- Enhancements of signal up to eight-fold have been tion method is in determining levels of antibody binding.
seen for progesterone using this method The differences in antibody binding responses are also To determine the best secondary antibody concentration reﬂected in the calculated afﬁnity and dissociation constants to use as a label for the binding, a plot of response ver- for the antibody binding to the sensor surfaces, The sus secondary antibody concentration at ﬁxed primary anti- afﬁnity constants increase signiﬁcantly when changing conju- body was prepared (This showed that primary anti- gation from 3-E2 to 4-E2 and then to 2-E2, showing increasing body at 0.5 g/mL had maximum binding with secondary antibody binding afﬁnity. Correspondingly, the dissociation antibody label at concentrations of about 200 g/mL. Use of constants decrease as the afﬁnity constants increase.
300 g/mL produced only approximately 4% additional bind-ing but could potentially signiﬁcantly increase non-speciﬁcbinding. To determine just how much signal enhancement hadbeen obtained and thus how low the primary antibody concen-tration could be reduced, a plot of response versus primaryantibody concentration at ﬁxed secondary antibody concen-tration was prepared By comparing the slopes of theplots 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
KA (107 M−1) Fig. 2 – Plot of response (RU) vs. primary antibody
concentration (g/mL) for 3-E
Errors quoted are standard errors, values are the average of ﬁve 2 (), 4-E2 () and 2-E2 ()
conjugates (error bars too small to see).
Fig. 3 – Plot of response (RU) vs. secondary antibody
Fig. 5 – Estradiol immunoassay curve plot for secondary
concentration (g/mL) for 4-E
antibody enhancement and 2-E2 conjugation showing the
2 () and 2-E2 () linked
conjugates (error bars too small to see).
logistic-log ﬁtted assay curve.
9.5-fold for the 2-E2 surface and 8.9-fold for the 4-E2 surface.
Sensitive SPR assay of 17ˇ-estradiol
These enhancements were slightly better than those seen pre-viously with progesterone support the previous obser- Having optimized the antibody concentrations that can be vation that secondary antibody can enhance primary antibody used, assays could be performed for 17␤-estradiol. The ﬁrst binding signal more than would be expected from simple one- assay was constructed without secondary antibody signal to-one binding These plot also show that the primary enhancement and so used primary antibody at 0.5 g/mL. The antibody concentration can be reduced to 25 ng/mL and still assay gave a limit of detection (LOD) of 96 pg/mL using the maintain about 100 RU of speciﬁc binding after secondary anti- 2-E2 surface (It was not possible to conduct assays body enhancement using the 3-E2 surface because the binding signals were toosmall.
The assay was then repeated but adding the secondary antibody signal enhancement and using a ﬁnal pri-mary antibody concentration of 25 ng/mL This assaygave a LOD of 25 pg/mL with the 2-E2 surface TheLOD for the unenhanced primary antibody at the same con-centration was 66 pg/mL (The IC50 for the assayhad dropped by approximately 75% upon secondary antibodyenhancement whilst the sensitivity had increased three-foldThese assays have the lowest LOD of reported SPRassays of estradiol and show how LOD for this steroid canbe obtained that are comparable to certain ELISA tests but much more rapid and simple. The only previous study ofestradiol SPR immunoassay demonstrated eight-fold higherLOD, 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 functionalgroup The in situ covalently immobilized sensor surface, like the previously reported progesterone surface has proved tobe quite stable over a large number of cycles (in excess of 200 Fig. 4 – Plot of response (RU) vs. primary antibody
cycles), with no noticeable decline in antibody binding capac- concentration (ng/mL) using 2-E2 conjugate and showing
ity, a major factor in a practical biosensor.
mAb binding only () and secondary antibody-enhanced
Production of stable biosensor surfaces that project small binding (). The linear portions of the curves are from 0 to
molecule antigens with conjugation at positions not bear- 100 ng/mL and the line equations are y = 0.574x + 3.98
ing functional groups is essential to retaining high speciﬁc (R2 = 1.00) for mAb only and y = 5.43x + 92.7 (R2 = 0.99) for
binding signal in ﬂow-through biosensors. A novel route secondary antibody enhancement (error bars too small to
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The new england journal of medicine established in 1812 october 29, 2009 A Homozygous CARD9 Mutation in a Family with Susceptibility to Fungal Infections Erik-Oliver Glocker, M.D., Andre Hennigs, Mohammad Nabavi, M.D., Alejandro A. Schäffer, Ph.D., Cristina Woellner, M.Sc., Ulrich Salzer, M.D., Dietmar Pfeifer, Ph.D., Hendrik Veelken, M.D., Klaus Warnatz, M.D.,
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