Immunoassay Reagents for Thyroid Testing. 3. Determination of the

Nov 15, 1997 - the Solution Binding Affinities of a T4 Monoclonal Antibody Fab ... anti-T4 Fab fragment were determined using a single high-density L-...
0 downloads 0 Views 229KB Size
Download PDF
Bioconjugate Chem. 1998, 9, 23−32

23

ARTICLES Immunoassay Reagents for Thyroid Testing. 3. Determination of the Solution Binding Affinities of a T4 Monoclonal Antibody Fab Fragment for a Library of Thyroxine Analogs Using Surface Plasmon Resonance Maciej Adamczyk,* Donald D. Johnson, Phillip G. Mattingly, Jeffrey A. Moore, and You Pan Diagnostics Division Organic Chemistry (9-NM), Abbott Laboratories, Building AP 20, 100 Abbott Park Road, Abbott Park, Illinois 60064. Received July 23, 1997; Revised Manuscript Received October 1, 1997X

A library of thyroxine analogs and tracers was prepared, and their solution binding affinities for an anti-T4 Fab fragment were determined using a single high-density L-T4 biosensor surface in a BIAcore surface plasmon resonance instrument. The high-density L-T4 analog biosensor was calibrated by determination of the initial binding rate was of known concentrations of free anti-T4 Fab fragment in solution to the biosensor surface. A range of individual thyroxine analog and tracer concentrations was subsequently mixed with a fixed concentration of anti-T4 Fab fragment. The concentration of free anti-T4 Fab fragment in each solution at equilibrium was determined, and the equilibrium dissociation constant (KD) for each case was derived. The KD values determined in solution are compared to values determined by a direct kinetic analysis on the BIAcore instrument using individual biosensor surfaces.

INTRODUCTION

The need for well-characterized immunoreagents utilized in immunoassays is widely recognized (1-6). While haptens, tracers, and antibodies are routinely characterized chemically to determine their identity and purity, the kinetic and thermodynamic properties of their binding interactions are generally only inferred from their performance in a particular assay format. These binding interactions are critical to all stages of assay development from selection of antibody through determination of the structural features important for tracer design. Unfortunately, determination of these binding parameters traditionally has been a laborious endeavor (7). The recent development of automated BIAcore (BIAcore, Inc.) surface plasmon resonance technology (SPR) has greatly simplified this task. We recently reported the utility of the automated BIAcore SPR technology in assessing the binding kinetics of an anti-T4 mAb and its Fab fragment to a library of surface-bound thyroxine analogs (2). In that study, individual thyroxine analogs were conjugated to a solidsupported hydrophilic dextran matrix of an SPR chip to form a biosensor, mimicking a heterogeneous assay format. A solution of the mAb or Fab fragment was then introduced at a constant rate to the surface of each biosensor, and binding was monitored continuously, providing real-time kinetic information. Dissociation of * To whom correspondence should be addressed. Telephone: (847) 937-0225. Fax: (847) 938-8927. E-mail: maciej.adamczyk@ abbott.com. X Abstract published in Advance ACS Abstracts, November 15, 1997.

bound mAb or Fab fragment was subsequently monitored after the sample had traversed the biosensor surface. By using a range of binding protein concentrations, a series of sensorgrams was produced for each thyroxine analog biosensor. Nonlinear regression analysis of the data provided the association and dissociation rate constants (ka and kd) from which the equilibrium association constant (KA) and the change in free energy (∆G) of binding were calculated. These values helped to define the structural features of the thyroxine analogs that were required for the binding interaction, but necessitated production of a different biosensor for each analog studied. Additionally, this format neither allowed for the direct measurement of antibody interaction with a native analyte (underivatized T4) or tracer nor served as a good model for homogeneous assay formats. These issues are addressed in this study. The BIAcore instrument was used to study the binding interactions between an anti-T4 Fab fragment and a library of thyroxine analogs and tracers in solution using a single thyroxine analog biosensor. Thus, anti-T4 Fab fragment is known to bind to a biosensor surface of thyroxine immobilized through an aminoalkyl linker (2). With high-density T4 biosensor surfaces, the binding observed is limited by the diffusion of the protein to the surface. Under such conditions, initial binding rates are proportional to the free anti-T4 Fab fragment concentration in solution and independent of the interaction kinetics or analyte-ligand affinity (8, 9). High-density T4 biosensor surfaces were prepared, and the initial binding rates were measured with known concentrations of anti-T4 Fab fragment to construct a calibration curve. A range of individual thyroxine analog and tracer con-

S1043-1802(97)00135-3 CCC: $15.00 © 1998 American Chemical Society Published on Web 01/12/1998

24 Bioconjugate Chem., Vol. 9, No. 1, 1998

centrations were subsequently mixed with a fixed concentration of antibody fragment in solution and allowed to reach equilibrium. The concentration of free anti-T4 Fab fragment in each solution was subsequently determined, and the equilibrium dissociation constant (KD) for each case was derived. MATERIALS AND METHODS

Sodium L-thyroxine pentahydrate (L-1) and all other reagents were obtained from Aldrich Chemical Co. (Milwaukee, WI) unless otherwise noted. Sodium D-thyroxine pentahydrate (D-1), sodium L-3,3′,5-triiodothyronine (L2), and L-3,5-diiodothyronine (3) were obtained from Sigma Chemical Co. (St. Louis, MO). D-3,3′,5-Triiodothyronine (D-2) was obtained from ICN Biochemicals (Cleveland, OH). Compounds 4-16 were prepared as reported earlier (2). 5-Carboxyfluorescein was obtained from Calbiochem (La Jolla, CA). Acridinium chemiluminescent label 39 was prepared following the general procedure of Mattingly et al. (10, 11). Synthesized compounds were purified by HPLC [Waters (Millford, MA) system consisting of a model 590 pump, a Lambda-Max 481 UV detector, a model 745B data module, and a 40 × 100 mm µBondapak C18 column; flow rate, 45 mL/min]. Analytical HPLC was performed on a Beckman Gold system with UV detection at 254 nm using a Waters 8 × 100 mm µBondapak C18 column eluting with aqueous acetonitrile (v:v, CH3CN/H2O) containing 0.05% trifluoroacetic acid at a flow rate of 2 mL/min. 1H NMR spectra were recorded at 300 MHz on a Varian Gemini spectrometer (Palo Alto, CA). Chemical shifts are reported in parts per million (δ) using tetramethylsilane (TMS) as the internal reference; coupling constants (J) are in hertz. Electrospray ionization mass spectrometry (ESI/MS) was carried out on a Perkin-Elmer (Norwalk, CT) Sciex API 100 Benchtop system employing the Turbo IonSpray ion source. Surface plasmon resonance measurements were carried out on a BIAcore 2000 (BIAcore, Inc., Piscataway, NJ) automated system using CM-5 four-channel sensor chips. Reagents for the BIAcore instrument consisted of HBS buffer [10 mM Hepes (pH 7.4), 150 mM NaCl, 3.4 mM EDTA, and 0.05% surfactant P-20] and a coupling kit containing N-hydroxysuccinimide (NHS), N-ethyl-N[3-(diethylamino)propyl]carbodiimide (EDAC), and 1 M ethanolamine hydrochloride (pH 8.5), all from BIAcore, Inc. Anti-thyroxine Fab fragment was prepared and purified as previously described (2). General Procedure for Preparation of 5-Carboxyfluorescein Tracers. (A) In Situ Activation of 5-Carboxyfluorescein. A solution of 5-carboxyfluorescein (250 mg, 0.681 mmol), EDAC (196 mg, 1.02 mmol), and N-hydroxysuccinimide (117 mg, 1.02 mmol) in DMF (6.8 mL) was stirred overnight. The reaction solution (0.1 M) was used without further purification. (B) Coupling of Succinimidyl 5-Carboxyfluorescein to Thyroxine Analogs. To a solution of the analog compound (9-16) (0.03 mmol) in DMF (3 mL) were added a 5-carboxyfluorescein-activated ester stock solution (0.34 mL, 0.1 M, 0.34 mmol) and N, N-diisopropylethylamine (100 µL). After stirring for 4 h, the reaction mixture was acidified with TFA and purified by preparative HPLC. Compound L-17 was obtained from compound L-9 (79%). HPLC (55:45): retention time, 5.8 min, 98%. 1H NMR (methanol-d4): 8.45 (1 H, s), 8.20 (1 H, d, J ) 8), 7.89 (2 H, s), 7.33 (1 H, d, J ) 8), 7.05 (2 H, s), 6.74 (2 H, s), 6.65 (2 H, d, J ) 8), 6.60 (2 H, d, J ) 8), 4.70 (1 H, m), 3.43 (2 H, m), 3.22 (1 H, m), 2.93 (1 H, m), 2.24 (2 H, m), 1.65 (4 H, m), 1.40 (2 H, m). ESI/MS m/z: 1247 (M H)-.

Adamczyk et al.

Compound D-17 was obtained from compound D-9 (59%). HPLC (55:45): retention time, 5.8 min, 96%. 1H NMR (methanol-d4): 8.48 (1 H, s), 8.19 (1 H, d, J ) 8), 7.80 (2 H, s), 7.34 (1 H, d, J ) 9), 7.05 (2 H, s), 6.78 (2 H, s), 6.71 (2 H, d, J ) 8), 6.65 (2 H, d, J ) 8), 4.72 (1 H, m), 3.42 (2 H, m), 3.24 (1 H, m), 2.90 (1 H, m), 2.24 (2 H, m), 1.67 (4H, m), 1.38 (2 H, m). ESI/MS m/z: 1247 (M H)-. Compound L-18 was obtained from compound L-10 (61%). HPLC (55:45): retention time, 4.4 min, 98%. 1H NMR (methanol-d4): 8.45 (1 H, s), 8.20 (1 H, d, J ) 8), 7.79 (2 H, s), 7.33 (1 H, d, J ) 8), 6.98 (1 H, d, J ) 3), 6.74 (2 H, s), 6.71 (3 H, m), 6.65 (2 H, d, J ) 8), 6.57 (1 H, dd, J ) 8, 3), 4.71 (1 H, m), 3.42 (2 H, m), 3.25 (1 H, m), 2.91 (1 H, m), 2.24 (2 H, m), 1.66 (4 H, m), 1.41 (2 H, m). ESI/MS m/z: 1123 (M + H)+. Compound D-18 was obtained from compound D-10 (42%). HPLC (55:45): retention time, 4.4 min, 99%. 1H NMR (methanol-d4): 8.48 (1 H, s), 8.19 (1 H, d, J ) 8), 7.79 (2 H, s), 7.35 (1 H, d, J ) 8), 6.98 (1 H, d, J ) 3), 6.80 (2 H, s), 6.76 (3 H, m), 6.64 (2 H, d, J ) 8), 6.56 (1 H, dd, J ) 8, 3), 4.68 (1 H, m), 3.42 (2 H, m), 3.25 (1 H, m), 2.88 (1 H, m), 2.24 (2 H, m), 1.64 (4 H, m), 1.38 (2 H, m). ESI/MS m/z: 1123 (M + H)+. Compound 19 was obtained from compound 11 (49%). HPLC (50:50): retention time, 4.6 min, >99%. 1H NMR (methanol-d4): 8.45 (1 H, s), 8.20 (1 H, d, J ) 9), 7.77 (2 H, s), 7.32 (1 H, d, J ) 9), 6.75 (2 H, s), 6.65 (4 H, m), 6.61 (2 H, d, J ) 9), 6.49 (2 H, d, J ) 9), 4.67 (1 H, m), 3.42 (2 H, m), 3.23 (1 H, m), 2.87 (1 H, m), 2.21 (2 H, m), 1.64 (4 H, m), 1.23 (2 H, m). ESI/MS m/z: 995 (M - H)-. Compound 20 was obtained from compound 12 (59%). HPLC (55:45): retention time, 5.5 min, >99%. 1H NMR (methanol-d4): 8.46 (1 H, s), 8.21 (1 H, d, J ) 8), 7.81 (2 H, s), 7.33 (1 H, d, J ) 8), 7.03 (1 H, d, J ) 3), 6.83 (1 H, d, J ) 3), 6.76 (2 H, s), 6.68 (2 H, d, J ) 9), 6.62 (2 H, d, J ) 9), 4.72 (1 H, dd, J ) 10, 4), 3.42 (2 H, m), 3.23 (1 H, m), 2.85 (1 H, m), 2.23 (2 H, m), 1.66 (4 H, m), 1.38 (2 H, m). ESI/MS m/z: 1201, 1203 (M + H)+. Compound 21 was obtained from compound 13 (47%). HPLC (55:45): retention time, 4.1 min, >99%. 1H NMR (methanol-d4): 8.46 (1 H, s), 8.21 (1 H, d, J ) 8), 7.79 (2 H, s), 7.34 (1 H, d, J ) 8), 6.77 (4 H, m), 6.72 (2 H, d, J ) 8), 6.63 (2 H, d, J ) 9), 6.54 (1 H, m), 4.69 (1 H, m), 3.44 (2 H, m), 3.22 (1 H, m), 2.88 (1 H, m), 2.24 (2 H, m), 1.66 (4 H, m), 1.57 (2 H, m). ESI/MS m/z: 1075, 1077 (M + H)+. Compound 22 was obtained from compound 14 (44%). HPLC (55:45): retention time, 5.1 min, 98%. 1H NMR (methanol-d4): 8.43 (1 H, s), 8.20 (1 H, d, J ) 8), 7.81 (2 H, s), 7.31 (1 H, d, J ) 8), 6.83 (2 H, s), 6.72 (2 H, s), 6.62 (2 H, d, J ) 9), 6.57 (2 H, d, J ) 9), 4.72 (1 H, m), 3.41 (2 H, m), 3.28 (1 H, m), 2.88 (1 H, m), 2.23 (2 H, m), 1.63 (4 H, m), 1.38 (2 H, m). ESI/MS m/z: 1151, 1153, 1155 (M - H)-. Compound 23 was obtained from compound 15 (58%). HPLC (55:45): retention time, 7.6 min, 98%. 1H NMR (methanol-d4): 8.44 (1 H, s), 8.21 (1 H, d, J ) 8), 7.83 (2 H, s), 7.37 (1 H, d, J ) 8), 7.05 (2 H, s), 6.71 (2 H, s), 6.62 (2 H, d, J ) 8), 6.58 (2 H, d, J ) 8), 5.07 (1 H, m), 3.42 (2 H, m), 3.02 (3 H, s), 2.96 (1 H, m), 2.91 (3 H, s), 2.85 (1 H, m), 2.23 (2 H, m), 1.66 (4 H, m), 1.35 (2 H, m). ESI/ MS m/z: 1276 (M + H)+. Compound 24 was obtained from compound 16 (60%). HPLC (55:45): retention time, 5.3 min, >99%. 1H NMR (methanol-d4): 8.46 (1 H, s), 8.22 (1 H, d, J ) 8), 7.81 (2 H, s), 7.31 (1 H, d, J ) 8), 6.94 (1 H, d, J ) 3), 6.72 (2 H, s), 6.61 (3 H, m), 6.58 (3 H, m), 5.07 (1 H, m), 3.42 (2 H, m), 3.03 (3 H, s), 2.98 (1 H, m), 2.92 (3 H, s), 2.85 (1 H,

Thyroxine Biosensors

m), 2.23 (2 H, m), 1.64 (4 H, m), 1.38 (2 H, m). ESI/MS m/z: 1150 (M + H)+. Compound 29 was obtained from compound D-1 (80%). HPLC (55:45): retention time, 6.9 min, >99%. 1H NMR (DMSO-d6): 9.39 (1 H, d, J ) 7), 8.44 (1 H, s), 8.15 (1 H, d, J ) 8), 7.89 (2 H, s), 7.36 (1 H, d, J ) 8), 6.99 (2 H, s), 6.66 (2 H, d, J ) 2), 6.54 (4 H, m), 4.67 (1 H, m), 3.03 (1 H, m), 2.95 (1 H, m). ESI/MS m/z: 1136 (M + H)+. L-Thyroxine 6-carboxyfluorescein tracer (25) was prepared from compound L-9 and 6-carboxyfluorescein according to the general procedure used for the 5-carboxyfluorescein tracers above (70%). HPLC (55:45): retention time, 5.4 min, >99%. 1H NMR (methanol-d4): 8.11 (2 H, d, J ) 3), 7.77 (2 H, s), 7.67 (1 H, s), 7.03 (2 H, s), 6.72 (2 H, s), 6.65 (2 H, d, J ) 9), 6.58 (2 H, d, J ) 9), 4.66 (1 H, m), 3.21 (3 H, m), 2.85 (1 H, m), 2.16 (2 H, m), 1.54 (4 H, m), 1.27 (2 H, m). ESI/MS m/z: 1247 (M - H)-. N-(Carboxymethyl)-L-thyroxine 4′-Aminomethylfluorescein Tracer (26). (A) N-(Carboxymethyl)-Lthyroxine tert-butyl ester (4) (38 mg, 0.04 mmol) and N-hydroxysuccinimide (5 mg, 0.04 mmol) were dissolved in DMF (500 µL) and stirred with DCC for 14 h under nitrogen. The crude active ester solution was filtered and then added to a solution of 4′-aminomethylfluorescein hydrochloride (17 mg, 0.04 mmol) and triethylamine (3 drops) in DMF (500 µL). The reaction mixture was stirred under nitrogen in the dark. After 14 h, the reaction solvent was removed in vacuo, and the residue was purified by preparative HPLC (78:22:0.4, CH3OH/ H2O/HOAc). Product fractions were pooled and evaporated in vacuo to give the tert-butyl ester-protected tracer (30 mg, 57%) as an orange powder. Analytical HPLC (78: 22:0.4, CH3OH/H2O/HOAc; 1 mL/min, 240 nm): retention time, 12.97 min, 99.7%. FAB/MS m/z: 1235 (M + H)+. (B) The tert-butyl ester-protected tracer (13 mg) was dissolved in 1:1 TFA/CH2Cl2 (1 mL). After the mixture was stirred for 4 h, the reaction solvent was removed in vacuo. The residue was dissolved in methanol (0.6 mL) and purified by preparative HPLC as above to give 26 (9 mg, 69%). Analytical HPLC (75:25:0.4, CH3OH/H2O/ HOAc; 1 mL/min, 240 nm): retention time, 11.2 min, 99%. FAB/MS m/z: 1179 (M + H)+. N-(Carboxymethyl)-L-thyroxine 5-aminomethylfluorescein tracer (27) was prepared from N-(carboxymethyl)L-thyroxine tert-butyl ester (4) and 5-aminomethylfluorescein hydrobromide (12) according to the procedure used for compound 26 (61%). Analytical HPLC (80:20: 0.4, CH3OH/H2O/HOAc; 1 mL/min, 240 nm): retention time, 6.1 min, 99%. FAB/MS m/z: 1179 (M + H)+. L-Thyroxine 4′-N-glycylaminomethylfluorescein tracer (28) was prepared from N-tert-(butoxycarbonyl)-L-thyroxine (2) and 4′-N-glycylaminomethylfluorescein (Molecular Probes, Eugene, OR) according to the procedure outlined for compound 26 (61%). Analytical HPLC (75: 25:0.4, CH3OH/H2O/HOAc; 1 mL/min, 240 nm): retention time, 10.3 min, 99%. ESI/MS m/z: 1178 (M + H) +. General Procedure for Preparation of 10-(3-Sulfopropyl)-N-tosyl-N-(3-carboxypropyl)acridinium9-carboxamide Tracers. To a solution of the analog compound (9-16) (0.03 mmol) in DMF (3 mL) were added the acridinium active ester (39) (0.03 mmol) and N,Ndiisopropylethylamine (100 µL). After being stirred for 6 h, the reaction mixture was acidified with TFA and purified by preparative HPLC. Compound L-31 was obtained from compound L-9 (52%). HPLC (50:50): retention time, 6.5 min, >99%. 1H NMR (methanol-d ): 8.92 (2 H, d, J ) 10), 8.43 (2 H, 4 m), 8.05 (2 H, d, J ) 10), 7.85 (2 H, m), 7.77 (2 H, s), 7.17 (4 H, bs), 7.03 (2 H, s), 5.73 (2 H, b), 4.67 (1 H, m),

Bioconjugate Chem., Vol. 9, No. 1, 1998 25

4.24 (2 H, m), 3.24 (5 H, m), 2.90 (1 H, m), 2.65 (2 H, m), 2.44 (2 H, m), 2.36 (3 H, s), 2.32 (2 H, m), 2.20 (2 H, m), 1.55 (4 H, m), 1.34 (2 H, m). ESI/MS m/z: 1457 (M + H)+. Compound D-31 was obtained from compound D-9 (51%). HPLC (50:50): retention time, 6.5 min, >99%. 1H NMR (DMSO-d /TFA-d): 9.01 (2 H, dd, J ) 12, 9), 6 8.42 (2 H, bt), 8.16 (1 H, d, J ) 7), 8.01 (2 H, m), 7.85 (2 H, m), 7.85 (1 H, t, J ) 8), 7.80 (2 H, d, J ) 6), 7.69 (1 H, d, J ) 9), 7.63 (1 H, d, J ) 9), 7.16 (1 H, d, J ) 8), 7.11 (1 H, d, J ) 8), 7.03 (2 H, d, J ) 2), 5.66 (2 H, b), 4.44 (1 H, b), 4.20 (2 H, b), 3.42 (1 H, m), 3.08 (2 H, m), 2.92 (2 H, m), 2.81 (1 H, m), 2.47 (2 H, m), 2.37 (1 H, m), 2.30 (3 H, s), 2.27 (1 H, m), 2.06 (2 H, m), 1.65 (1 H, b), 1.43 (4 H, m), 1.23 (2 H, m). ESI/MS m/z: 1457 (M + H)+. Compound L-32 was obtained from compound L-10 (63%). HPLC (50:50): retention time, 4.8 min, >99%. 1H NMR (DMSO-d /TFA-d): 8.96 (2 H, dd, J ) 14, 9), 6 8.40 (2 H, bt), 8.13 (1 H, d, J ) 9), 7.97 (2 H, m), 7.82 (1 H, t, J ) 7), 7.76 (2 H, d, J ) 6), 7.68 (1 H, d, J ) 9), 7.60 (1 H, d, J ) 9), 7.16 (1 H, d, J ) 8), 7.08 (1 H, d, J ) 8), 6.94 (1 H, t, J ) 3), 6.80 (1 H, dd, J ) 9, 3), 6.53 (1 H, m), 5.65 (2 H, b), 4.46 (1 H, m), 4.18 (2 H, b), 3.42 (2 H, m), 3.06 (4 H, m), 2.73 (1 H, m), 2.43 (2 H, m), 2.30 (1 H, m), 2.27 (3 H, s), 2.14 (1 H, m), 2.05 (2 H, m), 1.64 (1 H, m), 1.40 (4 H, m), 1.22 (2 H, m). ESI/MS m/z: 1331 (M + H)+. Compound D-32 was obtained from compound D-10 (55%). HPLC (50:50): retention time, 4.8 min, >99%. 1H NMR (DMSO-d /TFA-d): 9.01 (2 H, dd, J ) 13, 10), 6 8.41 (2 H, bt), 8.15 (1 H, d, J ) 8), 7.98 (2 H, m), 7.85 (1 H, bt), 7.78 (2 H, d, J ) 5), 7.66 (1 H, d, J ) 9), 7.62 (1 H, d, J ) 9), 7.16 (H, d, J ) 8), 7.12 (1 H, d, J ) 8), 6.95 (1 H, t, J ) 3), 6.78 (1 H, dd, J ) 9, 3), 5.66 (2 H, b), 4.45 (1 H, m), 4.20 (2 H, b), 3.45 (2 H, b), 3.07 (2 H, m), 2.94 (2 H, m), 2.86 (1 H, m), 2.47 (2 H, m), 2.31 (1 H, m), 2.29 (3 H, s), 2.15 (1 H, m), 2.03 (2 H, m), 1.65 (1 H, b), 1.42 (4 H, m), 1.27 (2 H, m). ESI/MS m/z: 1331 (M + H)+. Compound 33 was obtained from compound 11 (67%). HPLC (45:55): retention time, 9.2 min, >99%. 1H NMR (DMSO-d6/TFA-d): 9.87 (2 H, dd, J ) 13, 10), 8.42 (2 H, bt), 8.17 (1 H, d, J ) 8), 8.01 (2 H, m), 7.86 (1 H, t, J ) 8), 7.76 (2 H, d, J ) 5), 7.67 (1 H, d, J ) 9), 7.63 (1 H, d, J ) 9), 7.18 (1 H, d, J ) 8), 7.11 (1 H, d, J ) 8), 6.64 (2 H, d, J ) 9), 6.49 (2 H, d, J ) 9), 5.65 (2 H, b), 4.43 (1 H, m), 4.20 (2 H, b), 3.44 (2 H, b), 3.06 (2 H, m), 2.95 (2 H, m), 2.85 (1 H, m), 2.48 (2 H, m), 2.31 (1 H, m), 2.29 (3 H, s), 2.14 (1 H, m), 2.06 (2 H, m), 1.65 (1 H, b), 1.43 (4 H, m), 1.23 (2 H, m). ESI/MS m/z: 1205 (M + H)+. Compound 34 was obtained from compound 12 (57%). HPLC (50:50): retention time, 4.8 min, >99%. 1H NMR (DMSO-d6/TFA-d): 9.00 (2 H, dd, J ) 14, 10), 8.42 (2 H, bt), 8.14 (1 H, d, J ) 7), 7.97 (2 H, m), 7.83 (1 H, m), 7.78 (2 H, d, J ) 6), 7.68 (1 H, d, J ) 9), 7.62 (1 H, d, J ) 9), 7.15 (1 H, d, J ) 9), 7.10 (1 H, d, J ) 9), 7.03 (1 H, t, J ) 3), 6.85 (1 H, t, J ) 3), 5.65 (2 H, m), 4.48 (1 H, dd, J ) 10, 4), 4.19 (2 H, m), 3.44 (2 H, m), 3.05 (2 H, m), 2.93 (2 H, m), 2.81 (1 H, m), 2.46 (2 H, m), 2.30 (1 H, m), 2.28 (3 H, s), 2.15 (1 H, m), 2.01 (2 H, m), 1.64 (1 H, b), 1.381.23 (4 H, m). ESI/MS m/z: 1407, 1409 (M - H)-. Compound 35 was obtained from compound 13 (35%). HPLC (50:50): retention time, 4.5 min, >99%. 1H NMR (methanol-d4): 8.93 (2 H, d, J ) 9), 8.43 (2 H, dd, J ) 11, 9), 8.05 (2 H, d, J ) 9), 7.83 (2 H, m), 7.76 (2 H, s), 7.16 (4 H, s), 6.77 (2 H, m), 6.54 (1 H, m), 5.73 (2 H, b), 4.66 (1 H, m), 4.24 (2 H, b), 3.26 (5 H, m), 2.84 (1 H, m), 2.61 (2 H, m), 2.42 (2 H, m), 2.37 (3 H, s), 2.37 (2 H, m), 2.20 (2 H, m), 1.56 (4 H, m), 1.38 (2 H, m). ESI/MS m/z: 1283, 1285 (M + H)+.

26 Bioconjugate Chem., Vol. 9, No. 1, 1998

Compound 36 was obtained from compound 14 (51%). HPLC (50:50): retention time, 5.7 min, 97%. 1H NMR (methanol-d4): 8.93 (2 H, d, J ) 9), 8.44 (2 H, dd, J ) 11, 9), 8.07 (2 H, d, J ) 9), 7.87 (2 H, m), 7.81 (2 H, s), 7.17 (4 H, s), 6.83 (2 H, s), 5.76 (2 H, b), 4.67 (1 H, m), 4.25 (2 H, m), 3.22 (5 H, m), 2.87 (1 H, m), 2.61 (2 H, m), 2.44 (2 H, m), 2.37 (3 H, s), 2.29 (2 H, m), 2.18 (2 H, m), 1.60 (4 H, m), 1.32 (2 H, m). ESI/MS m/z: 1361, 1363, 1365 (M + H)+. Compound 37 was obtained from compound 15 (67%). HPLC (50:50): retention time, 8.6 min, 96%. 1H NMR (DMSO-d6/TFA-d): 9.04 (2 H, dd, J ) 12, 9), 8.41 (2 H, bt), 8.17 (1 H, d, J ) 7), 7.84 (1 H, m), 7.80 (2 H, d, J ) 3), 7.69 (1 H, d, J ) 9), 7.66 (1 H, m), 7.15 (1 H, d, J ) 9), 7.11 (1 H, d, J ) 9), 7.04 (2 H, s), 5.66 (2 H, b), 4.87 (1 H, m), 4.20 (2 H, m), 3.44 (2 H, m), 3.05 (2 H, m), 2.94 (3 H, s), 2.85 (2 H, m), 2.80 (3 H, s), 2.72 (2 H, m), 2.41 (2 H, m), 2.30 (3 H, s), 2.14 (2 H, m), 2.05 (2 H, m), 1.66 (1 H, m), 1.43 (4 H, m), 1.22 (2 H, m). ESI/MS m/z: 1484 (M + H)+. Compound 38 was obtained from compound 16 (41%). HPLC (50:50): retention time, 5.9 min, >99%. 1H NMR (DMSO-d6/TFA-d): 8.99 (2 H, dd, J ) 12, 9), 8.41 (2 H, bt), 8.18 (1 H, d, J ) 9), 8.01 (2 H, d, J ) 9), 7.87 (1 H, bt), 7.80 (2 H, d, J ) 3), 7.69 (1 H, d, J ) 8), 7.66 (1 H, b), 7.18 (1 H, d, J ) 8), 7.11 (1 H, d, J ) 8), 6.91 (1 H, t, J ) 3), 6.82 (1 H, dd, J ) 9, 4), 6.56 (1 H, m), 5.65 (2 H, b), 4.88 (1 H, m), 4.20 (2 H, b), 3.44 (2 H, m), 3.06 (2 H, m), 2.94 (3 H, s), 2.89 (2 H, m), 2.80 (3 H, s), 2.71 (2 H, m), 2.46 (2 H, m), 2.29 (3 H, s), 2.15 (2 H, m), 2.04 (2 H, m), 1.65 (1 H, m), 1.42 (4 H, m), 1.22 (2 H, m). ESI/MS m/z: 1358 (M + H)+. Preparation of a Biosensor Surface. Immobilization of L-T4 analog L-9 through the free amine to the CM-5 sensor chip was performed by a modification of the method previously described (2). Briefly, a continuous flow of HBS buffer at 20 µL/min was initiated over the biosensor surface. The carboxymethylated dextran matrix on the sensor surface was activated by a 6 min injection of a solution of 0.2 M EDAC and 0.05 M NHS. L-T4 analog L-9 (25 µg/mL in 10% EtOH, 50 mM NaHCO3, and 1 M NaCl at pH 8.5) was then immobilized (5 min injection), followed by a 6 min injection of 1 M ethanolamine hydrochloride to block remaining unreacted active ester groups. Typical immobilizations yielded 350-450 RU of L-9 bound to the biosensor surface. Solution Competition Analysis. (A) Construction of a Standard Curve. All BIAcore studies were carried out at 25 °C using the Fab fragment of an anti-T4 mAb which eliminated the complexity associated with the bivalency of the monoclonal antibody. Seven concentrations (0, 5, 10, 15, 20, 25, and 30 nM) of anti-T4 Fab fragment were serially diluted into HBS buffer and allowed to equilibrate for 1 h. Each sample was then injected over the L-T4 biosensor surface at 5 µL/min for 2 min. The biosensor surface was regenerated after each run with a 1 M formic acid wash for 1 min. Initial rates of binding of anti-T4 Fab fragment to the biosensor surface were measured over a 15 s window beginning 15 s postinjection using homogeneous association model type 3 (13). A standard curve was obtained from the linear fit of a plot of initial binding rates versus concentration of added anti-T4 Fab fragment. (B) Determination of Solution Affinities. Ten concentrations of each soluble T4 analog, determined from preliminary studies to completely define the competition curve, were serially diluted into HBS buffer and mixed with 20 nM anti-T4 Fab fragment. Samples were allowed to equilibrate (g2 h) and then injected over the calibrated

Adamczyk et al.

T4 biosensor surface at 5 µL/min for 2 min. The biosensor surface was regenerated after each run with a 1 M formic acid wash for 1 min. Sensorgrams for each solution were obtained in duplicate, and initial binding rates of free anti-T4 Fab fragment to the biosensor surface were measured as described above. The resulting free antiT4 Fab fragment concentrations in solution, measured as initial binding rates to the biosensor surface, were plotted against the concentration of added soluble T4 analog to generate competition curves. Equilibrium dissociation constants (KD) were obtained by nonlinear regression analysis of the data using the solution affinity model built into BIAevaluation 2.1 software (BIAcore, Inc.) with the slope and intercept obtained from the standard curve. RESULTS

Preparation of Soluble T4 Analogs. Thyroxine analogs with and without aminoalkyl linkers (1-16) were obtained from commercial sources or synthesized as previously described (2). Analogs 9-16 bearing an aminoalkyl linker were coupled with the NHS active ester of 5-carboxyfluorescein to give tracers 17-24 in 4279% yield after preparative HPLC (Scheme 1). Analog L-9 was coupled in an identical manner to the NHS active ester of 6-carboxyfluorescein to yield compound 25 in 70% yield. The fluorescein tracers 26 and 27 were prepared from the NHS active ester of N-(carboxymethyl)-Lthyroxine tert-butyl ester and 4′-aminomethylfluorescein or 5-aminomethylfluorescein in 39 and 61% overall yields, respectively, after deprotection with TFA. N-tert(Butoxycarbonyl)-L-thyroxine NHS active ester and 4′N-glycylaminomethylfluorescein were reacted to give the carboxy-substituted thyroxine tracer 28 after deprotection with TFA in 61% overall yield. Fluoresceinated compound 29 was prepared by the direct conjugation of D-thyroxine and 5-carboxyfluorescein NHS ester in 80% yield. Analogs 9-16 were also coupled with the NHS active ester of 10-(3-sulfopropyl)-N-tosyl-N-(3-carboxypropyl)acridinium-9-carboxamide (39) to give chemiluminescent tracers 31-38 in 35-67% yield after preparative HPLC (Scheme 1). Preparation of an L-T4 Biosensor. L-T4 containing an aminoalkyl linker (L-9, Figure 1) was immobilized on the activated carboxymethylated dextran surface of a CM-5 sensor chip through amine coupling and used for all experiments to determine the concentration of free anti-T4 Fab fragment in solution. To ensure that initial rates of binding of anti-T4 Fab fragment to the immobilized L-T4 biosensor surface were mass transport limited, a relatively high-density surface was prepared via a modification of the method utilized for the preparation of low-density thyroxine analog biosensors previously described (2). Thus, high-density L-T4 surfaces were obtained by increasing the flow rate across the biosensor surface from 5 to 20 µL/min for the activation (NHS/ EDAC), coupling (L-9), and blocking (ethanolamine) solutions. Additionally, the concentration of L-9 in the coupling solution was increased from 5 to 25 µg/mL. Typical immobilizations yielded 350-450 RU due to L-9 covalently bound to the biosensor surface. Initial binding rates obtained for a given concentration of anti-T4 Fab fragment (e30 nM, vida infra) to all biosensor surfaces generated during these studies were identical within experimental error, indicating that mass transport limiting binding conditions were met. Biosensors prepared in this manner were suitable for use in the solution affinity analyses described below. Construction of a Standard Curve. Typical sensorgrams obtained for different concentrations of anti-

Thyroxine Biosensors

Bioconjugate Chem., Vol. 9, No. 1, 1998 27

Scheme 1

T4 Fab fragment binding to a high-density L-T4 biosensor surface under mass transport limiting conditions are shown in Figure 2. Both the initial binding rates and the total response units obtained during the binding phase of the sensorgrams increase proportionately with the increase in binding protein concentration within the range examined (0-30 nM). The initial binding rate for each anti-T4 Fab fragment concentration was obtained

by averaging the binding rate observed over a 15 s window beginning 15 s postinjection. Data from the first 15 s of each sensorgram were omitted due to sample dispersion effects at the start of injections. A plot of initial binding rate versus concentration of anti-T4 Fab fragment yields a standard curve which can be used to determine the concentration of free Fab fragment in equilibrium with various T4 analogs from which KD

28 Bioconjugate Chem., Vol. 9, No. 1, 1998

Adamczyk et al.

Figure 1. Biosensor utilized for solution affinity analysis of T4 analogs for anti-T4 Fab fragment.

Figure 2. Overlay plot of sensorgrams generated with 0, 5, 10, 15, 20, 25, and 30 nM anti-T4 Fab fragment binding to the immobilized T4 biosensor surface. Initial binding rates were obtained from the shaded portion of each sensorgram.

Figure 4. Analysis of anti-T4 Fab fragment binding to L-T3Br fluorescein tracer in solution. (A) Overlay plot of sensorgrams generated from solutions of 0.5, 1, 4, 7, 10, 13, 16, 19, 22, and 25 nM L-T3Br fluorescein tracer with 20 nM anti-T4 Fab fragment. Initial binding rates were obtained from the shaded portion of each sensorgram. (B) Nonlinear regression plot of initial binding rate versus concentration of added L-T3Br fluorescein tracer.

Figure 3. Average standard curve obtained for binding of antiT4 Fab fragment to the immobilized T4 biosensor surface.

values in solution can be determined. Thirteen identical standard curves were generated during the course of these studies. The initial binding rates measured for each concentration of anti-T4 Fab fragment were averaged and plotted against the Fab fragment concentration, providing an average standard curve depicted in Figure 3. The standard curve is linear (r2 ) 0.998) over the Fab fragment concentration range of 0-30 nM with measured binding rates from 0 to 6 RU s-1. The slope and intercept for the average standard curve are 2.0 × 108 RU M-1 s-1 and -0.099 RU s-1, respectively. Determination of Solution Affinities of Soluble T4 Analogs for Anti-T4 Fab Fragment. Ten concentrations of each T4 analog tested were mixed with a fixed concentration of anti-T4 Fab fragment (20 nM) in HBS buffer. After the solutions had reached equilibrium, the samples were individually injected over the calibrated high-density T4 biosensor surface to generate a series of sensorgrams. A typical result is illustrated in Figure 4A for the solution affinity analysis of L-T3Br fluorescein tracer (20) with anti-T4 Fab fragment. As the concentration of T4 analog increases, the amount of free anti-T4

Fab fragment in solution available for binding to the biosensor surface decreases. Since the concentration of free binding protein is proportional to the binding rate of anti-T4 Fab fragment to the biosensor surface, the initial binding rate also decreases. A plot of initial binding rate, determined as above, versus total concentration of added L-T3Br fluorescein tracer (20) provides the competition curve depicted in Figure 4B. The data points represent the experimentally determined initial rates of binding of free Fab fragment to the biosensor surface at a given concentration of soluble T4 analog in solution. The curve represents the best nonlinear fit of the data using eq 1 (solution affinity model in BIAevaluation 2.1 software)

r0 ) r m -

(

)

rm - I k + KD + x+ 2 k rm - I 1 + KD k x+ 4 k

x(

) ( 2

-x

)

rm - I (1) k

where r0 is the initial binding rate, rm is the maximum initial binding rate where [T4 analog] ) 0, x is the total concentration of T4 analog or tracer, and k and I are the slope and intercept, respectively, determined from the standard curve above. KD is the equilibrium dissociation constant for the binding of the T4 analog to anti-T4 Fab fragment in solution. In the example depicted, values for KD of 122 ( 50 pM and an rm of 3.76 RU s-1 were obtained. Using the standard curve slope and intercept

Thyroxine Biosensors

Bioconjugate Chem., Vol. 9, No. 1, 1998 29

Table 1. Effect of T4 Analog Structure on the Binding Properties of T4 Analogs and Tracers with an Anti-T4 Fab Fragment equilibrium dissociation constant (KD) L-T4 D-T4 L-T3 D-T3 L-T2 L-T3Br L-T2Br L-T2Br2 L-T4NMe2 L-T3NMe2

X1

X2

R1

parent

parent with linker

fluorescein tracer

acridinium tracer

solid phasea

Ib Id Hb Hd H I H Br I H

I I I I H Br Br Br I I

OH OH OH OH OH OH OH OH N(CH3)2 N(CH3)2

e100 pMc e100 pM 194 ( 12 nM 282 ( 35 nM 16 ( 3.7 µM 417 ( 96 pM 3.6 ( 0.3 µM 831 ( 150 pM e100 pM 41 ( 12 nM

e100 pM e100 pM 44 ( 6.2 nM 31 ( 3.5 nM 12 ( 1.5 µM e100 pM 585 ( 57 nM 283 ( 55 pM e100 pM 20 ( 3.2 nM

e100 pM 924 ( 240 pM 28 ( 4.0 nM 18 ( 3.1 nM 6 ( 0.83 µM 122 ( 50 pM 298 ( 45 nM 336 ( 60 pM 145 ( 87 pM 55 ( 12 nM

e100 pM e100 pM 18 ( 3.0 nM 19 ( 3.4 nM 22 ( 3.7 µM e100 pM 214 ( 41 nM 329 ( 50 pM 241 ( 117 pM 142 ( 12 nM

75 ( 10.2 pM 225 ( 42 pM 0.82 ( 0.19 nM 1.3 ( 0.17 nM nde 225 ( 17 pM 0.89 ( 0.3 nM 367 ( 49 pM 60 ( 19 pM 7.0 ( 1.2 nM

a These data were obtained an from earlier study (2) in which the Fab interacted with each parent/linker structure conjugated to a separate sensor surface. Data were reported as equilibrium association constants (KA) in the original reference. b L-Enantiomer. c KD was too small to be reliably measured by solution affinity experiments. d D-Enantiomer. e Not determined.

Table 2. Effect of the Linker and Fluorescein Regioisomer on the Binding Properties of T4 Fluoresecein Tracers with Anti-T4 Fab Fragment

determined above and the equation for a straight line (y ) mx + b), an rm value of 3.76 RU s-1 corresponds to 19.3 nM free anti-T4 Fab fragment without any L-T3Br fluorescein tracer added which is very close to the predicted value of 20 nM. Solution affinity studies with all T4 analogs were conducted in a similar manner. Equilibrium dissociation constants obtained for several T4 analogs and tracers with the anti-T4 Fab fragment in solution are summarized in Table 1. For comparison, the table also contains KD values obtained previously for the same T4 analogs bound to the biosensor chip surface through aminoalkyl linkers (compounds 9-16) using the direct kinetic analysis protocol on the BIAcore instrument (2). In general, the KD values obtained in solution were comparable to those determined by direct kinetic analysis. The largest differences are seen in the T3 analog series (L- and D-T3, L-T2Br, and L-T3NMe2) with KD values obtained in solution being higher. As seen previously, the T4 analogs (L- and D-T4, L-T3Br, and L-T4NMe2 series) bind exceedingly tight with several KD values being outside the range for which the BIAcore instrument can accurately provide values from solution competition studies (e100 pM) (14). Conversely, the L-T2 analogs

bind much weaker with KD values in the micromolar range. There is no preference for the stereochemistry of the R-amino group. Significantly, incorporation of fluorescein or acridinium moieties into any of the T4 analogs for generation of tracers had little effect on the observed K D. To determine the effect of the linker position and the fluorescein regioisomer utilized for a tracer on the KD values, five additional fluorescein tracers were studied. The results are summarized in Table 2. 5-Carboxyfluorescein tracers (27 and 29) bind the anti-T4 Fab fragment the tightest with KD values being outside the range for which the BIAcore instrument can accurately provide values from solution affinity studies (e100 pM). For the tracers linked through the 5-position of fluorescein studied here, there is no apparent preference for the stereochemistry of the R-amino group or the length of the linker arm. Similar KD values were obtained for analogs containing aminoalkyl linkers attached to fluorescein through the 4′- and 6-positions (25 and 26). However, the tracer bearing a 4′-N-glycylaminomethylfluorescein moiety linked through the carboxyl group of L-T4 (28)

30 Bioconjugate Chem., Vol. 9, No. 1, 1998

showed a considerable decrease in affinity for the antiT4 Fab fragment. DISCUSSION

Surface plasmon resonance technology allows for the direct real-time observation of molecular interactions between an analyte in solution and a binding partner immobilized on a biosensor surface. The SPR phenomenon measures small changes in the refractive index at the biosensor surface which is directly proportional to the mass of the bound analyte (8, 15). The larger the mass of the analyte used, the larger the instrumental response. Thus, the majority of studies which have utilized BIAcore SPR technology have involved binding interactions between two macromolecules. In such cases, the choice of which macromolecule to conjugate to the biosensor surface is largely arbitrary since binding of either macromolecule to the immobilized partner will result in an easily measurable instrument response. However, the observed kinetics of the interaction may be different for each approach because the process of immobilization of a macromolecule is generally nonspecific and can effect the conformation and reactivity of the species involved (16). Relatively few studies have been reported in which one of the binding partners is a low-molecular weight ligand or analyte. In this case, the design of the experiment is critical to the reliability of the information obtained. Conjugation of the macromolecule member of the binding pair to the biosensor surface is again nonspecific, and a subsequent binding interaction with the low-molecular weight analyte would generate a minimal instrument response. The complementary experiment in which the low-molecular weight species is conjugated to the biosensor surface is more advantageous with regard to generation of an instrument response upon binding of the macromolecular analyte. However, there are limitations in the conjugation of low-molecular weight ligands to a biosensor surface. Small molecules generally have fewer functional groups available for coupling to a biosensor surface and in many cases require chemical modification for incorporation of a linker functionality (2, 14, 17). In fact, the acridinium and fluorescein tracers described here contain no functionalities which would allow them to be coupled to a biosensor surface. Low-molecular weight ligand immobilizations also cannot be followed directly, since the instrument response obtained for small molecules is too low. A subsequent binding experiment with saturating amounts of the macromolecular analyte is required to obtain a large and easily quantitated instrument response. Moreover, the amount of a lowmolecular weight ligand immobilized on the biosensor surface is difficult to control and often represents an excessively high binding capacity for the macromolecular analyte. The need to study binding interactions involving lowmolecular weight ligands or analytes using SPR technology is still of considerable importance since many ligandreceptor interactions of biological significance involve at least one low-molecular weight interactant. Several approaches to the study of binding interactions involving a low-molecular weight ligand or analyte have been described. One approach involves the direct, simultaneous analysis of a low-molecular weight analyte binding to multiple sensing surfaces with varying amounts of an immobilized ligand (multispot sensing) (18). The methodology was shown to be applicable to analytes as small as 180 Da and particularly useful for low-affinity interac-

Adamczyk et al.

tions. Drawbacks to this approach include a requirement for the most sensitive BIAcore instrument, BIAcore 2000, and the need to use several sensor surfaces for a single binding study. Furthermore, the kinetic information obtained from this method is more qualitative than quantitative. A more quantitative analysis of binding interactions involving a low-molecular weight analyte can be obtained from a competitive study in which the lowmolecular weight analyte competes with a larger macromolecule for the same immobilized ligand (surface competition) (8). The method provides both the rate and affinity constants in a one-step procedure that monitors binding directly. In a qualitative manner, the method allows for a rapid affinity ranking of different lowmolecular weight analytes interacting with the same immobilized ligand. However, the mathematics used to characterize the surface competition experiment are much more complex than that required for a direct binding experiment. The instrument response for a given low-molecular weight/high-molecular weight analyte combination binding to an immobilized ligand is dependent upon the association and dissociation rate constants of both the low- and high-molecular weight analytes and their respective molecular weights. The approach also requires the additional preparation and characterization of the high-molecular weight analyte to be used in the competition studies. An alternative and simpler competitive experiment involves an immobilized ligand and a soluble ligand free in solution competing for binding sites on a single analyte (solution competition) (8, 19, 20). For the solution competition experiment, if high-density immobilized ligand surfaces are employed and the association rate between the analyte and immobilized ligand is g1 × 105 M-1 s-1, the observed binding will be limited by mass transfer. Under such conditions, initial binding rates are directly proportional to the amount of free analyte (not bound to soluble ligand) in solution and independent of the interaction kinetics or analyte-ligand affinity. Measuring concentrations of free analyte in equilibrium solutions with different concentrations of soluble ligand allows one to determine equilibrium dissociation constants. This was the method utilized in these studies. The advantage to the solution competition experiment is that a single biosensor surface can be utilized for determination of binding affinities for a series of related analogs, making direct comparison of results more reliable. Unfortunately, the method is indirect and does not provide individual association and dissociation rate constants. Solution competition experiments require the generation of a standard curve from which the concentration of free analyte in equilibrium solutions with a soluble ligand can be determined. Several high-density L-T4 surfaces, ranging from 350 to 450 RU of immobilized L-9, were prepared during the course of these studies, and a standard curve was generated at the start of each experiment. As depicted in the average standard curve (Figure 3), the measured initial binding rate for a given concentration of anti-T4 Fab fragment binding to the different immobilized L-T4 surface densities was quite reproducible, indicating a mass transport limiting situation. The high reproducibility of initial binding rates also supports the accuracy of the slope and intercept obtained from the average standard curve which are critical to evaluation of solution competition data by the solution affinity model (cf. eq 1), particularly for highaffinity interactions. For low-affinity interactions (micromolar concentrations), KD values obtained are largely independent of the slope and intercept values from the

Thyroxine Biosensors

standard curve (8). The standard curves and solution competition studies described here were all carried out with the Fab fragment of an anti-T4 mAb. Anti-T4 mAb contains two identical binding sites and under the conditions described for solution competition experiments can bind zero, one, or two T4 antigens (immobilized and/ or soluble). Under such conditions, the observed binding rate is no longer proportional to free antibody binding sites in solution. Equations accounting for mAb bivalency have been derived, but they do not necessarily correct the problem (8, 14). The Fab fragment contains a single binding site and eliminates the problems associated with mAb bivalency. In general, structural requirements important for the anti-T4 Fab fragment-T4 analog binding interaction in solution were the same as those identified from the direct analysis of Fab fragment interaction with individual T4 analog biosensors (2). Specifically, the diphenyl ether ring system, including the degree and type of halogenation, was the major determinant in binding affinity. The binding interaction became less favorable with the loss of each halogen from the terminal aryl ring (KD for a series, L,D-T4 ≈ L-T4NMe2 < L-T3Br < L-T2Br2 < L,D-T3 ≈ L-T3NMe2 < L-T2Br < L-T2). Iodo-bromo exchanges in the terminal aryl ring system also resulted in slight decreases in binding affinity. These observations have been attributed to the acidity of the phenolic hydroxyl group and the effect the different halogenation patterns have on the pKa of this moiety (2). As previously observed in the direct kinetic analysis, the binding interaction in solution is relatively insensitive to modifications to the amino acid portion of the T4 analogs. Specifically, there is no preference for the stereochemistry of the R-amino chiral center (L-T4 series ≈ D-T4 series) or a requirement for an ionizable carboxyl group (L-T4 series ≈ L-T4NMe2 series). Binding interactions were generally more favorable for the analogs bearing an aminoalkyl linker relative to those of the native analytes. This was not unexpected since the immunogen used to elicit the antibody was prepared by conjugating N-acetyl-L-T4 to the carrier protein. With an immunogen thus prepared, the animal’s immune system was presented a hapten in which both the R-amino and carboxylic acid groups had been converted into amides, unlike the native analyte. Incorporation of the acridinium or 5-carboxyfluorescein labels via an aminohexyl linker also had little effect on the observed binding affinity in solution (Table 1). However, the linker, linker position, and regioisomer of the label incorporated can effect the binding interaction as revealed by the particular combinations that were tested (Table 2). In absolute terms, some significant differences were observed between KD values determined by the solution competition method and those determined by direct kinetic analysis (solid phase) on the BIAcore instrument (Table 1), with the disparity being more pronounced for the weaker binding interactions. In these cases, the KD value determined by the solution competition method was always higher than the KD value determined by direct kinetic analysis. Previous literature reports applying BIAcore technology to the study of binding interactions have noted similar differences between solution competition studies and direct kinetic analysis studies (14, 21, 22). The disparity has been primarily attributed to the different experimental designs of the two methods. A direct kinetic analysis of binding interactions involves the determination of association and dissociation rate constants (ka and kd) for an analyte free in solution binding to individual immobilized ligands. Equilibrium

Bioconjugate Chem., Vol. 9, No. 1, 1998 31

dissociation constants (KD) can then be calculated from the measured rate constants. However, the density of the immobilized ligand can greatly effect the measured rate constants due to mass transport effects and rebinding of analyte to the immobilized ligand. Rebinding can become particularly problematic with rapid association rates which will favor reassociation of analyte to the immobilized ligand during the dissociation phase of the study. Rebinding of analyte to the immobilized ligand will artificially slow the observed dissociation rate constant. This phenomenon will become more pronounced as the true dissociation rate becomes faster and can result in an artificially low equilibrium dissociation constant (KD ) kd/ka) for weaker binding interactions. Association rates measured by direct kinetic analysis of the anti-T4 Fab fragment-T4 analog binding interaction were all rapid, ranging from 1 to 7 × 106 M-1 s-1 (2). Dissociation rates measured by the direct kinetic analysis method were much more varied, ranging from 6 to 920 × 10-5 s-1. It would not be surprising if some of the antiT4 Fab fragment-T4 analog binding affinities previously determined by direct kinetic analysis were artificially low. Thus, due to the solution-surface reaction format, the values can only be regarded as apparent rate and equilibrium constants. In contrast, the KD values determined here by the solution competition method involve equilibrium mixtures in solution which are independent of the solution-surface interactions described above for the direct kinetic analysis method. Equilibrium dissociation constants measured in solution can be regarded as true affinity constants. CONCLUSION

Direct kinetic analysis of binding interactions using BIAcore SPR technology provides individual association and dissociation rate constants from which equilibrium constants can be calculated. However, the biosensor optimization process for minimizing mass transport effects and analyte rebinding during the dissociation phase of the sensorgram can be time-consuming and subjective. Furthermore, direct comparison of binding affinities determined for a series of related analogs with a single binding protein is difficult since a different biosensor must be generated for each analog studied. In contrast, the solution competition format utilized in these studies provides only equilibrium constants but allows for a reliable, direct comparison of binding affinities using a single biosensor surface. The binding affinities of an anti-T4 Fab fragment for a series of T4 analogs and tracers were determined using the solution competition format and compared to results obtained by the direct kinetic analysis method on the BIAcore instrument. Qualitatively, results obtained from solution competition studies of the anti-T4 Fab fragmentT4 analog binding interaction followed the same trend as that observed in the direct kinetic analysis. Primary T4 analog structural features important for the binding interaction include the diphenyl ether ring system and the ionizable phenolic hydroxyl moiety. Incorporation of fluorescein or acridinium labels to generate tracers had little effect on the observed binding affinities. Quantitatively, some significant differences were observed between results determined by the solution competition method and those determined by direct kinetic analysis. We attribute the differences to the different experimental designs (solution-surface versus solution phase) and the considerations affecting these interactions.

32 Bioconjugate Chem., Vol. 9, No. 1, 1998 LITERATURE CITED (1) Henry, C. (1996) FDA, reform, and the well-characterized biologic. Anal. Chem. 68, 674A-677A. (2) Adamczyk, M., Gebler, J. C., Gunasekera, A. H., Mattingly, P. G., and Pan, Y. (1997) Immunoassay reagents for thyroid testing. 2. Binding properties and energetic parameters of a T4 monoclonal antibody and its Fab fragment with a library of thyroxine analog biosensors using surface plasmon resonance. Bioconjugate Chem. 8, 133-145. (3) Adamczyk, M., Johnson, D. D., Mattingly, P. G., Clarisse, D. E., Tyner, J. D., and Perkowitz, M. M. (1994) Reagents and methods for the detection and quantitation of thyroxine in fluid samples. U.S. Patent 5,359,093; Chem. Abstr. 122, 124579. (4) Adamczyk, M., Fino, L., Fishpaugh, J. R., Johnson, D. D., and Mattingly, P. G. (1994) Immunoassay reagents for thyroid testing. 1. Synthesis of thyroxine conjugates. Bioconjugate Chem. 5, 459-462. (5) Adamczyk, M., Fishpaugh, J. R., Harrington, C., Johnson, D. D., and Vanderbilt, A. (1993) Immunoassay reagents for psychoactive drugs. II. The method for the development of antibodies specific to imipramine and desipramine. J. Immunol. Methods 163, 187-197. (6) Adamczyk, M., Fishpaugh, J. R., Harrington, C., Hartter, D., Johnson, D. D., and Vanderbilt, A. (1993) Immunoassay reagents for psychoactive drugs. I. The method for the development of antibodies specific to amitryptyline and nortriptyline. J. Immunol. Methods 162, 47-58. (7) Van Regenmortel, M. H. V., and Azimzadeh, A. (1994) Determination of antibody affinity. In Immunochemistry (C. J. Van Oss and M. H. V. Van Regenmortel, Eds.) pp 805824, Marcel Dekker, Inc., New York. (8) Karlsson, R. (1994) Real-time competitive kinetic analysis of interactions between low-molecular-weight ligands in solution and surface-immobilized receptors. Anal. Biochem. 221, 142-151. (9) Karlsson, R., Fa¨gerstam, L., Nilshans, H., and Persson, B. (1993) Analysis of active antibody concentration. Separation of affinity and concentration parameters. J. Immunol. Methods 166, 75-84. (10) Mattingly, P. G. (1991) Chemiluminescent 10-methylacridinum-9-(N-sulfonylcarboxamide) salts. Synthesis and kinetics of light emission. J. Biolumin. Chemilumin. 6, 107114. (11) Mattingly, P. G., and Bennett, L. (1995) Chemiluminescent acridinium salts. U.S. Patent 5,468,646.

Adamczyk et al. (12) Mattingly, P. G. (1992) Preparation of 5- and 6-(aminomethyl)fluorescein. Bioconjugate Chem. 3, 430-431. (13) BIAevaluation Software Handbook (1995) pp A1-A16, BIAcore, Inc., Piscataway, NJ. (14) Stenberg, E., Persson, B., Roos, H., and Urbaniczky, C. (1991) Quantitative determination of surface concentration of protein with surface plasmon resonance by using radiolabeled proteins. J. Colloid Interface Sci. 143, 513-526. (15) Karlsson, R., Mo, J. A., and Holmdahl, R. (1995) Binding of autoreactive mouse anti-type II collagen antibodies derived from the primary and the secondary immune response investigated with the biosensor technique. J. Immunol. Methods 188, 63-71. (16) Nieba, L., Krebber, A., and Plu¨ckthun, A. (1996) Competition BIAcore for measuring true affinities: large differences from values determined from binding kinetics. Anal. Biochem. 234, 155-165. (17) Minunni, M., and Mascini, M. (1993) Detection of pesticide in drinking water using real-time biospecific interaction analysis (BIA). Anal. Lett. 26, 1441-1460. (18) Karlsson, R., and Stahlberg, R. (1995) Surface plasmon resonance detection and multispot sensing for direct monitoring of interactions involving low-molecular-weight analytes and for determination of low affinities. Anal. Biochem. 228, 274-280. (19) Sternesjo¨, A., Mellgren, C., and Bjorck, L. (1995) Determination of sulfamethazine residues in milk by a surface plasmon resonance-based biosensor assay. Anal. Biochem. 226, 175-181. (20) Zeder-Lutz, G., Van Regenmortel, M. H. V., Wenger, R., and Altschuh, D. (1994) Interaction of cyclosporin A and two cyclosporin analogs with cyclophilin: relationship between structure and binding. J. Chromatog. B 662, 301-306. (21) Morelock, M. M., Ingraham, R. H., Betageri, R., and Jakes, S. (1995) Determination of receptor-ligand kinetic and equilibrium binding constants using surface plasmon resonance: application to the lck SH2 domain and phosphotyrosyl peptides. J. Med. Chem. 38, 1309-1318. (22) Payne, G., Shoelson, S. E., Gish, G. D., Pawson, T., and Walsh, C. T. (1993) Kinetics of p56lck and p60src Src homology 2 domain binding to tyrosine-phosphorylated peptides determined by a competition assay or surface plasmon resonance. Proc. Natl. Acad. Sci. U.S.A. 90, 4902-4906.

BC9701353