Anal. Chem. 1994,66,1683-1689
Investigation of Specific Binding of Antifluorescyl Antibody and Fab to Fluorescein Lipids in Langmuir-Blodgett Deposited Films Using Quartz Crystal Microbalance Methodology Hlroshl Ebato,t Christine A. Gentry,* James N. Herren,* Wolfgang Muller,§ Yoshlo Okahata,t Helmut Rlngsdorf,§ and Peter A. Suc1'~ll Department of Polymer Chemistry, Tokyo Institute of Technology 2- 12- 1 Ookayama, Meguro-ku Tokyo 152, Japan, Department of Bioengineering, University of Utah, Salt Lake City, Utah 84 1 12, Institut fur Organische Chemie der Universitat Mainz, J. J. Becherweg 18-20, 0-6500 Mainz 1, Germany, and Center for Biofilm Engineering, Montana State University, Bozeman, Montana 597 17
Antifluorescyl IgC antibody and Fab binding to two fluoresceinconjugated lipids was measured using the quartz crystal microbalancemethodology. By use of the Langmuir-Blodgett technique, the fluorescein lipids, which were diluted to 5%in a L-cy-dipalmitoyl phosphatidylethanolamine(DPPE) matrix, were deposited directly onto one gold electrode of the quartz crystal. Binding to films containing the fluorescein hapten was significantly enhanced compared to films of the pure DPPE matrix lipid, indicatingthat binding occurred primarily through a specific interaction. Association constants were 40-300 times less than for binding to haptens free in solution. Binding of IgC to the lipid in which the hydrocarbon chains and the fluorescein hapten were linked via a hydrophilic spacer was -7 times as great as to the lipid containing no spacer. IgC binding to the lipid containing the spacer was increased 1.54.4 times compared to Fab binding for the same lipid. Equilibrium binding curves and kinetic measurements are analyzed quantitatively and compared. Many naturally occurring antibody/antigen interactions involve recognition and binding to membrane-associated antigens or receptors. This generalization is valid for both the immunogenic and histocompatibility responses and has motivated various investigations at the air/water interface,' with vesicle~,~-s and on supported planar membra ne^.^^ It can be presumed that the presentation of the hapten to the antibody binding sitecan significantly alter the binding affinity. For example, the attachment of the hapten to a membrane + Tokyo Institute of Technology. t University of Utah.
1 Institut fur Organische Chemie der Universitit Mainz. 1 Montana State-University.
(1) Ahlers,M.; Muller, W.;Reichert,A.;Ringsdorf,H.;Venzmer, J. AngewChem. 1990, 29, 1269-1285. (2) Kalb, E.; Engel, J.; Tamm, L. K. Biochemistry 1990, 29, 1607-1613. (3) Petrossian, A.; Owicki, J. C. Biochim. Biophys. Acta 1984, 776, 217-227. (4) Balakrishnan, K.; Mehdi, S. Q.; McConnell, H. M. J. Biol. Chem. 1982,257, 6434-6439. ( 5 ) Tamm, L. K.; Bartoldus, I. Biochemistry 1988, 27, 7453-7458. (6) Timbs, M. M.; Poglitsch, C. L.; Pisarchick, M. L.; Sumner, M. T.; Thompson, N. L. Biochim. Biophys. Acta 1991, 1064, 219-228. (7) Poglitsch, C. L.; Sumner, M. T.; Thompson, N. L. Biochemistry 1991, 30, 6662-667 1. (8) McConncll, H. M.; Watts, T. H.; Weis, R. M.; Brian, A. A. Biochim. Biophys. Acta 1986, 864, 95-106. (9) Pisarchick, M. L.; Thompson, N. L. Biophys. J. 1990, 58, 1235-1249.
0003-2700/94/0366-1683$04.50/0
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1994 American Chemical Soclety
can hinder mobility, introduce steric constraints, or limit accessibility to various extents, depending on the quality of the molecular linkage between the,membrane anchor and the hapten.1° Characteristics of this "spacer" which may be significant are its length, flexibility, solvent compatibility, and affinity for the supporting membrane.3,4J1J2 Interactive factors in this respect are the extent of mobility, and the density,& of the anchors in the membrane. Studies involving binding to surface-bound haptens provide an opportunity to examine possible cooperative interactions which are either not apparent or nonexistent when both species are in solution. For example, on surfaces in which the haptens have sufficient density and mobility, binding of a complete antibody molecule possessing multiple binding sites is expected to be enhanced compared to a fragment (Fab) possessing only a single active site. Solution binding properties of a group of antifluorescyl monoclonal antibodies which comprise a particular idiotypic family have been studied i n t e r ~ s i v e l y ~and ~ - thus ~ ~ provide an ideal model system for investigating the effects of presentation of the hapten (fluorescein) on antibody binding. Binding of 4-4-20 monoclonal IgG and Fab to fluorescein-conjugated phospholipids incorporated into vesicles3 and to a series of synthetic fluorescein-derivatized lipids16 incorporated into monolayers at the air/water interface, micelles and giant vesicles, has been investigated.' Here we extend this type of study to include investigation of antibody binding to fluoresceinderivatized lipids incorporated into supported lipid (L-W dipalmitoyl phosphatidylethanolamine, DPPE) multilayers. (10) Ringsdorf, H.; Schlarb, B.; Venzmer, J. Angew. Chem. 1988, 100, 117-162. (1 1) Ahlers, M.; Grainger, D. W.; Herron, J. N.; Lim, K.; Ringsdorf, H.; Salesse, C. Biophys. J. 1992, 63, 823-838. (12) Cooper, A. D.; Balakrishnan, K.; McConnell, H. M. J. Biol. Chem. 1981,256, 9379-9381. (13) Herron, J. N.; He, X.-M.; Mason, M. L.; Voss, E. W., Jr.; Edmundson, A. B. Proteins 1989, 5 , 271-280. (14) Gibson, A. L.; Herron, J. N.; He, X.-M.; Patrick, V. A.; Mason, M. L.; Lin, J.-N.; Kranz, D. M.; Voss, E. W., Jr.; Edmundson, A. E. Proteins 1988, 3, 155-160. (15) Herron, J. N. Equilibrium and kinetic methodology for the measurement of binding properties in monoclonal and polyclonal populations of antifluorescylIgG antibodies. In Fluorescein Hapten: An Immunological Probe; Voss, E. W., Jr., Ed.; CRC Press: Boca Raton, FL 1984; p 49. (1 6) Ahlers, M. Funktionelle Amphiphile zur Simulation Biomembranprozessen: Stabilisierung, spezifischeErkennung und Proteinkristallisation. Dissertation, Mainz, 1990.
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used as a model system in these studies. It exhbits an affinity of 1.7 X 1Olo M-I at 2 OC2' which drops to 5 X lo9 M-I at 25 0C.28The hybridoma cell line which secretes this antibody was obtained from Prof. Edward W. Voss, Jr. (University of Illinois at Urbana-Champaign) and stored in liquid nitrogen. Hybridoma cells were grown in" tissue culture (Delbecco's minimal essential medium with 15% fetal calf serum) for 1 week and then injected ip (106 cells/mouse) into BALB/c mice, which had been previously injected with pristane (2,6,10,14-tetramethylpentadecane), Ascites fluid was collected 14-21 days after inoculation, and the IgG fraction was prepared by precipitation in 50%saturated ammonium sulfate followed by anion exchange chromatography (DEAE-cellulose, Pierce Chemical). The IgG fraction was applied to a chromatofocusing column (PBE 9 1, Pharmacia/LKB) and Mab 4-4-20 was eluted at pH -7.5 using Polybuffer 96 (Pharmacia). Antibodies were hydrolyzed with papain to produce Fab fragments. Mab 4-4-20 (10-15 mg/mL) was dialyzed into a digestion buffer which contairied 50 mM Tris-HC1 (pH 7.5),0.015 M NaCl, 2 mM ethylenediaminetetraacetic acid (EDTA) and 1 mM dithioerythritol (DTE). Papain was added to the antibody at a ratio 1:lOO (w/w) and digested for 0.5 h at 37 OC. The reaction was terminated by the addition of iodoacetamide (to a final concentration of 2 mM). Reducing agent and iodoacetamide were removed by dialysis. Papain digests were applied to a chromatofocusing column (PBE 94, These films were deposited via the Langmuir-Blodgett Pharmacia), and Fab fragments wereeluted at pH -7.3 using technique directly to a gold surface. Binding of antifluorescyl Polybuffer 96 (Pharmacia). IgG and Fab to two fluorescein-derivatized lipids, which were Synthesis of Fluorescein Lipids. The strwtures of 1 and synthesized especially for this type of study, was investigated 2 are presented in Chart 1. The synthesis was as follows. (Chart 1). In one of the lipids (1) the fluorescein hapten is Lipid 1: 115 mg (0.3 mmol) of fluorescein isothiocyanate directly bound to the hydrocarbon membrane anchor. For (FITC) was added to an ice-cooled suspension of 160 mg (0.3 lipid 2 this connection is made via an intervening hydrophilic mmol) of N,N-dioctadecylamine (DODA) in 5 mL of CHC13/ spacer. The tenacity of binding of antifluorecyl IgG to 2 was DMSO (9: 1) and reacted for 3 h. Completion of the reaction considerably greater than that to 1, implying that binding was checked by TLC (CHCl3/MeOH 12:l). The solvent sites were relatively more accessible. This prompted experiwas evaporated and the product was isolated by flash ments to investigate comparison of IgG with Fab binding to chromatography to yield 244 mg (88%) of 1. supported films containing 2. Equilibrium binding curves as Lipid 2: N,N-dioctadecyldiglycolic acid monoamide (DOwell as kinetic data were obtained. DA-GSA) was prepared according to a published procedure.16 The measurement of protein binding to the surface was A 4-g sample of DODA-GSA (6.3 mmol) was reacted with performed using the quartz crystal microbalance meth1.1 g of carbonyldiimidazole (6.8 mmol) in dry T H F in the ~ d o l o g y . ' ~ This - ~ ~ technique has been used previously to presence of sodium for 2 h under reflux to yield the monitor antibody/antigen interactions on The corresponding imidazole active ester. The resulting solution measurement is sensitive e s ~ e n t i a l l y I ~ Jto ~ -adsorbed ~' mass was then added dropwise to a stirred solution of 8 g of 1,8(nanograms). Thedata can be taken in real time, thus making diamino-3,6-dioxaoctane (54 mmol) in 50 mL of dry THF. analysis of the kinetics of binding possible. Thereaction was followed by TLC (CHC13/MeOH 5:l). After completion (20 min) the solvent was evaporated and the residue MATERIALS AND METHODS taken and purified with chromatography to yield 2.8 g of Antibodies. A murine monoclonal IgG2, ( K ) antibody (Mab 1-(N,N-dioctadecy1amido)carbox-13-amin0-4-carbox-5-aza4-4-20) which binds the fluorescent hapten, fluorescein, was 2,8,11-trioxatridecane (DODA-EO2-NH2). FITC (41 mg, (17)Sauerbrey, G. 2.Phys. 1959, 15S3206-222. 0.1 1 mmol) was added to an ice-cooled suspension of 6 1 mg (18)Walton,P.W.;Butler,M.E.;OFlaherty,M.R.Biochem.Soc.Trans.Biosens. (0.08 mmol) of DODA-EOz-NHt in 7 mL of CHC13/DMSO 1991, 19, 44-41. (19)Kanazawa, K. K.; Gordon, J. G., I1 Anal. Chem. 1985, 57, 1770-1771. (9: 1). The mixture was stirred until the reaction was completed (20)Kanazawa, K. K.; Gordon, J . G., I1 Anal. Chim. Acta 1985, 175, 99-105. (8 h). The solvent was evaporated, and the product was isolated (21)Rajakovi'c, L. V.; Cavi'c-Vlasak, B. A,; Ghaemmaghami, V.;Kallury, K. M. R.; Kipling, A. L.; Thompson, M. Anal. Chem. 1991, 63, 615-621. by chromatography to yield 70.6 mg (70%) of 2. Chart 1. Fluorescein Llpld Structures
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(22)Okahata, Y.;Ebato, H. Anal. Chem. 1991, 63, 203-207. (23)Okahata, Y.;Ebato, H. Anal. Chem. 1989, 61, 2185-2188. (24)Ebersole, R. C.; Ward, M. D. J. Am. Chem. Soc. 1988, 110, 8623-8628. (25)Muramatsu, H.; Dicks, J. M.; Tamiya, E.; Karube, I. Anal. Chem. 1987, 59, 2760-2763. (26)Davis, K. A.;Leary, T. R. Anal. Chem. 1989, 61, 1227-1230.
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(27)Kranz, D. M.; Herron, J. N.; Voss, E. W., Jr. J. B i d . Chem. 1982, 257, 6987-6995. (28)Herron, J. N.,Department of Bioengineering, University of Utah, unpublished results.
The purity of the final compounds (1 and 2) was verified by TLC. Elemental analysis was performed by Microanalysis Laboratories, Universitat, Mainz, and the structures were confirmed by 'H-NMR spectroscopy (400 MHz). Characterization of 1. Anal. Calcd for C57HssNzOsS (910.74):C,75.10;H,9.52;N,3.08;0,8.78;S,3.52. Found: C, 74.63; H, 9.19; N, 3.33; 0, 8.88; S , 3.97. 'H NMR (400 MHz, CDC13/MeOD) 6 (ppm) 0.88 (t, CH?CH3,6H), 1.151.45 (m, alkyl-CH2, 60H), 1.68 (m, (CH2CH2)2NCS, 4H), 3.61 (m, (CH2)2NCS, 4H), 6.21 (dd, ar-H, 2H), 6.36 (d, ar-H, 2H), 6.40 (d, ar-H, 2H), 6.80 (d, ar-H, lH), 7.45 (d, ar-H, lH), 7.58 (s, ar-H, 1H). Characterization of 2. Anal. Calcd for C67H104N4010S (1 156.89): C, 69.50; H, 9.06; N, 4.84; 0, 13.83; S,2.77. Found: C, 68.25; H, 8.88; N, 4.65; 0, 14.56; S , 3.66. IH NMR (400 MHz, CDC13/MeOD) 6 (ppm) 0.81 (t,CH2CH3,6H), 1.05-1.35 (m,alkyl-CH2,60H), 1.40-1.60 (m (CH2CH2)2NCO, 4H), 3.02 (t, (CH2)2NCO, 2H), 3.22 (t, (CH&NCO, 2H), 3.35-3.85 (m, CONHCH2 CH20CH2 CHzNHCS, 8 + 2 + 2H), 3.97 (s, COCH20, 2H), 4.17 (s, COCH20, 2H), 6.45 (dd, ar-H, 2H), 6.58 (d, ar-H, 2H), 6.62 (d, ar-H, 2H), 6.80 (d, ar-H, lH), 7.03 (d, ar-H, lH), 7.47 (m, NH, 1H) 7.64 (m, NH, lH), 7.90 (m, ar-H, 2H). G B Deposition. Langmuir deposition of 1 and 2 on the gold electrode was performed as follows: A 5% molar mixture (5% fluorescein lipid, 95% DPPE) was spread on the subphase (Millipore water, 20 "C, Lauda film balance) from a M chloroform solution and compressed to 40 mN/m. Gold surfaces were precleaned with methanol. Deposition was directly onto the gold at 10 mm/min vertical dipping speed and was performed in two dips. Deposition occurred on the down and up stroke of the first dip (Y-type) and only on the up stroke of the second dip (Z-type). Between subsequent dips the film was allowed to dry for 1 h. The area changes on the first up and out strokes, as well as the last up stroke, were always positive, whereas the area change for the second down stroke was zero. As described below, one gold electrode was insulated from the solution by a protective cover which entered the subphase on each dip. Variable deposition onto this cover, which consisted of variable amounts of exposed silicone and glass, prevented precise correlation of the area changes recorded on the film balance with the depositionwhich occurred exclusively onto the gold surface. Accurate estimates of deposition which occurred on the exposed gold electrode could be obtained from the frequency shifts (measured in air). Preliminary experiments using 5% mixtures of both biotin and fluorescein lipids in a DPPE matrix, in which the mass (in air) was measured after each dip, indicated that two monolayers were transferred after the first dip and one monolayer was transferred after the second dip. In addition, for 14 films deposited as described above using 5% fluorescein lipid 1in a DPPE matrix, the measured mass change after the two dips was 117 f 38 ng and the calculated mass change for the proposed three-layer structure was 122 ng. This indicates an average transfer ratio close to 1 for deposition of the three monolayers. Deposited monolayers were stable for a period of several hours when exposed to phosphate buffer as indicated by the stable frequency response which varied by only f4Hz. Quartz Crystal Microbalance Measurements. The quartz crystal microbalance apparatus used in these experiments is
+
+
described e l s e ~ h e r e . Briefly, ~ ~ , ~ ~it consists of a 9-MHz, ATcut crystal, (8-mm diameter), sandwiched between twovapordeposited gold electrodes (4.5" diameter) (Kyushu Deutsu Co. Ltd., Tokyo, Japan). In order to eliminate frequency drift caused by charge flow between the electrodes, which has been observed to result in instabilities,25 one electrode was completely insulated from the test liquid. This was accomplished by mounting a glass coverslip over one side of the quartz crystal which was held in place by two silicon O-rings. The O-rings were large enough (8 mm) that they made contact only with the exposed quartz peripheral to the centrally located electrode and not with the electrode itself. Thps, the covered electrode was in contact only with air. This covering assembly was held in place using silicone glue. The electrical contacts were insulated from the test solution using silicon tubing and silicone glue. This general approach (i.e., exposing only one electrode to the test solution) is typically employed for measurements in liquids using QCM devices.26329-31 Using an equivalent circuit model, an equation which predicts the change in resonance frequency (Af>of an AT-cut crystal under both mass and liquid loading has been derived.29
where f is the fundamental resonance' frequency, N is the overtone number ( N = 1 for the fundamental frequency), cq is the quartz elastic constant (2.947 X 10" dyn/cm2), pq is the density of quartz (2.651 g/cm3) ps is the density of the adsorbed film, p is the liquid mass density, and q is the liquid shear velocity. This equation predicts that liquid and mass loading will have an additive effect on the resonance frequency and that the effect of the mass loading will follow the Sauerbrey e q ~ a t i 0 n . lThe ~ second term in parenthesis accounts for liquid loading, assuming a no-slipconditionat the i n t e r f a ~ e . l ~ * ~ ~ T h e r e is some controversy about the validity of this assumption, and it has been suggested that a more complex model which incorporates the effects of interfacial free energy and surface roughness is more a p p r ~ p r i a t e .Experimentally ~~ it has been found that the change in resonance frequency is linear with respect to quantity of protein adsorbed on the surface.26 For the electrode used in these experiments eq 1 predicts a frequency change of 1 Hz for 1 ng of adsorbed lipid or protein. For this calculation the area of the electrode exclusively, and not the larger area of the quartz crystal, is used. The field is localized between the electrodes, and thus mass loading on the portion of the exposed quartz surrounding the electrodes is generally considered to have a negligable Possible edge effects have been studied in detai1.34 The actual change in frequency will probably be less than that predicted by eq 1 since there will a moderation in the frequency change attributed to shear deformation of the adsorbed protein.33 The calculation of binding constants and kinetic rate constants depends only on the weak assumption that the
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(29) Martin, S.J.; Granstaff, V. E.; Frye, G. C. Anal. Chem. 1991,63.2272-2281. (30) Thompson, M.; Arthur, C. L.; Dhaliwal, G. K. Anal. Chem. 1986.58, 12061209. (31) Kurosawa, S.; Tawara, E.; Kamo, N.; Kobatake, Y. Anal. Chim. Acta 1990, 230, 41-49. (32) Duncan-Hewitt, W. C.; Thompon, M. Anal. Chem. 1992, 64, 94-105. (33) Deakin, M. R.; Buttry, D. A. Anal. Chem. 1989,61, 1147A-1154A. (34) Hiller, A. C.; Ward, M. D. Anal. Chem. 1992, 64. 2539-2554.
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frequency change is proportional to the adsorbed mass of protein. This assumption is supported by eq 1 and by experimental results.26 The estimation of valence of binding relies on the less certain assumption that the protein experiences negligable shear deformation. Significant shear deformation will result in an underestimation of the valence. Absolute resonance frequency varies considerably for different crystals. Therefore, only differences between resonance frequencies are reported, always in hertz. With respect to the change in frequency produced by exposure to a particular solution concentration of protein, the response of different crystals was quite consistent, with a maximum standard deviation of 20 Hz for three to four independent measurements (lipid films prepared using an identical protocol on different crystals exposed to the same bulk protein concentration and allowed to come to equilibrium). Note that this relatively small variation in frequency differences for independent measurements also implies that the reproducibility of the L-B deposition, inoculation of protein into the sample chamber, and other measurement procedures was very good. For measurement of the binding curves the electrode was placed in a 2.5-mL solution of stirred buffer (50 mM Na2HP04, pH 7.4) and allowed to equilibrate until the frequency signal stabilized. Protein was added to the stirred solution from a more concentrated protein solution (1.77 X lo4 or 1.37 X lo-' M), by using an Eppendorf pipet. All measurements were performed in a temperature-controlled atmosphere (22 f 0.5"C). The addition of protein to the buffer solution produced in all cases a decrease in the frequency of the immersed crystal. For kinetic measurements, the forward reactions (Figures 4a and 5a) were initiated by exposing the surface to a concentrated protein solution: 2.5 X 10-* M for the IgG and 5 X lo-* M for the Fab. The reverse reactions (Figures 4b and 5b) were initiated by rapid transfer (
