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Langmuir 1996, 12, 4292-4298
Site-Specific Immobilization of Monoclonal Antibodies Using Spacer-Mediated Antibody Attachment Shao-Chie (Patrick) Huang,† Karin D. Caldwell,† Jinn-Nan Lin,‡,§ Hsu-Kun Wang,‡ and James N. Herron*,‡ Department of Chemical and Fuels Engineering and Department of Pharmaceutics and Pharmaceutical Chemistry, Center for Biopolymers at Interfaces, University of Utah, Salt Lake City, Utah 84112 Received December 11, 1995. In Final Form: June 10, 1996X Site-directed and random coupling of antibodies (Abs) was performed using aminated silica surfaces as substrates. The site-directed coupling linked the Ab, a monoclonal anti-fluorescein IgG1 (9-40), to the surface via 3400 Dalton (Da) poly(ethylene oxide) (PEO) spacers. The hydrazide end groups of these spacers were attached to aldehyde groups in the hinge region of the oxidized Ab to yield surfaces with an Ab concentration of 2.1 pmol/cm2. The random coupling, in turn, linked the Ab via its available primary amines directly to the glutaraldehyde-activated surface with a yield of 3.8 pmol/cm2. Despite a nearly twofold difference in Ab concentration, the two surfaces bound similar amounts of the FL-BSA antigen (0.56 vs 0.55 pmol/cm2), while their nonspecific uptake of protein was 0.11 vs. 0.21 pmol/cm2, respectively, reflecting the protein repellent quality of PEO-coated surfaces. Fab′ fragments of the 9-40 Ab were also linked to the same tethers. Here, the succinimidyl 4-(N-maleimidomethyl)cyclohexane-1-carboxylate (SMCC) difunctional coupling reagent was attached to the PEO-hydrazide via its succinimide end and to the carboxy-terminal thiol of the Fab′ via its maleimide end. The concentration of reactive groups was varied by mixing difunctionalized PEO (PEO-(HZ)2) with monofunctionalized polymer (CH3O-PEO-HZ) prior to surface attachment. At 100% PEO-(HZ)2 the FL-BSA (Ag) binding was 0.77 pmol/cm2 while the nonspecific binding was 0.058 pmol/cm2. Progressive dilution of the reactive PEO chains to 25% led to the remarkable binding of 0.87 pmol/cm2 of Ag with 0.037 pmol/cm2 of nonspecific binding. Competitive release from these various surfaces showed more favorable kinetics for the Fab′ surface with 25% active tethers.
Introduction The immobilization of biomolecules to solid phase materials is a frequently used procedure to confer a specific affinity or enzymatic activity on the surface. In performing these immobilization reactions, the aim is generally to attach the often fragile ligands in such a way as to leave them structurally intact with their active sites easily accessible to specifically binding reactants present in the surrounding fluid phase. This strategy is the basis for production of a wide array of materials, ranging from biospecific adsorbents for protein purification to analytical devices used in biosensing and diagnostics. In the particular case of immunoassays, antibodies (Abs) are immobilized on insoluble supports where they are made to contact and bind their specific antigen (Ag) in proportion to its concentration in the fluid phase. If the quantification of bound antigen is based on measurements of fluorescence, using strategies collectively referred to as fluoroimmunoassays (FIAs), detection sensitivities of up to femtomolar levels can be achieved.1-4 Although associated with some disadvantages, most conjugation procedures couple Abs to solid supports via the -amino groups of one or more of their lysine residues. Using this immobilization strategy, the potential for multiple attachments, with resulting conformational * To whom correspondence should be addressed. Telephone: (801) 581-7303. Fax: (801) 585-5151. E-mail:
[email protected]. utah.edu. † Department of Chemical and Fuels Engineering. ‡ Department of Pharmaceutics and Pharmaceutical Chemistry. § Present address: Diagnostics Products Corp., Los Angeles, CA 10045. X Abstract published in Advance ACS Abstracts, August 1, 1996. (1) North, J. R. Trends Biotechnol. 1985, 3, 180. (2) Meadows, D.; Schultz, J. C. Talanta 1988, 35, 145. (3) Komives, C.; Schultz, J. S. Med. Des. Mater. 1991, April, 24. (4) Plowman, T. E.; Reichert, W. M.; Peters, C. R.; Wang, H. K.; Christensen, D. A.; Herron, J. N. Biosens. Bioelectron. 1996, 11, 149.
S0743-7463(95)01532-0 CCC: $12.00
distortions, is high. More problematical yet is the likelihood of an attachment at the readily accessible and reactive amino terminals, which are the very sites of Ag binding. In either case, a reduction in the Ab’s specific ability to bind Ag is a likely outcome of a random coupling to available amines. To avoid this dilemma, researchers have been attempting to use an attachment chemistry which assures coupling to a site far removed from the Ag binding sites.5 One approach to achieving this goal is to perform the coupling via the carbohydrate moieties located in the Ab molecule’s Fc region, close to the “hinge” between this C-terminal domain and the two N-terminal Fab domains. This requires an oxidation of the Ab to produce aldehyde groups which can then be used to link the molecule to primary amines or hydrazides on the surface.6-9 Another possibility for directed coupling is offered by the mildly reduced Fab′ fragment, whose free thiol at the C-terminal can be made to bind with high specificity to reactive groups like a maleimide or a pyridyl disulfide-activated thiol.10 In the present work both approaches will be carried out, and their relative merits will be compared with that of the customary random attachment via the protein’s surface amine groups. As the detection limits for bound Ag are pushed to ever lower levels, there will be an increasingly important error introduced by nonspecific adsorption. This is particularly serious in sandwich immunoassays, where the binding of Ag is measured through the adsorption of a second Ab (5) O’Shannessy, D. J. J. Chromatogr. 1990, 510, 13. (6) O’Shannessy, D. J.; Hoffman, W. L. Biotechnol. Appl. Biochem. 1987, 9, 488. (7) Little, M. C.; Ciebert, C. J.; Matson, R. S. BioChromatography 1988, 3, 156. (8) Cress, M. C.; Ngo, T. T. ABL 1989, February, 16. (9) Zara, J. J.; Wood, R. D.; Boon, P.; Kim, C. H.; Pomato, N.; Bredehorst, R.; Vogel, C. W. Anal. Biochem. 1991, 194, 156. (10) Carlsson, J.; Drevin, H.; Axen, R. Biochem. J. 1978, 173, 723.
© 1996 American Chemical Society
Site-Specific Immobilization of Monoclonal Antibodies
carrying the quantifiable label. Although the signal intensity of this label may be amplified to almost limitless levels, e.g. by counting for extended periods of time in cases where the label is a radioisotope or by allowing extended times for the development of an optically detectable product in cases where the label is an enzyme, it is clear that any nonspecific adsorption of label will limit the levels of Ag that can be accurately measured. In recent years, much evidence has accumulated regarding the protein-repelling nature of surface-attached poly(ethylene oxide) (PEO).11-13 In studying the plasma protein and platelet adhesion to PEO-grafted surfaces, Nagaoka et al.14 were able to show a clear molecular weight dependence of the repulsion efficiency of the polymer, with longer PEO chains being more effective at least up to a chain length of 100 monomer units. Against this background an immobilization strategy which would link the Ab to the solid phase by means of a PEO tether appeared to have considerable merit. By this arrangement, the “immobilized” Ab would be relatively free to move and engage the Ag in a three-dimensional collision pattern. If properly carried out, the attachment of PEO to the surface would effectively shield it from potential nonspecific adsorbates. While only a portion of the PEO would serve as Ab tethers, the surface as a whole would show high specific and low nonspecific binding characteristics. Frequently, continuous measurement of the Ag concentration is desired. One such approach, which eliminates the need for binding of a second Ab and the subsequent development of its associated activity, is based on the competitive displacement of labeled ligand from the surface-attached Ab. In this case, the surface would be saturated with a fluorescently labeled Ag analog, and an exposure to unlabeled Ag in the unknown sample would result in a displacement reaction, in turn, causing a reduction in the emission intensity in proportion to its concentration. The success of this approach hinges on the rapid elimination of a released ligand from the surface before it reattaches itself to a neighboring Ab. Given that the local concentration of Ab is high while the unstirred layer immediately above the surface is depleted with respect to Ag, there is significant probability for reattachment and a resulting loss of the intended signal reduction. The design of surfaces for displacement-based Ag detection, therefore, requires the careful balance of a desire for low surface concentrations of Ab to minimize reattachment and a desire for high concentrations to provide a measurable signal. In this work we examine the relative merits of sitedirected Ab attachment via PEO tethers to aminated silica surfaces, as compared to direct, random attachment. The comparison is based both on the surface’s ability to specifically bind Ag and on the level of nonspecific protein binding recorded on the surface. In addition, the small Fab′ fragment is attached via PEO tethers to the same silica surfaces whose specific abilities to bind and competitively release Ag are being determined as a function of the Fab′ surface concentration. Materials and Methods Silica Chips and Particles. Fused silica chips from ESCO (CO grade, dimensions 1.0 × 0.9 × 0.1 cm3), whose edges had been smoothened with a grit polishing wheel from Buehler, Ltd, (11) Lee, J. H.; Kopecek, J.; Andrade, J. D. J. Biomed. Mater. Res. 1989, 23, 351. (12) Gombotz, W. R.; Wang, G.; Horbett, T. A.; Hoffman, A. S. J. Biomed. Mater. Res. 1991, 25, 1547. (13) Kishida, A.; Mishima, K.; Corretge, E.; Konishi, H.; Ikada, Y. Biomaterials 1992, 13, 113. (14) Nagaoka, S.; Mori, Y.; Tanzawa, H.; Kikuchi, Y.; Inagaki, F.; Yokota, Y.; Noishiki, Y. ASAIO 1987, 10, 76.
Langmuir, Vol. 12, No. 17, 1996 4293 were cleaned in hot chromic acid and deionized (DI) water of Milli-Q quality prior to derivatization. Nonporous silica particles with an average diameter of 1.25 µm were the generous gift of Professor Klaus Unger of the Johannes Gutenberg Universita¨t, Mainz, Germany. These particles were washed with 10 mL of ethanol three times and then dried by freeze drier before use. Production and Purification of Monoclonal Antibodies. A murine monoclonal IgG1 (κ) antibody (9-40) which binds the fluorescent hapten fluorescein was used as a model system in these studies. It exhibits an affinity of about 1 × 107 M-1 at 25 °C.15 The hybridoma cell line which secretes this antibody was obtained from Prof. Edward W. Voss, Jr. (University of Illinois at Urbana-Champaign). Hybridoma cells were grown in tissue culture (Dulbecco’s minimal essential medium with 20% fetal calf serum) for approximately 1 week and then injected I.P. (106 cells/mouse) into BALB/c mice which had been previously injected with pristane. 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 Chemicals). Protein A (Pharmacia) chromatography was used as the final purification step. The monoclonal antibody eluted in 0.1 M glycine, pH 3.02, and was g95% pure. Preparation of Antigen Binding Fragments (Fab′). Purified 9-40 was digested with pepsin to produce F(ab′)2 fragments,16 which were then reduced to Fab′ fragments using dithiothrietol (DTT).17 Specifically, 33 mg of purified Mab 9-40 and 1 mg of pepsin (Sigma) were dissolved in 0.1 M sodium acetate buffer (pH 4.2) and the digestion was carried out at 37 °C for 16 h. The digestion was terminated by adjusting the pH of the reaction mixture to 8.0 with 2 M tris base. The F(ab′)2 fraction was separated by gel permeation chromatography (Superdex HiLoad, Pharmacia) using phosphate buffer saline (PBS), pH 7.7, as eluent. Fab′ fragments were prepared by reducing the F(ab′)2 fragments (1 mg/mL) with 1.75 mM DTT and 3.5 mM ethylenediaminetetraacetate (EDTA) in 0.17 M tris buffer (pH 7.4), for 45 min at room temperature. After reduction, excess DTT was removed by gel permeation chromatography using a Sephadex G-25 column (Pharmacia) equilibrated in 0.1 M sodium phosphate buffer (pH 6.0) containing 5 mM EDTA. Antigens. Two different antigen systems were employed in these studiessthe fluorescein hapten (FL) and fluorescein conjugated to bovine serum albumin (FL-BSA). The latter Ag was used to emulate the behavior of proteinaceous antigens in our solid phase assay systems. Both reagents were obtained from Molecular Probes, and the fluorescein-BSA conjugate exhibited a labeling ratio of 9:1 (FL:BSA). Other Reagents. The poly(ethylene oxides) (Aldrich) were either the monofunctional CH3-(OCH2CH2)n-OH (MW 5000) or the difunctional H-(OCH2CH2)n-OH (MW 3400). They were functionalized through activation with p-nitrophenyl chloroformate (C7H4NO4Cl, Aldrich) followed by treatment with hydrazine (N2H4, Aldrich). Amination of the silica surfaces was performed through reaction with (3-aminopropyl)triethoxy silane (NH2(CH2)3Si(OC2H5)3 (APS)) from Aldrich, while quantification of the introduced amine groups involved reaction with succinimidopropyldithiopyridine (SPDP) from Pierce; the latter was reduced by dithiothreitol (DTT, Bio-Rad). The surface-attached amine groups were activated prior to further coupling by reaction with glutaraldehyde (E. M. grade) from Polysciences.18 Isotope labeling involved the introduction of 125I (100 mCi/ mL, Amersham) through oxidation of protein tyrosines with chloramine-T (Kodak), while the oxidation of the IgG carbohydrate moiety was carried out by means of sodium metaperiodate (NaIO4) from Mallinckrodt.19 This and all other chemicals were reagent grade. Amination of Silica Surfaces. Silica samples, in the form of chips or solid spheres, were treated with hot (80 °C) dichro(15) Bates, R. M.; Ballard, D. W.; Voss, E. W. Mol. Immunol. 1985, 22, 871. (16) Grey, H. M.; Kunkel, H. G. J. Exp. Med. 1964, 120, 253. (17) Wilson, M. B.; Nakane, P. K. In Immunofluorescence and Related Staining Techniques; Knapp, W., Holubar, K., Wick, G., Eds.; Elsevier: Amsterdam, 1978; p 247. (18) Monsan, P. J. Mol. Catal. 1977/78, 3, 371. (19) Lee, S. H.; Ruckenstein, E. J. Colloid Interface Sci. 1988, 125, 365.
4294 Langmuir, Vol. 12, No. 17, 1996 mate-sulfuric acid (25 mL of Monostat Chromerge in 4.1 kg of concentrated sulfuric acid) for 30 min and were then thoroughly rinsed in Milli-Q grade deionized water. After drying for 3 h in a desiccator at 120 °C, the silica was ready for silanization. For this purpose, a solution of 5% (v/v) APS in DI water was prepared and immediately allowed to react with the chosen silica surface for 15 min at room temperature. After removal of the solution and careful rinsing in DI water, followed by absolute ethanol, the sample was dried for 1 h at 120 °C in a vacuum oven, repeatedly flushed with nitrogen. The level of surface amination was quantified for the uniform silica spheres, which provided a large and accurately quantifiable amount of surface area. In this process, ca. 75 mg of dry solid support was suspended in 0.5 mL of dry ethanol and reacted at room temperature with 0.5 mL of a solution containing 12 mg/ mL SPDP under gentle tumbling.20 The rate of acylation of the solid support was promoted by the addition of 3 mg of the catalytically active 4-methylaminopyridine. At the end of the 30 min reaction period the sample was centrifuged and the supernatant removed by Pasteur pipets. The particles were then washed consecutively with ethanol, DI water, 1 M NaCl, and 0.1 M NaHCO3. After tumbling for another 30 min in 4 mL of 0.1 M NaHCO3, the sample was suspended in 4 mL of a 50 mM DTT solution to accomplish a reductive release of the pyridine-2-thione. This reaction was again carried out at room temperature under tumbling for 15 min. After centrifugation, the supernatant was diluted 20 times with 0.1 M NaHCO3, and the absorbance was read at 343 nm against a 50 mM DTT blank. On the basis of a molar extinction coefficient for the thione of 8080 M-1 cm-1 the amount of SPDP molecules attached to surface amines was calculated. The reaction was assumed to be quantitative, so that the molar amount of released thione could be considered equal to the amine content on the beads. Derivatization of Poly(ethylene oxide). The surface attachment of PEO was envisioned to take place via the formation of a hydrazone with aldehydes on the silica surface. This necessitated the introduction of hydrazide groups at the end hydroxyls of the polymer. For this purpose, either monofunctional methoxy-PEO (M5000) or difunctional PEO (3400) (5 g) was reacted with p-nitrophenyl chloroformate (1.77 g) in 5 mL of benzene at room temperature for 24 h under tumbling. Dry ethyl ether (less than 0.01% water) was used to precipitate the PEO(o-NP)2 (or CH3O-PEO-(o-NP)) from the benzene solution. This dissolution-precipitation was repeated twice, and the sample was then submitted to drying under vacuum overnight. To determine the extent of derivatization, a given amount of polymer was weighed and dissolved in 0.1 M NaOH.21 A yellow color developed as the p-nitrophenol was set free; the absorbance was read at 402 nm, and the degree of conversion was calculated on the basis of an extinction coefficient of 18 400 M-1 cm-1. The synthesis of PEO-hydrazide (PEO-(Hz)2 or CH3O-PEO(Hz)) was accomplished by dissolving 5 g of the appropriate (oNP) derivative in 2 mL of methanol and adding this solution to 2 mL of hydrazine. The mixture was allowed to react under tumbling for 3 h at room temperature, whereupon the polymer was precipitated by the addition of 500 mL of dry ethyl ether. Dissolution in methanol followed by precipitation with ether was repeatedly carried out until all yellow color had disappeared. After the final precipitation the wet polymer powder was allowed to dry under vacuum overnight. Surface Modification with PEO-Hydrazide. APS-treated silica chips were soaked for 2 h in a 2.5% glutaraldehyde solution in 0.1 M carbonate-bicarbonate (CB) buffer at pH 9.2. Portions of 24 mg of PEO-Hz powder were dissolved in 1.2 mL of either 0.15 M phosphate-buffered saline (PBS), pH 7.4, or 11% (w/v) K2SO4 in sodium acetate buffer of pH 5.2. Following a DI water rinse, glutaraldehyde-treated chips were immersed in polymer solutions made up from the two buffers and incubated in a water bath at 60 °C for 24 h. After careful rinsing in DI water, the level of coupling was determined qualitatively by electron spectroscopy for chemical analysis (ESCA) analysis (see below) and quantitatively by the SPDP reaction with available hydrazide groups on solid silica spheres derivatized in a process paralleling that used for the chips. The quantification of reactive hydrazides (20) Ngo, T. T. J. Biochem. Biophys. Meth. 1986, 12, 349. (21) Cabrera, K. E.; Wilchek, M. Anal. Biochem. 1986, 159, 267.
Huang et al. was performed as described above in conjunction with the determination of amine groups on APS-derivatized silica beads. Prior to the coupling of Ab to these surfaces, free aldehyde groups left behind after grafting of the PEO were passivated by reaction with 0.2 M aqueous ethanolamine. In this process, each chip was immersed in 2 mL of the amine solution and left on a shaker for 30 min at room temperature. The stability of the PEO-coated silica surfaces was then examined by soaking the coated chips in DI water for varying periods of time and subsequently determining their elemental composition by ESCA. During the soaking, the samples were left immersed in DI water without stirring. At sampling, the chips were removed, washed carefully for 3 min with DI water, and dried in a desiccator. ESCA Analysis of PEO on Silica Surfaces. Following PEO derivatization, the silica surfaces were examined by ESCA to determine the relative surface composition of the elements nitrogen, oxygen, carbon, and silica.22,23 In these determinations, each elemental peak was normalized by division with its specific Scofield cross-section. Thus, the relative amount of carbon in the surface layer was considered a measure of the amount of PEO found on the surface. The carbon signal was resolved into the two carbon 1s peaks, corresponding to aliphatic and ether carbon, respectively, and a value of approximately 3 for the ratio of carbon in these two binding states indicated the origin of the surface carbon to be PEO. Oxidation of Antibody. A 3 mg aliquot of the 9-40 Ab was dissolved in 1 mL of 0.15 M sodium acetate buffer, pH 5.2, to which was added 1 mL of a 50 mM solution of NaIO4. The reaction was allowed to take place for 1 h at room temperature with shaking. Unreacted NaIO4 was then separated from the Ab by passing the reaction mixture through a desalting column (PD10, Pharmacia) that had been equilibrated with the acetate buffer prior to use. Activity of Oxidized Antibody. The fluorescein hapten is highly fluorescent when free in solution, but when bound to the 9-40 Ab its fluorescence is significantly quenched. Furthermore, the reduction in emission intensity for a given fluorescein concentration is a sensitive measure of this binding for soluble preparations of Ab. A fluorescence-quenching assay based on this phenomenon was used to examine the antigen-binding activity of the oxidized 9-40 Ab. In a typical assay, a series of 10 µL aliquots of a fluorescein solution (4.1 × 10-6 M) were added to 1 mL of Ab solution (4 × 10-7 M) placed in a stirred cuvette in a PC1 photon-counting spectrofluorometer (ISS, Champaign, IL). The sample was excited at 485 nm, and emission was measured at 515 nm. The affinity of the Ab-Ag reaction was calculated from the observed levels of quenching using Scatchard and Sips analysis.24,25 Immobilization of Antibody. Two types of immobilization chemistry were employed for the intact Ab: (1) direct attachment of Ab (0.35 mL of a 1 mg/mL solution in PBS buffer, pH 7.2) to glutaraldehyde-treated silica surfaces was carried out in CB buffer of pH 9.2 (7.5 mL added to the Ab solution) for 6 h at room temperature; (2) site-directed immobilization of oxidized Ab (1 mg/mL) to surface-linked PEO-Hz was performed in acetate buffer of pH 5.2 for 3 days at 4 °C. After either of the coupling reactions the surface was thoroughly rinsed in PBS buffer. The Fab′ fragments were linked to the surface with the aid of a PEO tether. In this case the hydrazide end group was first reacted with the bifunctional reagent SMCC for 1 hour at room temperature in a PBS buffer at pH 7.2. This produced reactive maleimide groups at the end of the PEO chains which were readily available attachment sites for the free thiols at the C-terminal ends of the Fab′ fragments. The attachment of Fab′ was carried out for 20 h in 5 mM EDTA, pH 6.0, at 4 °C. The surface concentration of Ab or Fab′ resulting from each of these coupling procedures was determined employing radiolabeled proteins in the conjugation reactions. (22) Andrade, J. D. In Surface and Interfacial Aspects of Biomedical Polymers; Andrade, J. D., Ed.; Plenum Press: New York, 1985. (23) Winters, S. Ph.D. Thesis, Department of Pharmaceutics, University of Utah, 1987. (24) Rossignol, M.; Thomas, P.; Grignon, C. Biochim. Biophys. Acta 1982, 684, 195. (25) Herron, J. N. In Fluorescein Hapten: An Immunological Probe; Voss, E. W., Jr., Ed.; CRC Press: Boca Raton, FL, 1984; p 49.
Site-Specific Immobilization of Monoclonal Antibodies Radiolabeling of Antibodies and Antigens. Antibodies, antigen-binding fragments and antigens were radiolabeled with 125I using the method of Chuang et al.26 In brief, 0.5 mL of protein solution (1 mg/mL) was mixed with 3 mL of carrier-free 125I (100 mCi/mL, Amersham) and 50 mL of chloroamine T solution (4 mg/mL) and then allowed to react at room temperature for 1 min. The reaction was quenched by adding 50 mL of sodium metabisulfite solution (4.8 mg/mL) for 2-3 min. Unreacted iodide was removed by gel permeation chromatography (Sephadex G-25, Pharmacia). The concentrations of iodinated proteins were measured using a UV-visible spectrophotometer (Beckman, 0.1% Model 35) at 278 nm. Extinction coefficients (278nm ) of 1.35 and 0.67 were used for the 9-40 Ab and BSA, respectively. Radiolabeling efficiency was determined by precipitating proteins with 20% trichloroacetic acid (TCA, Sigma) in the presence of BSA as the carrier protein. The labeling efficiency was equal to (b a)/b, where a was the number of counts in 5 mL of supernatant and b was the number of counts in 5 mL of the labeled protein solution. The efficiency was always greater than 95%. Antigen-Binding Activity of Immobilized Antibodies. The antigen-binding activity of immobilized Ab cannot be assayed using the aforementioned fluorescence quenching assay, and instead, the selected method is based on the binding of fluoresceinlabeled, radioiodinated BSA. The nonspecific uptake of protein was similarly determined from the binding of radioiodinated BSA (without fluorescein). Because the immobilized antibody exhibits stringent specificity for the fluorescein hapten, radioiodinated BSA without fluorescein can only interact nonspecifically with it.
Langmuir, Vol. 12, No. 17, 1996 4295 Table 1. Immobilization Scheme for Site-Directed Conjugation of Antibodies to Silica Surfaces using Poly(ethylene oxide) (PEO) Spacersa
Results and Discussion The derivatization of a silica surface is conveniently begun by a silanization procedure which introduces amine groups onto the surface.27,28 This is commonly done by a reaction of carefully cleaned silica surfaces with 3-aminopropyltriethoxysilane in an aqueous environment. Because of their limited surface area, it is difficult to assess the degree of derivatization of the small silica chips selected as substrates in this work. Instead, the surface concentration of amines is conveniently determined through a parallel experiment in which nonporous silica spheres of uniform size and surface area have been similarly aminated. Assuming identical levels of derivatization for the two substrates, one can determine the surface concentration after quantification of the larger molar amounts of amine groups available on the high surface area particulates. In the process selected here, surface concentrations of 2 × 10-10 mol/cm2 were routinely observed (see Table 1). Having introduced these anchor groups on the surface, attention turned to the effective surface derivatization with PEO. This polymer, in difunctionalized form, would serve as a spacer for the subsequent attachment of Ab or Fab′ molecules. While allowing these proteins to bind their specific ligand, the polymer would reduce nonspecific binding in its immediate vicinity. In monofunctional form, the PEO would attach to the surface as a coating agent, suppressing nonspecific adsorption of protein at sites uncovered by the actual ligand-binding tethers. Surface Derivatization with PEO. After a glutaraldehyde treatment of the aminated silica surface, the now aldehyde-functionalized surface was available for reaction with PEO hydrazide. The amine terminal of the hydrazide group has a pK of 2.629 and is therefore (26) Chuang, H. Y. K.; King, W. F.; Mason, R. G. J. Lab. Clin. Med. 1978, 92, 483. (27) Royer, G. P.; Liberatore, F. A. In Silylated Surfaces; Leyden, D. E., Collins, W. T., Eds.; Gordon and Breach Science Publishers: London, 1980; p 189. (28) Plueddemann, E. P. Silane Coupling Agents; Plenum Press: New York, 1991. (29) Smith, P. A. S. Derivatives of Hydrazine and Other Hydronitrogens Having N-N Bonds; Benjamin/Cummings: Reading, MA, 1983.
a Asterisks (*) denote 125I-labeled antibodies (Ab) or antigens (Ag). NA denotes “not applicable”. Abs were immobilized using either (a) site-directed or (b) random conjugation methods. BSA, bovine serum albumin; FL-BSA, fluorescein conjugated to bovine serum albumin.
deprotonated even at relatively acidic pH. In this regard, the group differs significantly from the primary aliphatic amine, whose pK lies somewhere around 9-10, depending on its environment. Since either group in its deprotonated form reacts with aldehydes to form, respectively, a hydrazone or a Schiff base, the choice of pH governs the reaction path in cases where the two groups are present simultaneously. This strategy was later relied on in conjunction with the site-directed attachment of oxidized Ab. In a detailed study of the PEO derivatization of aminated, glutaraldehyde-treated silica,30 comparisons were made between attachments of the PEO-amine at pH 9.2 and the PEO-hydrazide at pH 5.2. Since, under otherwise identical reaction conditions, the two coupling chemistries proved to yield comparable results, the choice was made to base the present study exclusively on the surface linking of PEO-hydrazide. The attachment was attempted both in ordinary PBS of pH 7.4 and in an acetate buffer, pH 5.2, which was made 11% with respect to K2SO4. The high sulfate concentration was thought to salt out the polymer onto the surface, thereby increasing its local concentration and promoting coupling.31 From the carbon contents listed in Table 2, it is clear that the high salt concentration gives the best coupling yields. Using this same medium, the coupling was carried out in a variety of PEO concentrations. The limited data set included in the table shows no gain in PEO attachment by coupling at concentrations above 20 mg/mL. In order to test the stability of the hydrazone bond with the surface, the derivatized chips (30) Huang, S. C. MS Thesis, Department of Chemical and Fuels Engineering, University of Utah, 1992. (31) Kiss, E.; Golander, C. G.; Eriksson, J. C. Prog. Colloid Polym. Sci. 1987, 74, 113.
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Huang et al.
Table 2. Electron Spectroscopy Analysis (ESCA) of Silica Surfaces Derivatized with PEO-(Hz)2a
1 mg/mL 20 mg/mL 100 mg/mL 20 mg/mL (30 days later)
clean surface APS surface Glu surface 11% K2SO4d 0% K2SO4c 11% K2SO4d 11% K2SO4d 11% K2SO4d
{
C%
C1%
C2%
C2/C1
6 16 30 38 35 42 44 37
NAb NA NA 14.5 16.1 10.5 11.8 10.4
NA NA NA 23.5 18.9 31.5 32.2 26.6
NA NA NA 1.7 1.2 3.0 2.7 2.5
a The carbon percentage after each step was used as a measure of the amount of PEO-(Hz)2 coupled to the surface. b NA denotes “not applicable”. c Conjugation reactions performed in PBS. d Conjugation reactions performed in acetate buffer.
Figure 1. Reaction chemistry for site-directed immobilization of antibodies (Ab) via poly(ethylene oxide) (PEO) spacers.
were immersed in water for periods of up to a month without substantial reduction in the amount of surface carbon, to judge from Table 2. For all entries in the table, the C2/C1 ratio is close to 3, as expected for a PEO coating. On the basis of the results in this table, coupling conditions were standardized to 20 mg of PEO per milliliter of acetate buffer, pH 5.2, with an 11% K2SO4 additive, which yielded a surface concentration of PEO equal to 3 × 10-11 mol/cm2 (see Table 1), as determined by the SPDP method using silica spheres as substrates. At that surface concentration, there is an average of 24 Å between attachment points for the PEO chains. This is approximately the radius of gyration (Rg) of a 4000 MW PEO in solution, and the surface must therefore be considered to be fully covered with PEO chains which, due to their close-packing, form a thicker coating (“brush” as opposed to “mushroom” configuration)32 than would a collection of unconstrained polymer molecules (distance between attachment points >2Rg) on the surface.33 Attachment of Antibody. The site-directed attachment of Ab to these PEO tethers was to be accomplished by hydrazone formation between aldehyde groups in the carbohydrate moieties of the Ab and the terminal hydrazides of the PEO spacer arms on the surface, as shown in Figure 1. For this purpose, the Ab had to be oxidized in a potentially destructive treatment with sodium periodate. Following removal of the oxidant, the Ab’s ability to bind its fluorescein Ag was assayed and found to have been reduced by 25%, i.e. from an original activity level of 95% down to 70% following oxidation. As the coupling was to take place for an extended period of time (3 days) at the rather low pH of 5.2, the oxidized Ab was stored under these same conditions and was then again assayed for its activity. This second assay showed no further deterioration in Ag binding beyond that suffered in the oxidation process.30 (32) de Gennes, P. G. Adv. Colloid Interface Sci. 1987, 27, 189. (33) Li, J. T. Ph.D. Thesis, Department of Chemical and Fuels Engineering, University of Utah, 1993.
As mentioned above, the activity of the Ab was decreased from 95% to 70% after the step of oxidation with sodium metaperiodate. Although the 25% activity reduction is substantial, it does not alone account for the experimentally observed specific Ag binding which was found to be 25% of the theoretical activity based on one Ag molecule bound per Ab. From this relatively low binding, one must conclude that the PEO silica surfaces harbor Ab molecules which are so closely packed as to protect a large fraction of the binding sites from interacting with the Ag. In the binding studies presented here, it was assumed that, at the end of 2 h of interaction between Ab and Ag, the plateau value concentration has been achieved. Thus, the loss of Ag binding activity may result from the effect of steric hindrance, as discussed later.30 The coupling pH of 5.2 was selected, as discussed above, to direct any reaction with the newly formed aldehyde groups away from Schiff base formation with -amine groups on neighboring Abs and toward formation of hydrazones with the PEO tethers. By coupling 125I-labeled Ab in this manner, the surface concentration of protein could be quantified and was reproducibly found to be around 2 × 10-12 mol/cm2 (see Table 1). These Abcontaining surfaces were found to be quite stable chemically and showed no loss of radiolabel during immersion in PBS at 4 °C for 130 h. For the sake of comparison, unoxidized 125I-labeled Ab was nonspecifically attached directly to the glutaraldehyde-activated APS-silica through coupling in CB buffer at pH 9.2. As expected, the Ab surface concentration following this procedure was nearly twice as high (3.75 × 10-12 mol/cm2) as that obtained in the site-directed process. Immunoglobulin G is a disk-shaped molecule with a diameter of around 150 Å. A monolayer coverage of IgG in a flat arrangement can therefore be assumed to contain 1.1 × 10-12 mol/cm2, or about half of the amount found for the PEO-tethered attachment or less than a third of that found in the case of random coupling. The crowded surface is therefore likely to cause some steric hindrance, particularly to the binding of large antigens, and it might well prove advantageous to dilute the Ab on the surface for most efficient Ag binding. This strategy will be discussed in detail below. Binding of Antigen. The important question of these surfaces’ abilities to specifically bind Ag, while minimizing the nonspecific uptake of protein, was addressed through binding experiments involving FL-BSA and BSA, respectively. The fluorescein hapten was attached to the larger protein in order to give a more realistic picture of the reactivity of a typical surface-bound Ab with its bulky Ag. In both cases, the BSA was radioiodinated for quantification purposes, while the surface-linked Ab was unlabeled. As seen in Table 1, exposure to the FL-BSA Ag for 30 min resulted in a surface concentration of 5.5 × 10-13 mol/cm2 for the random-attached Ab, while the site-directed Ab bound comparable levels (5.6 × 10-13 mol/ cm2). The results also indicate the nonspecific uptake of labeled BSA, which turned out to be about twice as high for the surface with direct linking of Ab (2.1 × 10-13 mol/ cm2), as in the case where linking was mediated by the PEO tether (1.1 × 10-13 mol/cm2). Since there was nearly twice as much Ab on the surface with random attachment, the specific Ag (difference between surface concentrations of FL-BSA and BSA) binding ability of the Ab immobilized through Schiff base formation to surface aldehydes is nearly one third of that found for the Ab attached through site-directed coupling. From the practical standpoint of surface activity, the latter is showing an Ag binding comparable to that of the former, with about half the level of nonspecific uptake.
Site-Specific Immobilization of Monoclonal Antibodies
Langmuir, Vol. 12, No. 17, 1996 4297
Table 3. Effect of the Relative Amount of PEO-(Hz)2 on Specific and Nonspecific Binding in a Solid Phase Radioimmunoassay for FL-BSAa % PEO-(Hz)2
total binding (FL-BSA,b ×10-12 mol/cm2)
nonspecific binding (BSA,b ×10-12 mol/cm2)
0 25 50 75 100
0.034 ( 0.003 0.871 ( 0.060 0.859 ( 0.048 0.864 ( 0.031 0.766 ( 0.024
0.037 ( 0.002 0.037 ( 0.003 0.047 ( 0.003 0.052 ( 0.005 0.058 ( 0.003
a BSA, bovine serum albumin; FL-BSA, fluorescein conjugated to bovine serum albumin. b 125I-labeled antigens (Ag).
Coupling of Fab′ Fragments. In order to examine the question of steric hindrance to Ag binding and the possibility of obtaining a higher specific affinity of the surface, attachment of the whole Ab was abandoned in favor of site-directed attachment of the much smaller Fab′ fragment to PEO tethers. The availability of a free thiol near the C-terminal of the fragment suggests a coupling chemistry based on its addition to the maleimide group of the SMCC reagent, in turn bound to the surface PEOHz via its succinimide end. In addition to reducing the crowding at the surface by reducing the size of the affinity ligand, the surface concentration of these ligands was controlled by varying the concentration of coupling sites on the surface. This could be accomplished by mixing monofunctionalized CH3O-PEO-Hz with the difunctionalized analog PEO-(Hz)2 in the desired stoichiometric ratio prior to surface derivatization. The subsequent introduction of maleimide groups and addition of Fab′ fragments would then take place in proportion to the availability of hydrazides on the surface. Surfaces derivatized in this manner were assayed both for their ability to specifically bind the 125I-labeled FL-BSA, as described earlier for the intact Ab, and for their nonspecific binding of 125I-BSA. The results, summarized in Table 3, give a clear indication of a steric hindrance to Ag binding in the case of the most crowded ligand arrangement. This case also shows the highest level of nonspecific binding of all the surfaces listed in the table. By diluting the surface concentration of Fab′ fragments to 25% of its maximum value, one actually increases the surface’s ability to specifically bind Ag, while simultaneously reducing the nonspecific uptake to levels comparable to those characteristic of surfaces coated with underivatized PEO. In reference to the results in Table 1, the nonspecific binding in the case of these surfaces is less than 20% of that seen in random, direct coupling of the Ab. Clearly, the often voiced protein immobilization goal of maximizing the
ligand density to achieve the best specific binding characteristics should be re-examined in light of the present results. Kinetics of Antigen Displacement. As discussed in the introduction section, diagnostic assays based on the displacement of a labeled Ag or Ag analogs are affected by the rate of Ag release. Just as the surface concentration of Ab is affecting the equilibrium binding of Ag, it is likely to affect its competitive release. Therefore, a study was undertaken to determine the rates of displacement of radiolabeled FL-BSA from the Fab′-covered surfaces described above, using either the free fluorescein hapten or the bulkier FL-BSA as competitive displacers. The concentration of the displacer, regardless of type, was fixed at 1.5 × 10-7 M. For the sake of comparison, displacement was also performed from a surface containing whole Ab, coupled to PEO via the oxidized carbohydrate moiety, as described above. After the surfaces had been allowed to bind FL-BSA for 5 min, they were rinsed and immersed in the displacer solution from which they were removed, after given lengths of time, and analyzed by γ-counting. The results shown in Table 4 are a clear indication of enhanced release from the surface with the lowest Fab′ concentration (25% PEO-(Hz)2). After 12 min of displacement with FL-BSA, there is 81% of the original Ag left on the Ab (100%) surface, as opposed to 47% on the Fab′ (25%). The displacement reaction shows signs of mass transfer limitation, since the more rapidly diffusing fluorescein hapten is more effective as a displacer during shorter exposure times, while this edge has virtually disappeared after 50 h of exposure. The cumulative effect of the data in Table 4 is disturbing insofar as only a fraction (70%) of bound Ag can be displaced at all from its surfaceattached ligand. This suggests that highly sensitive displacement assays are best performed using labeled Ag analogs, as opposed to intact Ag, whose binding constants might be a couple of orders of magnitude lower than that of the true Ab-Ag complex. Conclusions Site-directed immobilization of Ab to silica can be performed through coupling via the carbohydrate moieties in its hinge region. When the oxidized Ab is attached to the surface by means of highly close-packed PEO spacers, it is found to bind Ag with a specific binding activity per bound Ab molecule which is nearly twice that observed for random attachment. In terms of Ag bound per unit area of surface, the two attachments gave similar results, although the random, direct coupling showed nonspecific
Table 4. Kinetics of Antigen Displacementa from Either Intact Oxidizied Ab or Fab′ Fragments Immobilized to Silica Surfaces Containing Different Relative Amounts of PEO-(Hz)2 intact Ab
12 min % left 30 min % left 1 h % left 50 h % left a
{FL BSA-FL {FL BSA-FL {FL BSA-FL {FL BSA-FL
Fab′ fragments
100% PEO-(Hz)2
100% PEO-(Hz)2
75% PEO-(Hz)2
50% PEO-(Hz)2
25% PEO-(Hz)2
75.0 81.0 67.0 74.0 60.0 63.0 NA NA
59.7 ( 2.2 67.4 ( 1.7 51.0 ( 3.0 56.5 ( 2.5 49.5 ( 3.0 52.7 ( 1.9 39.5 ( 3.1 41.1 ( 2.2
45.1 ( 1.5 48.5 ( 2.0 36.7 ( 1.5 38.7 ( 1.8 34.0 ( 0.6 36.0 ( 1.5 31.1 ( 0.8 31.2 ( 0.5
42.5 ( 1.2 47.9 ( 0.9 36.6 ( 1.0 38.7 ( 0.9 34.1 ( 0.6 35.1 ( 1.5 30.9 ( 1.2 31.0 ( 0.5
38.6 ( 0.3 46.9 ( 0.4 32.6 ( 0.3 34.6 ( 1.5 31.8 ( 0.2 33.1 ( 0.7 29.6 ( 0.3 30.7 ( 0.4
Displacement reaction shown as follows (asterisks denote
125I-labeled
antigens):
4298 Langmuir, Vol. 12, No. 17, 1996
binding of protein which was twice the level of that observed for the PEO-tethered Ab. Maximizing the amount of ligand on the surface was shown to be less advantageous than is frequently assumed. Reducing the concentration of active PEO tethers by mixing them with monofunctionalized, and thus nonbinding PEO, prior to surface coating and the subsequent attachment of protein ligand, yielded surfaces with high capacity for binding Ag at a much reduced level of nonspecific protein binding. Site-directed coupling of Fab′ fragments via their C-terminal thiols was shown to produce the most ideal Ag-binding surfaces of the ones examined in this study, both from the point of Ag binding per unit area and from the point of release kinetics in a displacement situation. Thus, surfaces where only 25% of the PEO ligands had
Huang et al.
the ability to tether to a Fab′ fragment showed higher Ag binding than those covered to 100% with potential tethers and saturated with whole Ab. The competitive displacement of Ag from the former was shown to be significantly more rapid and efficient than displacement from the latter. The surfaces produced here are likely to prove useful in immune detection based on optical signal transduction, where attachment to a silica surface is essential. Acknowledgment. The authors would like to thank AKZO Corporate Research America, Inc. for the financial support of this work. Portions of this article report technology licensed to HCP Diagnostics, L.P., of Salt Lake City, UT. LA951532+
