Oriented Surface Immobilization of Antibodies at the Conserved

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Oriented Surface Immobilization of Antibodies at the Conserved Nucleotide Binding Site for Enhanced Antigen Detection Nathan J. Alves,† Tanyel Kiziltepe,†,‡ and Basar Bilgicer*,†,‡,§ †

Department of Chemical and Biomolecular Engineering, ‡Advanced Diagnostics and Therapeutics, and §Department of Chemistry and Biochemistry, University of Notre Dame, Notre Dame, Indiana 46556, United States S Supporting Information *

ABSTRACT: The conserved nucleotide binding site (NBS), found on the Fab variable domain of all antibody isotypes, remains a not-so-widely known and unutilized site. Here, we describe a UV photo-cross-linking method (UV-NBS) that utilizes the NBS for oriented immobilization of antibodies onto surfaces, such that the antigen binding activity remains unaffected. Indole-3-butyric acid (IBA) has an affinity for the NBS with a Kd ranging from 1 to 8 μM for different antibody isotypes and can be covalently photo-cross-linked to the antibody at the NBS upon exposure to UV light. Using the UV-NBS method, antibody was successfully immobilized on synthetic surfaces displaying IBA via UV photo-cross-linking at the NBS. An optimal UV exposure of 2 J/cm2 yielded significant antibody immobilization on the surface with maximal relative antibody activity per immobilized antibody without any detectable damage to antigen binding activity. Comparison of the UVNBS method with two other commonly used methods, ε-NH3+ conjugation and physical adsorption, demonstrated that the UVNBS method yields surfaces with significantly enhanced antigen detection efficiency, higher relative antibody activity, and improved antigen detection sensitivity. Taken together, the UV-NBS method provides a practical, site-specific surface immobilization method, with significant implications in the development of a large array of platforms with diverse sensor and diagnostic applications.

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INTRODUCTION The conserved nucleotide binding site (NBS), which is found in the variable region of the Fab arm of all antibody isotypes, remains a not-so-widely known and unutilized site. This paper describes a method for UV photo-cross-linking antibodies onto surfaces at the NBS, in an oriented manner such that the antigen binding activity is preserved. Antibodies are conjugated to surfaces in a large array of platforms including sensor and diagnostic applications developed for the detection of pathogens, disease biomarkers, water contaminants, drug discovery, and laboratory-based immunoassays.1−4 For all of these applications, antibody’s antigen binding activity is a critical parameter that governs the sensitivity, dynamic range, and reproducibility of the detection tools. The current standard method of immobilizing antibodies to surfaces involves noncovalent physical adsorption to a detection surface through nonspecific hydrophobic interactions (physical adsorption method).5−12 This method, however, results in randomly oriented antibody molecules on the detection surface and yields ∼90% antibody that is in an inactive orientation due to steric blocking of the antigen binding sites.13−15 A common alternate method involves nonspecific chemical immobilization to amine reactive surfaces by utilizing the lysine side chain amino groups present on the surface of antibodies (ε-NH3+ method).16−23 In this method, control over sites of conjugation is not possible, which results in an inhomogeneous surface with © 2012 American Chemical Society

reduced antibody activity similar to that of the random physical adsorption method.24,25 Despite resulting in a significant loss of antibody activity, these methods are still commonly used due to their simplicity of execution. Site-specific immobilization methods are also currently available; however, these methods have either complicated chemical procedures or are detrimental to the antibody activity. For example, one method involves selective reduction of the disulfide bonds between the two heavy chains and uses the available thiol side chains as reactive sites for conjugation to gold or maleimide functionalized surfaces.26−30 This method, albeit site specific, yields monovalent capture antibodies and inactive antibody fragments due to unintentional reduction of the disulfide bonds that maintain the structure of the antigen binding framework regions. An alternate method utilizes the carbohydrate chains present on antibodies originating from post-translational protein modifications. These side chains can be oxidized by various chemical approaches to form reactive aldehyde groups that can be used to selectively couple to hydrazine functionalized surfaces.31−35 This technique provides a high coupling yield, but the denaturing conditions and oxidative chemicals used can result in a loss of antibody activity.36 In addition, vastly different degrees of post-translational modifications on Received: January 25, 2012 Published: May 21, 2012 9640

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Figure 1. (A) Location of the nucleotide binding site (NBS) in antibodies is shown in a cartoon representation and on the crystal structure of the antibody Fv region. (B) This is a schematic representation of the method for UV photo-cross-linking of antibodies onto surfaces at the NBS. IBA functionalized surfaces are generated by reacting IBA-EG11-amine with maleic anhydride-coated plates. Antibodies associate with IBA moiety at the NBS, and upon UV exposure a covalent bond forms between IBA and the antibody, permanently tethering the antibody to the surface. The oriented, site-specific conjugation of the antibody through its NBS preserves antibody’s antigen binding activity.

with aromatic side chains. When aromatic rings are exposed to a specific wavelength (254 nm) of UV light and are in close proximity, reactive radicals form, resulting in the formation of new covalent bonds between the aromatic rings.42,43 Therefore, the NBS provides a useful site for selective conjugation of antibodies to small molecule ligands that contain aromatic rings. To identify such small molecule ligands with a high binding affinity and selectivity for the NBS, we performed an in silico screening by docking various small molecules from the ZINC database at the NBS.39 The top scoring molecules(2(2-benzimidazolylamino)-1-ethanol, 7-methyltryptamine, sinefugin, indole-3-butyric acid, tryptophan, and 5-methylindole-3carboxaldehyde)were then experimentally investigated for their binding affinity to the NBS. Indole-3-butyric acid (IBA) emerged as the highest affinity binding nucleotide analogue, with Kd values ranging between 1 and 8 μM depending on the antibody, and was therefore chosen for the application described in this study. The UV-NBS method necessitates IBA conjugated surfaces for antibody cross-linking. When antibody is introduced to the well, surface conjugated IBA associates with the antibody via binding to its NBS. Upon UV exposure, a covalent bond forms between the IBA and the antibody, permanently tethering the antibody to the surface and thereby generating the antibody functionalized surface (Figure 1B). The described method, being site-specific, enables immobilization in an oriented manner such that antigen binding activity of the antibody is preserved for enhanced antigen detection efficiency of the functionalized surface.

antibodies cause this approach to deliver highly variable outcomes from antibody to antibody.37,38 The primary concern with many of the currently available antibody immobilization methods is the loss of antigen recognition due to steric hindrance of the antigen binding sites or partial denaturation of the antibody due to chemically harsh reaction conditions.13,24,25 These outcomes result in a loss of antibody activity that directly equates to a loss in antigen capture efficiency of the functionalized surface. Taken together, these points highlight the need for the development of a practical and reproducible method for site specific immobilization of antibodies to surfaces such that the antigen binding activity remains unaffected. Here we describe a photochemistry-based NBS specific antibody immobilization (UV-NBS) method for oriented immobilization of antibodies onto surfaces. In an earlier publication, we extensively characterized the NBS using molecular modeling and showed that it is a highly conserved binding pocket located in the “conserved” region of the variable domain of all antibody isotypes from various species (Figure 1A).39,40 This characterization was achieved by performing a least-squares root-mean-square deviation superposition of all Fab domain crystal structures of >260 immunoglobulins in the RCSB Protein Data Bank.39 Specifically, our analysis revealed that four residues, namely two tyrosine residues on the variable region of the light chain [framework region 2 (FR2), position 42, and FR3, position 103 based on IMGT numbering]41 and one tyrosine and one tryptophan residue on the variable region of the heavy chain [FR3, position 103, and junction region, position 118, respectively], are conserved. In some instances a phenylalanine is observed in place of the tyrosine at either of the conserved positions on the VL (Figure S1 in Supporting Information). Hence, the NBS is rich with amino acid residues 9641

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(Invitrogen) was used to detect the location where IBA-biotin was covalently conjugated on the antibody. To verify transfer of all protein content to the membrane, both the SDS-PAGE gel (post transfer) and nitrocellulose membrane were Coomassie blue stained in a solution of 10% acetic acid, 20% methanol, 0.15% Coomassie R-250 (EMD Chemicals) for 30 min and destained in a solution of 20% acetic acid, 20% methanol, 60% DI water for 1.5 h. Control experiment performed in the absence of UV exposure or in the absence of IBA-biotin did not yield any detectable bands. Similarly, control experiments performed with only biotin did not yield any detectable bands. Mouse anti-FITC IgG1 (clone: DE3) was purchased from Millipore, rat anti-DNP IgG1 (clone: LO-DNP-2) was purchased from Invitrogen, and mouse antiStreptavidin IgG1 (clone: S8C12) was purchased from Meridian Life Science, Inc. UV-NBS Antibody Immobilization Method. IBA-coated 96-well plates were generated as described above and were blocked with 50 mM Tris, 100 mM NaCl, pH 8.0 buffer for 1 h. Antibody was added to IBA-coated wells in a total volume of 100 μL PBS buffer with 0.1% Tween20 at pH 7.4 for 1.5 h at RT and was then exposed to the indicated amount of UV light using a Spectroline Select Series UV cross-linker (Figure S5). Unbound antibody was then washed using an automated plate washer (MDS Aquamax 2000), three cycles of 200 μL PBS with 0.05% Tween20 at pH 7.4. Antibody immobilized wells were blocked with BSA blocking buffer (2.5 g BSA in 50 mL of PBS buffer with 0.05% Tween20 at pH 7.4) for 1 h to prevent nonspecific adhesion/interactions. ε-NH3+ Antibody Immobilization Method. Antibody was incubated in an amine reactive maleic anhydride 96-well plate for 2 h at RT in PBS buffer at pH 8.0. Unbound antibody was washed using an automated plate washer (three cycles of 200 μL PBS with 0.05% Tween20 at pH 7.4). Any remaining reactive sites on the plate surface were then quenched using 50 mM Tris buffer with 100 mM NaCl at pH 8.0 for 1 h. The surface was then blocked using BSA blocking buffer for 1 h. Physical Adsorption Method. Antibody was adsorbed to a high binding 96-well plate (Costar) in 0.05 M carbonate−bicarbonate coating buffer at pH 9.6 for 2 h at RT (Figure S6). Unbound antibody was washed using an automated plate washer (three cycles of 200 μL PBS with 0.05% Tween20 at pH 7.4). Plate surface was then blocked with BSA blocking buffer for 1 h. Determination of Antigen Detection Efficiency of Antibody Immobilized Surfaces. The antigen detection efficiency of the antibody immobilized surfaces generated using the above-described methods were determined by an enzymatic assay, where biotin conjugated versions of antigens (e.g., DNP-biotin, FITC-biotin) were synthesized to enable detection using streptavidin-HRP. Briefly, IgG immobilized surfaces were incubated with saturating concentrations of antigen−biotin in a total volume of 100 μL PBS with 0.05% Tween20 at pH 7.4 for 1.5 h. After washing unbound antigen−biotin, the wells were incubated with a 1:10 000 dilution of streptavidin−HRP (1.0 mg/mL stock) in BSA blocking buffer for 1 h. HRP substrate (Amplex Red, Invitrogen) was added, and fluorescent product formation was observed on a Molecular Devices SpectraMax M5 plate reader (ex 570 nm, em 592 nm) (Figure S7). The results are reported as relative fluorescence units (RFU). Control experiments performed without antigen−biotin, and immobilized antibody were used as background for the antigen detection measurements. Determination of Total Antibody Content of Antibody Immobilized Surfaces. Quantification of the total surface immobilized antibody for each of the three immobilization methods was performed using HRP conjugated Fc domain specific secondary antibodies from goat purchased from Jackson ImmunoResearch (Figure S8). Briefly, wells were incubated with a 1:5000 dilution of anti-Fc antibody (1.0 mg/mL stock) in BSA blocking buffer for 1 h. After washing unbound anti-Fc antibody, HRP substrate (Amplex Red, Invitrogen) was added, and fluorescent product formation was observed (ex 570 nm, em 592 nm). The results are reported as RFU. Control experiments performed without immobilized antibody were used as background.

EXPERIMENTAL DETAILS

Synthesis of IBA-EG11-amine Conjugates and Generation of IBA-Coated Surfaces. The IBA-EG11-amine ligand was synthesized by coupling indole-3-butyric acid (IBA, Sigma) to mono-N-t-bocamido-dPEG11-amine (Quanta Biodesign) following activation using 2-(1H-benzotriazole-1-yl)-1,1,3,3-tetramethyluronium hexafluorophosphate (HBTU, Novabiochem) in dimethylformamide (DMF, Sigma) and N,N-diisopropylethylamine (DIEA, Sigma) at room temperature for 3.5 h while agitating. After rotate evaporating DMF, the t-boc protecting group was removed in a solution of 4% triisopropylsilane (Sigma), 4% DI water (Millipore Integral 10 Milli-Q system), and 92% trifluoroacetic acid (TFA, Sigma) for 45 min at room temperature. IBA-EG11-amine was purified via reverse phase high pressure liquid chromatography (RP-HPLC) on a Zorbax C18 (Agilent) column, and its mass was verified via matrix-assisted laser desorption ionization time-of-flight mass spectrometry (MALDI-TOF MS) using 2,5dihydroxybenzoic acid (DHB, Sigma) as matrix (Figure S2). The purity was confirmed using RP-HPLC on an analytical Zorbax C18 column (>95%), and the yield was 75%. IBA functionalized plate surface was generated by coupling IBA-EG11-amine to amine-reactive, maleic anhydride polymer-coated 96-well plates (Thermo Scientific). Coupling was achieved by incubating 100 μL of 0.625 μM IBA-EG11amine in each well for 2 h, in PBS at pH 8.0, at room temperature. Any remaining reactive maleic anhydride sites were then blocked by incubating the plate surface with 50 mM Tris buffer with 100 mM NaCl at pH 8.0 for 1 h. Synthesis of IBA-Biotin. IBA-biotin was synthesized using standard solid phase synthesis protocols on a NovaPEG Rink Amide resin (Novabiochem) and Fmoc chemistry. First, HBTU activated Fmoc-Trp(Boc)-OH in DMF, and DIEA was coupled to the resin for 3 h while agitating. Fmoc was deprotected using 20% piperidine in DMF. Fmoc-N-amido-dPEG2-acid (Quanta Biodesign) was then activated using HBTU and coupled in the next step. After Fmoc deprotection, HBTU activated Biotin (Sigma) was coupled to the resin conjugated molecule. Kaiser tests were performed between coupling steps to monitor synthesis progress. IBA-biotin was cleaved from the resin in 95% TFA, 2.5% TIS, and 2.5% DI water, purified via RPHPLC on a Zorbax C18 column, and characterized using MALDITOF MS (Figure S3). The purity was confirmed using RP-HPLC on an analytical Zorbax C18 column (>95%), and the yield was 82%. Synthesis of DNP-Biotin. DNP-biotin was synthesized using standard solid phase synthesis protocols on a NovaPEG Rink Amide resin and Fmoc chemistry. Fmoc-Lys(ivDde)-OH (Novabiochem) was activated with HBTU in DMF and DIEA, followed by coupling to the resin using agitation for 3 h. The Fmoc protecting group was removed with 20% piperidine in DMF. Boc-Lys(Fmoc)-OH (Novabiochem) was then activated and coupled to the deprotected amine followed by Fmoc deprotection and coupling of biotin. The ivDde protecting group on lys was then removed using 2% hydrazine in DMF followed by activation and coupling of Fmoc-N-amido-dPEG2-acid (Quanta Biodesign). The ethylene glycol linker was then deprotected and incubated with 2,4-dinitro-1-fluorobenzene (Aldrich) in DMF with DIEA. Kaiser tests were performed between coupling steps to monitor synthesis progress. Product was cleaved from the resin in 95% TFA, 2.5% TIS, and 2.5% DI water and purified via RP-HPLC on a Zorbax C18 column. The product was characterized using MALDI-TOF MS (Figure S4). The purity was confirmed using RP-HPLC on an analytical Zorbax C18 column (>95%), and the yield was 40%. Western Blot Analysis of UV-NBS Photo-Cross-Linking of IBA to the Antibody. IgG antibody (1.7 μM) was incubated with excess IBA-biotin (100 μM) in PBS buffer at pH 7.4 and then exposed to increasing UV (254 nm) energy in a Spectroline UV Select Series Cross-linker (Spectronics). The samples were run on a 10% SDSPAGE gel with a Tris-glycine running buffer under reducing conditions at 110 V for 1 h and were transferred to a nitrocellulose membrane at 110 V for 90 min in a 10% MeOH transfer buffer (Boston Bioproducts). The membrane was blocked with 10% dry milk in Tris buffered saline (TBS) for 1 h and was then blotted with 1:10 000 dilution of Streptavidin-HRP (Jackson ImmunoResearch) 1 h at room temperature (RT). A chemiluminescent HRP substrate 9642

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Figure 2. UV-NBS method for surface immobilization of IgGDNP antibody to IBA-functionalized surface using increasing UV energy. (A) Amount of surface immobilized antibody was measured using an HRP linked anti-Fc secondary antibody. (B) Antigen detection efficiency of the surface was measured with DNP−biotin as the antigen and streptavidin−HRP as the reporter. In both experiments, y-axis is reported as the relative fluorescence units (RFU). (C) Relative antibody activity (the ratio of the signals from antigen detection efficiency to the total surface immobilized antibody) is plotted. All data represents means (±SD) of triplicate experiments. Determination of Antigen Detection Sensitivity. A dose response curve was created using each of the immobilization techniques by varying the antigen concentration while keeping the total surface immobilized antibody constant. The resulting plots (antigen detection signal vs antigen concentration) were fit by linear regression. Sensitivity was determined from the slope of the linear regression.

antibody cross-linking was observed in control experiments performed using plates that were not coated with IBA or in the absence of UV exposure (results not shown). Furthermore, antibody cross-linking was dramatically inhibited in control experiments performed in the presence of competitively binding soluble IBA (results not shown). It is noteworthy that antigen detection efficiency reached a plateau at 2 J/cm2 despite an increase in total surface immobilized antibody (Figure 2A,B). This plateau effect could be a reflection of a shortcoming of the HRP-based enzymatic assay at the higher antigen concentrations, since the rate of the enzymatic reaction is limited by substrate diffusion from the bulk solution to the surface. Alternatively, it could result from increased damage to the antigen binding site when compared to the Fc region beyond 2 J/cm2. To confirm that no significant damage to the antibody’s antigen binding activity was induced at the UV doses used, we performed additional experiments to test the antibody’s structural integrity post-UV exposure. For this, IgG antibody was cross-linked to a plate surface using the common ε-NH3+ immobilization method and exposed to increasing amounts of UV energy (0−10 J/cm2). The damage to the antigen binding site was determined by incubating IgG immobilized surfaces with saturating levels of antigen−biotin conjugates, which was quantified by streptavidin−HRP in an ELISA assay. To evaluate the structural integrity of the Fc domain, a secondary anti-Fc antibody was used. The anti-Fc antibody only recognizes the intact Fc structure; therefore, loss in its ability to bind to UV exposed IgG correlates directly to Fc damage. Our results suggested that a UV dose of up to 2 J/cm2 did not have detectable impact on the antibody’s integrity based on (i) its ability to recognize its antigen and (ii) the secondary antibody’s ability to bind to the Fc domain (Figure S10). We further confirmed this result by challenging the antibody’s antigen binding activity by exposing it to increasing UV energies in solution and observing its ability to recognize its surface immobilized antigen in an ELISA assay. With this method, we did not detect any significant damage to either the antigen binding activity or the Fc recognition up to 5 J/cm2 (Figure S11). Finally, in a separate experiment we evaluated if UV exposure caused any higher order aggregates or cross-linked products between antibody molecules by dynamic light scattering and did not detect any structural changes after UV exposure at 2 J/cm2 (Figure S12). Combined, these results suggest 2 J/cm2 as a well-tolerated UV dose that preserves structural integrity and antigen binding activity of the antibody.

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RESULTS AND DISCUSSION The UV-NBS method for surface immobilization of antibodies requires an IBA functionalized surface (Figure 1). Therefore, we synthesized an IBA derivative, IBA-EG11-amine, and coupled it to an amine reactive maleic anhydride polymer coated 96-well plate to yield IBA functionalized surfaces (for optimization see Figure S9). To generate antibody immobilized surfaces, IgGDNP antibody (0.1 pmol) was incubated in the wells for 1.5 h to allow association of NBS and surfaceconjugated IBA. The plate was then exposed to UV light for covalent bond formation between IBA and the NBS residues. The site-specificity of the UV-NBS method leaves the antigen binding site unaffected and the Fc domain available for secondary antibody binding. Quantification of the total surface immobilized antibody, using an HRP linked anti-Fc secondary antibody, demonstrated that the UV-NBS method successfully generated antibody immobilized surfaces in a UV energy dependent manner, reaching up to ∼4.5 × 103 RFU (Figure 2A). Next, we evaluated the antigen detection efficiency of the antibody immobilized surface with an ELISA assay. Briefly, IgGDNP immobilized surfaces were incubated with saturating levels of DNP−biotin conjugate, and captured DNP-biotin was quantified using streptavidin−HRP as a reporter. Our results demonstrated that the antibody immobilized surfaces, generated via the UV-NBS method, were effective in antigen detection. Maximum antigen detection efficiency was achieved with surfaces produced using a UV exposure between 2 and 5 J/cm2, reaching up to ∼21 × 103 RFU (Figure 2B). Importantly, the relative antibody activity, which is the ratio of the antigen detection efficiency signal to the total surface immobilized antibody signal, reached a maximum at 2 J/cm2 (Figure 2C). The results of these experiments established the optimal UV energy for the UV-NBS method as 2 J/cm2, where we observed (i) a high yield of antibody photo-cross-linking, (ii) the maximum antigen detection efficiency of the surface, and (iii) the maximum relative antibody activity. We also achieved similar results with an alternate antibody−antigen system (mouse anti-FITC; IgGFITC) (results not shown). No 9643

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driven by nonspecific hydrophobic interactions between the antibody and plate surface. Therefore, for physical adsorption, the antibody was incubated on a high-binding ELISA plate surface. Similar to the ε-NH3+ method, the physical adsorption method also yields randomly oriented antibodies on the surface. Both of these commonly used methods were compared to the UV-NBS method for (i) antigen detection efficiency of the functionalized surface, (ii) total antibody immobilized to the surface, and (iii) relative antibody activity. For this comparison, we functionalized 96-well plates using the three respective methods with initial antibody amounts of 0.01, 0.05, or 0.1 pmol of IgGDNP to generate the antibodycoated surfaces. Antigen detection efficiency of each surface was determined via the aforementioned method by using saturating levels of DNP−biotin followed by HRP−streptavidin. Total immobilized antibody content in the wells was determined by an HRP conjugated Fc specific secondary antibody. When comparing the three methods, UV-NBS immobilization method yielded functionalized surfaces with significantly higher antigen detection efficiency than both of the other methods (Figure 4A). Specifically, the enhancement reached up to ∼10-fold when compared to the ε-NH3+ method and up to ∼122-fold

Furthermore, the Fc domain is also preserved, making it possible for quantification of the total surface immobilized antibody. Therefore, UV exposure of 2 J/cm2 was used in producing antibody immobilized surfaces for antigen detection in the rest of the study. Next, we investigated if the UV-NBS method yielded IBA cross-linking selectively at the NBS by analyzing the products of a UV-cross-linking reaction using Western Blot analysis. Briefly, we synthesized an IBA−biotin conjugate for easy detection by streptavidin. IBA−biotin was incubated with IgG antibody in PBS buffer to allow binding and was then photo-cross-linked to the NBS by exposure to a range of UV energies (0−2 J/cm2). The product, antibody conjugated IBA−biotin, was run on an SDS-PAGE under reducing conditions and transferred to a nitrocellulose membrane, and biotinylated fragments were probed by using a streptavidin−HRP reporter molecule. While the NBS is located between the light and heavy chains our results demonstrated a preferential covalent insertion of the IBA−biotin molecule selectively to the antibody light chain in a UV energy dependent manner as indicated by an increase in band intensity with increasing amounts of UV exposure (Figure 3). This is likely due to the orientation of the IBA when

Figure 3. UV-NBS photo-cross-linking site on the antibody was investigated using Western Blot analysis. IBA−biotin was cross-linked to the antibody (IgGDNP) by exposure to UV energy from 0 to 2 J/cm2 in PBS buffer. SDS-PAGE was run under reducing conditions, and the proteins were transferred to a nitrocellulose membrane. Both the gel (SDS−gel post-transfer) and membrane (nitrocellulose membrane) were stained by coomassie blue to verify efficiency of transfer. Streptavidin−HRP was used to probe for covalently attached IBA− biotin to the antibody. Blotted film shows that biotin tag only appears on the antibody light chain. Similar results obtained using IgGFITC (data not shown).

associated with the NBS prior to UV exposure. Control experiments performed with just biotin did not yield any bands in the blotted membrane (results not shown). Although this result does not necessarily ensure that IBA cross-linking takes place precisely at the NBS, it does confirm insertion of the IBA strictly on the antibody light chains, indicating that the photocross-linking is not a random event and takes place at a specific site. This result was confirmed by repeating this assay using various antibodies from different species with different antigen specificities (results not shown). We further evaluated the UV-NBS method by comparing it to two other commonly employed antibody immobilization methods: the ε-NH3+ and the physical adsorption methods. For ε-NH3+ immobilization, antibody was allowed to react directly with an amine reactive maleic anhydride functionalized plate. The ε-NH3+ method conjugates the antibody to the plate surface arbitrarily through multiple lysine side chains, resulting in randomly oriented antibody molecules on the surface. Immobilization via the physical adsorption method is primarily

Figure 4. Evaluation of the UV-NBS method in comparison to ε-NH3+ and physical adsorption methods using the rat IgGDNP/DNP antibody/antigen system. The 96-well plates were functionalized with IgGDNP using all three methods. (A) Antigen detection efficiency of all three surfaces was detected with DNP−biotin as the antigen and streptavidin−HRP as the reporter. (B) Total surface immobilized antibody was quantified using an HRP linked anti-Fc secondary antibody. In both experiments, y-axis is reported as relative fluorescence units (RFU), and the x-axis shows the starting amount of antibody used to generate the surface. (C) Relative antibody activity is plotted. Data represent means (±SD) of triplicate experiments. 9644

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when compared to physical adsorption method at 0.01 pmol of antibody. From another perspective, surfaces generated using only 0.01 pmol of antibody via the UV-NBS method delivered better or comparable antigen detection efficiency (10.2 × 103 RFU) when compared to surfaces generated using even 10-fold more antibody via the other two methods (2.4 × 103 and 11.0 × 103 RFU for physical adsorption and ε-NH3+ methods, respectively). Combined, these results demonstrate that the UV-NBS method provides us with an effective antibody immobilization method to generate functionalized surfaces with the highest antibody detection efficiency. Next, we compared the amount of surface immobilized antibody in all three methods as determined by a secondary anti-Fc antibody. Interestingly, the amount of surface immobilized antibody was significantly higher with the εNH3+ method when compared to the physical adsorption and UV-NBS methods (Figure 4B). Despite the lesser amount of surface immobilized antibody with the UV-NBS method, antigen detection efficiency was significantly higher (Figure 4A), demonstrating that the UV-NBS method yields enhanced preservation of antigen binding activity per surface immobilized antibody. This result strongly suggests that the UV-NBS method highly preserves antibody’s antigen binding activity as a result of the site-specific immobilization of antibody on the surface. It is noteworthy to mention that quantification of surface immobilized antibody, via an anti-Fc secondary antibody, in both ε-NH3+ and physical adsorption methods is an underestimate. This results from immobilization at random sites, which leaves the Fc domain unavailable for binding in a fraction of antibody molecules. Therefore, the enhancement observed for relative antibody activity with the UV-NBS method is an underestimate and the actual enhancement is likely even higher. To further emphasize this point, we calculated the relative antibody activity for all methods by using the ratio of the signal from antigen detection efficiency to the total surface immobilized antibody signal (Figure 4C). As expected, UVNBS resulted in a much higher relative antibody activity than the other two methods, reaching up to ∼43-fold and ∼91-fold enhancements in relative antibody activity when compared to the ε-NH3+ and physical adsorption methods, respectively. Taken together, these results strongly suggest that the UV-NBS method highly preserves antibody’s antigen binding activity as a result of oriented immobilization onto surfaces, while activity is significantly lost with the ε-NH3+ and physical adsorption methods as a result of randomly orientated antibodies. These results are summarized in Tables S1 and S3. Similar trends, with even more dramatic enhancement in surface antigen detection efficiency and relative antibody activity, were observed with the IgGFITC/FITC antibody/ antigen system (Figure 5). The UV-NBS method yielded functionalized surfaces with significantly higher antigen detection efficiency, enhancements reaching up to ∼3.6-fold when compared to the ε-NH3+ method at 0.01 pmol antibody and up to ∼280-fold when compared to physical adsorption method at 0.05 pmol of antibody. No antigen binding was detectable with the physical adsorption method at 0.01 pmol of antibody. Despite lower amounts of surface immobilized antibody, UV-NBS also resulted in a much higher relative antibody activity than the other two methods, reaching to ∼94fold and ∼674-fold enhancements when compared to the εNH3+ and physical adsorption methods, respectively. These results are summarized in Tables S2 and S3. These experiments

Figure 5. Evaluation of the UV-NBS method in comparison to ε-NH3+ and physical adsorption methods using the mouse IgGFITC/FITC antibody/antigen system. The 96-well plates were functionalized with IgGFITC using each respective method. (A) Antigen detection efficiency of the surfaces was determined with saturating levels of FITC−biotin as the antigen and streptavidin−HRP as the reporter. The y-axis shows the relative fluorescence units (RFU) obtained using an HRP substrate, and the x-axis shows the starting amount of antibody used to generate the surface. (B) Total surface immobilized antibody was quantified using an HRP linked anti-Fc secondary antibody. (C) Relative antibody activity is plotted. Data represent means (±SD) of triplicate experiments.

were also performed with a protein-based antigen detection system, IgGstreptavidin/streptavidin, which yielded similar trends (Figure S13). Finally, we evaluated the antigen detection sensitivity of the surfaces generated by the three immobilization methods. Sensitivity (S) is defined as the slope of the linear regression line obtained by plotting detection signal versus antigen concentration.35 A larger slope indicates a higher degree of sensitivity. Sensitivity was calculated and compared for all three immobilization techniques using IgGDNP (0.1 pmol) as the capture antibody, DNP−biotin conjugate as the antigen, and streptavidin−HRP as the reporter. A fluorescent HRP substrate was employed to determine the dose−response curve of a range of standard antigen concentrations from 0 to 10 μM. The linear regression equation was obtained for all three methods with regression coefficients (R2) of 0.9511, 0.9486, and 0.9576 for the UV-NBS, ε-NH3+, and physical adsorption methods, respectively (Figure 6). The UV-NBS immobilization method displayed overall higher signal intensity with the highest sensitivity (S = 1.796), ε-NH3+ immobilization method with lower signal intensity and lower sensitivity (S = 1.224), and physical adsorption with significantly lower signal intensity and the lowest sensitivity (S = 0.227). The difference in the slope of 9645

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CONCLUSION The results presented in this study establish the UV-NBS method as a practical, gentle, and reproducible method for site specific antibody immobilization on surfaces. The site specific cross-linking at the conserved NBS provides preservation of antibody’s antigen binding activity and has the potential to be applied to various immunosensor platforms. In our experiments, surfaces functionalized with the UV-NBS method displayed significantly enhanced antigen detection efficiency, higher relative antibody activity, and improved antigen detection sensitivity in comparison to two other commonly used antibody immobilization methods. Finally, the UV-NBS method can be adapted to different detection modalities other than enzyme-based assays such as colorimetric, fluorescence, impedance, refractive index, and plasmon modalities. With the development of micro- and nanofluidic diagnostic chips the physical detection areas in these devices are approaching length scales comparable to the size of the detection molecules themselves.45,46 Because of the limited detection area, and therefore decreased number of antibodies that can be presented, it is critical that every antibody on the detection surface maintains its ability to recognize and bind its specific antigen. The UV-NBS method provides a great advantage over other immobilization techniques by providing that each immobilized antibody preserves its antigen binding activity, which is essential to provide for reliable outcomes. Taken together, the UV-NBS method provides a site-specific, practical, and flexible surface immobilization method, with significant implications in the development of a large array of immunosensor platforms.

Figure 6. Linear regression analysis of dose−response curves comparing the UV-NBS, ε-NH3+, and physical adsorption methods. The 96-well plates were functionalized with 0.1 pmol of IgGDNP using the respective methods. Antigen capture was detected using increasing concentrations of DNP−biotin (0−10 μM) as the antigen and streptavidin−HRP as the reporter.

the regression curves can be explained by the enhanced binding efficiency of the UV-NBS surface, which results from enhanced antibody activity. Combined, these results demonstrate that surfaces generated by the UV-NBS method produce a higher sensitivity in antigen detection than the ε-NH3+ and the physical adsorption methods, 1.5- and 7.9-fold enhancement in sensitivity, respectively. Despite the enhanced sensitivity achieved with the UV-NBS method, an improvement at the lower limits of detection (LLD, 3SD to the mean of the zero standard35) was not detectable employing this antibody/antigen system. The LLD were comparable at ≤10 nM for all three methods after the 20 min reaction time. It is noteworthy that this estimated LLD value is in the same range as the dissociation constant of the antibody/antigen interaction (IgGDNP/DNP; Kd = 8 nM). For this reason, a reduction in the detection of analyte at concentrations below the Kd value can be attributed to the loss in the antibody’s ability to bind to its antigen and is not a reflection of a shortcoming of the UV-NBS method. On the other end of the spectrum, the upper limit of detection is governed by the inherent limitations of the HRP enzyme based assay used in the UV-NBS method. Although the intrinsic kinetic parameters of the enzyme and the substrate are not altered, substrate diffusion through the bulk solution to the surface where the enzyme is immobilized can become the ratelimiting factor.44 As a result, increasing the amount of HRP on the surface beyond a certain threshold will not increase the rate of product formation, yielding a plateau in the signal intensity. This is a possible explanation for the plateau observed in Figure 2B. The UV-NBS method may be further improved by optimizing the conditions to increase the total amount of antibody immobilized on the surface. IBA is a hydrophobic molecule, which may cause it to associate with the maleic anhydride polymer, rendering it partially inaccessible for binding to the antibody. Hence, a more hydrophilic ligand and a more uniform surface would make the NBS ligand more accessible to the antibody and may improve the yield of the surface immobilized antibody. However, a ligand with a higher affinity for the NBS and increased photoreactivity would have a much greater impact on enhancing the coupling efficiency of the antibody to the surface immobilized ligand. These studies are currently ongoing in our laboratory.

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ASSOCIATED CONTENT

* Supporting Information S

Figures S1−S15 and Tables S1−S3. This material is available free of charge via the Internet at http://pubs.acs.org.

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AUTHOR INFORMATION

Corresponding Author

*Tel 574-631-1429, Fax 574-631-8366, e-mail bbilgicer@nd. edu. Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS The authors thank the Center for Environmental Science and Technology for usage of the DLS and the Notre Dame Mass Spectrometry and Proteomics Facility for usage of mass analysis instrumentation.

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REFERENCES

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