Affibody Molecules in Protein Capture Microarrays - ACS Publications

Affibody Molecules in Protein Capture Microarrays: Evaluation of Multidomain Ligands and Different Detection Formats Bjo1 rn Renberg,† Jon Nordin,† Anna Merca,† Mathias Uhle´ n,‡ Joachim Feldwisch,§ Per-A° ke Nygren,† and Amelie Eriksson Karlstro1 m*,† School of Biotechnology, Division of Molecular Biotechnology, Royal Institute of Technology, AlbaNova University Center, SE - 106 91 Stockholm, Sweden, School of Biotechnology, Division of Proteomics, Royal Institute of Technology, AlbaNova University Center, SE - 106 91 Stockholm, Sweden, and Affibody AB, Box 20137, SE - 161 02 Bromma, Sweden Received June 27, 2006

The importance of the ligand presentation format for the production of protein capture microarrays was evaluated using different Affibody molecules, produced either as single 6 kDa monomers or genetically linked head-to-tail multimers containing up to four domains. The performances in terms of selectivity and sensitivity of the monomeric and the multidomain Affibody molecules were compared by immobilization of the ligands on microarray slides, followed by incubation with fluorescent-labeled target protein. An increase in signal intensities for the multimers was demonstrated, with the most pronounced difference observed between monomers and dimers. A protein microarray containing six different dimeric Affibody ligands with specificity for IgA, IgE, IgG, TNF-R, insulin, or Taq DNA polymerase was characterized for direct detection of fluorescent-labeled analytes. No cross-reactivity was observed and the limits of detection were 600 fM for IgA, 20 pM for IgE, 70 fM for IgG, 20 pM for TNF-R, 60 pM for insulin, and 10 pM for Taq DNA polymerase. Also, different sandwich formats for detection of unlabeled protein were evaluated and used for selective detection of IgA or TNF-R in human serum or plasma samples, respectively. Finally, the presence of IgA was determined using detection of directly Cy5-labeled normal or IgA-deficient serum samples. Keywords: Affibody molecule • protein microarray • protein capture • multidomain proteins • sandwich assay

1. Introduction The microspot assay for detection of proteins in minute sample volumes, described by Ekins1,2 already in 1989, has in recent years emerged as a valuable tool for proteomic research. Analysis of prostate cancer markers,3 cytokines,4 and protein levels in human plasma5 represent only a few of the many published proteomic studies based on the use of protein microarrays. Still, the field of protein microarrays is far less mature compared to the field of DNA microarrays. Two striking features that separate the two disciplines are the ability to amplify and simultaneously label low concentrations of analyte nucleic acids, i.e., by PCR, and the possibility to produce highaffinity binding elements by predictions based on primary sequence alone, as for DNA oligonucleotides. A challenge for protein microarray work is thus to generate defined highaffinity capture reagents that can bind their cognate analytes with high selectivity. Technical aspects of protein microarrays are also important, such as the optimization of production * To whom correspondence should be addressed. Telephone: +46 (8) 5537 8333; Telefax: +46 (8) 5537 8481; E-mail: [email protected]. † Division of Molecular Biotechnology. ‡ Division of Proteomics. § Affibody AB. 10.1021/pr060316r CCC: $37.00

 2007 American Chemical Society

parameters6 and the development of signal enhancement systems that push the detection limits further down toward the dream of single molecule detection.4,7 To date, antibodies are the most prominent capture agents used in microarrays, and attempts to produce antibodies against all human proteins are currently ongoing .8 However, antibodies have some inherent drawbacks as capture agents in microarrays. Cross reactivity has been observed,9,10 and antibodies have been reported to lose activity when used in microarrays; for example, in studies by Haab et al. and Macbeath, only 20% of 11511 and 5% of 10012 evaluated antibodies, respectively, retained their specificity and sensitivity. Furthermore, the generation of monoclonal antibodies by hybridoma technology is both time consuming and expensive, and therefore, minimized antibody domains, like scFv13 or Fab fragments,14 and alternative scaffolds, like aptamers,15 and ankyrin repeats,16 have been considered as capture agents on protein microarrays. In vitro selection of novel binders by phage display,17 SELEX,18 or other systems19,20 and subsequent recombinant production or chemical synthesis in high yields are typical benefits of using these classes of affinity ligands. One such alternative class of affinity reagents consists of the Affibody molecules; 58 amino acid non-cysteine three-helix Journal of Proteome Research 2007, 6, 171-179

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research articles bundle proteins created by combinatorial protein engineering of the Z domain derived from staphylococcal protein A. Through randomization of 13 of the surface-exposed amino acid residues in helices one and two, libraries have been constructed from which Affibody molecules with specificity for a wide range of targets including human, viral, and bacterial proteins have been selected.21-25 Affibody molecules have been used in a variety of biotechnological applications based on molecular recognition,26-30 also including harsh basic regeneration conditions.31 Apart from recombinant production in high yield, monomeric Affibody molecules are also possible to produce by chemical synthesis,32 which allows for site-specific incorporation of affinity handles, unnatural amino acids, or nonproteinaceous organic groups. Such monomeric, synthetic Affibody molecules have been used for the evaluation of random and specific immobilization on surfaces, via amines, a C-terminal cysteine or a biotin moiety, on SPR (Biacore) sensor surfaces and on microarray slides.33 The aim of the present study was to further evaluate the use of Affibody molecules as capture ligands on protein microarrays. To investigate the importance of the ligand presentation format, the performances of Affibody molecules produced either as single domains or genetically linked multimers containing up to four identical binding domains were compared. The Affibody molecules were immobilized on microarray slides by directed or random immobilization and studied by incubation with fluorescent-labeled target proteins. Furthermore, different sandwich formats were evaluated for the detection of unlabeled analytes in complex samples, including human serum or plasma. Finally, the analysis of directly fluorescent-labeled serum samples was performed.

2. Materials and Methods Production and Purification of Affibody Molecules. For evaluation of the effect of multimerization, mono- di, and trimers of the Affibody ZIgA1 binding human IgA27 and mono-, di-, tri- and tetramers of the Affibody ZInsulinA binding insulin (M. L., unpublished) were used. These proteins are here denoted His6-ZIgA-Cys, His6-(ZIgA)2-Cys, and His6-(ZIgA)3-Cys; His6-ZIns-Cys, His6-(ZIns)2-Cys, His6-(ZIns)3-Cys, and His6-(ZIns)4Cys. For the other experiments, dimers of the Affibody Zwt binding human IgG,34 the Affibody ZIgE:7 binding human IgE (A. S., unpublished), the Affibody ZTNFR:185 binding tumor necrosis factor alpha (TNF-R) (A. J., unpublished), and the Affibody ZTaq4:5 binding Taq DNA polymerase35 were also used. These proteins are here denoted (ZIgG)2-Cys, His6-(ZIgE)2-Cys or (ZIgE)2-Cys, His6-(ZTNFR)2-Cys, and His6-(ZTaq)2-Cys, respectively. All proteins were produced in E. coli with a C-terminal cysteine residue for directed surface immobilization. Out of the 12 proteins, 10 were produced with an N-terminal hexahistidine tag for affinity purification. Target Protein Labeling. One tube of Cy3 (Cy3 Monoreactive Dye Pack, Amersham Biosciences, Buckinghamshire, UK) was dissolved in DMSO (Fluka Chemie, GmbH, Buchs, Switzerland) and divided into aliquots, which were dried under vacuum (Speedvac concentrator, Savant Instruments, USA) and stored airtight in the dark at 4 °C, with silica, until used. Monomeric IgA (100 µg, Bethyl Laboratories, Texas) was labeled with an amount of Cy3 corresponding to 1/10 of the original Cy3 tube in PBS (pH 7.4) for 30 min after which excess dye was removed by gel filtration on a protein desalting spin column (Pierce, Rockford, IL). Recombinant insulin (100 µg) 172

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(Roche Diagnostics, GmbH, Germany) was labeled with an amount of Cy3 corresponding to 1/10 of the original Cy3 tube in 100 mM Na2CO3 (pH 8.9) for 30 min after which excess dye was removed by gel filtration on a protein desalting spin column (Pierce). Human IgG (100 µg) obtained from Kabi Pharmacia (Stockholm, Sweden) was labeled with an amount of Cy3 corresponding to 4/10 of the original Cy3 tube in PBS (pH 7.4) for 1 h, and excess dye was removed with a NAP5 gel filtration column (Amersham Biosciences, Uppsala, Sweden). Taq DNA polymerase, produced as a fusion protein between two Z domains34 and Taq DNA polymerase,36 was labeled with an amount of Cy3 corresponding to 1/10 of the original Cy3 tube for 1 h, and excess dye was removed with a NAP5 gel filtration column. Recombinant tumor necrosis factor alpha (10 µg, TNF-R) (Nordic Biosite AB, Ta¨by, Sweden) and human IgE (10 µg), from monoclonal hybridoma, (Diatec.com, Oslo, Norway) were each labeled with an amount of Cy3 corresponding to 4/100 of the original Cy3 tube for 70 and 90 min, respectively, and excess dye was removed with a NAP5 gel filtration column. All labeled proteins were stored in 1 × PBS (pH 7.4) refrigerated in the dark until used. Antibody Biotinylation. The secondary antibodies directed against TNF-R (Abcam, ab9813) and human IgE (Fitzgerald Industries Inc, Mab M604199) were biotinylated. Anti-TNF-R antibody (20 µg, Abcam) and anti-IgE antibody (40 µg) (Fitzgerald) were biotinylated with a 50 times molar excess of EZ-Link Sulfo-NHS-LC-LC-Biotin (Pierce) in a total volume of 12.2 µL for TNF-R and 14.4. µL for IgE in 1 × PBS (pH 7.4) + 0.1% NaN3 for 45 min at room temperature prior to gel filtration on a protein desalting spin column (Pierce) to remove unreacted biotin. The antibody directed toward human IgA (SigmaAldrich Chemie, GmbH, Germany, I1010) was biotinylated upon purchase. Antibodies were stored at 4 °C in 1 × PBS (pH 7.4), and experiments were carried out within a month of biotinylation. Activation of Thiol Dextran-Coated Slides. Thiol dextran slides (XanTec Bioanalytics, Muenster, Germany) were washed 1-2 times with water for 10-30 min and treated with 100 mM DTT (Sigma-Aldrich) in 0.1 M Na2HPO4 pH (8.1) for 20 min to reduce disulfide bonds on the slide surface. After two quick water washes, the slides were activated by a 20 min treatment with 10 mM 2,2′-dipyridyl disulfide (Lancaster Synthesis, Morecambe, UK) in Na2PO4 (pH 8.1) containing 20% ethanol. After activation, slides were washed with water 3-5 times before being spun dry on a Minicentrifuge (Merck Eurolab, Stockholm, Sweden). Slides were spotted within 6 h. Activation of Carboxymethyl Dextran Slides. Carboxymethyl dextran slides (XanTec Bioanalytics) were washed 1-2 times with water for 10-30 min before addition of the activation mix consisting of 0.5 M N-hydroxysuccinimide (NHS) (Bachem, Bubendorf, Switzerland) and 1-ethyl-3-(3-dimethylaminopropyl)-carbodiimide (EDC) (Bachem) in 0.5 M 2-morpholinoethanesulfonate (MES) buffer (pH 5.0) (BDH Laboratory Supplies, Poole, UK). Slides were activated for 7-15 min before two washes in 2 mM HOAc and three quick water washes. Slides were spun dry and spotted within 6 h. Microarray Slide Spotting. Because all Affibody molecules were produced with a C-terminal cysteine residue, they were reduced in 30 mM of DTT (Sigma-Aldrich) in 1 × PBS pH 7.4 for 30 min. Unreacted DTT was removed, and the buffer was changed with NAP5 gel filtration columns (Amersham Biosciences) to 2 mM NaOAc (pH 4.5) for all Affibody molecules except His6-(ZIns)n-Cys (n ) 1-4) and His6-(ZIgE)2-Cys, for which

Affibody Molecules in Protein Capture Microarrays

the buffers were changed to 1 × PBS pH 7.4, as they were found to precipitate in the NaOAc buffer. All proteins were spotted from 1 mg/mL solutions onto activated thiol dextran- or carboxymethyl dextran-coated slides with a GMS 417 arrayer (Affymetrix, Santa Clara, CA) using 4 hits/spot in triplicates, with buffer spotted after each protein to verify that there was no carry-over, in 16 arrays per slide. Microarray slides were incubated in 70-80% humidity over night and subsequently blocked in Superblock Dry Blend (Boule Nordic, Huddinge, Sweden) for 3-4 h at room temperature before being spun dry and stored airtight with silica gel at 4 °C until used (within 12 months from spotting). Estimation of the Amount of Immobilized Affibody Molecules on the Spotted Microarrays. To verify that equal amounts of multimeric Affibody molecules of the same target specificity were immobilized, the Affibody microarray slides were treated with SYPRO Ruby Protein Blot Stain (Molecular Probes, Leiden, Netherlands) according to the manufacturer’s instructions for western blot analysis. The slides were scanned, and the spot intensities were compared. Because SYPRO Ruby stains Affibody molecules differently depending on the primary sequence of the Affibody molecule, the array staining of Affibody dimers were compared to SYPRO Ruby stained Phast gels (Amersham Biosciences), loaded with equal amounts of the different Affibody dimers. The amount of immobilized dimers was also evaluated with a goat anti-Affibody antibody (a kind gift from Affibody AB). Two arrays containing the six different dimers were incubated with 90 µL of goat anti-Affibody antibody at a final concentration of 1 µg/mL in 1 × PBS (pH 7.4) + 0.25% casein (SigmaAldrich) for 1 h with agitation at room temperature. Arrays were washed in a mask 5-10 times in 2 × PBS (pH 7.4) for a total of 30 min and were incubated with 90 µL of a 1:1000 dilution of anti-goat IgG (whole molecule) (R)-Phycoerythrin-conjugated antibody (Sigma-Aldrich, P9787) in 1 × PBS (pH 7.4) + 0.25% casein for 1 h with agitation at room temperature. The arrays were washed 3-5 times in 2 × PBS (pH 7.4), the silicone mask was removed from the slide with the mask holder submerged in 2 × PBS (pH 7.4), and the slide was washed 3-4 times 5 min each in 2 × PBS (pH 7.4) and spun dry on a Minicentrifuge. Slides were scanned as described below. General Procedure for Labeled Target Protein Incubation. The microarray slides were incubated with 90 µL of Cy3-lableled protein in 1 × PBS + 0.25% casein from bovine milk (SigmaAldrich) over night at room temperature. A 96-well silicone mask with an in-house fabricated slide holder, using four screws to hold the mask in place, was used to separate the individual arrays and prevent leakage. After over night incubation with agitation, arrays were washed 3-5 times in 2 × PBS (pH 7.4), the silicone mask was removed from the slide with the mask holder submerged in 2 × PBS (pH 7.4), the slide was washed 3-4 times for 5 min in 2 × PBS (pH 7.4), and spun dry on a Minicentrifuge. Slides were scanned as described below. For the incubation with Cy3-labeled target protein on mono-, di-, tri-, and tetrameric Affibody microarray slides, thiol dextran slides with microarrays printed of the three multidomain Affibody molecules binding human IgA: His6-ZIgA-Cys, His6(ZIgA)2-Cys, and His6-(ZIgA)3-Cys and the four multidomain Affibody molecules binding insulin: His6-ZIns-Cys, His6-(ZIns)2Cys, His6-(ZIns)3-Cys, and His6-(ZIns)4-Cys were incubated with Cy3-labeled human IgA at a final concentration of 0.1-100 ng/ mL (600 fM-600 pM) or Cy3-labeled insulin at a final concentration of 0.1-100 ng/mL (20 pM-20 nM), as described above.

research articles For the incubation with Cy3-labeled target protein on dimeric Affibody microarray slides, thiol dextran slides with microarrays printed of the six Affibody dimers binding human IgG, human IgA, human IgE, insulin, TNF-R, and Taq DNA polymerase: (ZIgG)2-Cys, His6-(ZIgA)2-Cys, His6-(ZIgE)2-Cys, or (ZIgE)2-Cys, His6-(ZIns)2-Cys, His6-(ZTNFR)2-Cys, and His6-(ZTaq)2Cys, respectively, were incubated with Cy3-IgA at a final concentration of 33 pg/mL-10 ng/mL (200 fM-60 pM); Cy3insulin at a final concentration of 0.1-10 ng/mL (20 pM-2 nM); Cy3-IgG at a final concentration of 3.3 pg/mL-1 ng/mL (20 fM-7 pM), Cy3-IgE at a final concentration of 0.33-100 ng/ mL (500 fM-500 pM); Cy3-Taq DNA polymerase at a final concentration of 0.1-33 ng/mL (900 fM-300 pM); or Cy3TNF-R at a final concentration of 100 pg/mL-10 ng/mL (6600 pM), as described above. Sandwich Detection of Unlabeled Target Protein on Dimeric Affibody Microarray Slides. Carboxymethyl dextran slides with microarrays printed of the four Affibody dimers binding IgA, IgE, Taq DNA polymerase, and TNF-R: His6-(ZIgA)2-Cys, His6-(ZIgE)2-Cys, His6-(ZTaq)2-Cys, and His6-(ZTNF-R)2-Cys, respectively, were incubated with 90 µL of IgA at a final concentration of 33 pg/mL-10 ng/mL (200 fM-60 pM), or 90 µL of TNF-R at a final concentration of 3.3 pg/mL-3.3 ng/mL (200 fM-200 pM), in 1 × PBS + 0.25% casein over night at room temperature. Arrays were separated with the silicone mask described above. After over night incubation with agitation, arrays were washed in the mask 5-10 times in 2 × PBS (pH 7.4) for a total of 30 min. The arrays were then incubated with 90 µL of target-specific, biotinylated antibody: mouse antiTNF-R or goat anti-IgA, at a final concentration of 1 µg/mL in 1 × PBS + 0.25% casein. After 1 h of incubation with agitation at room temperature, arrays were washed in the mask 5-10 times in 2 × PBS (pH 7.4) for a total of 30 min, and incubated with 90 µL of Alexa Fluor 555-labeled goat anti-mouse antibody (Molecular Probes) at a final concentration of 1 µg/mL for TNF-R and 90 µL of of Cy3-Streptavidin (Sigma-Aldrich, S6402) at a final concentration of 1 µg/mL for IgA, in 1 × PBS + 0.25% casein. In a separate set of experiments, the arrays were incubated with 90 µL of Streptavidin: Sensilight PBXL-1 (Martek Biosiences, Columbia) at a final concentration of 0.150 µg/mL in 1 × PBS + 0.25% casein instead of the anti-mouse antibody or streptavidin. After 1 h of incubation with agitation at room temperature in the dark, arrays were washed in the mask 5-10 times in 2 × PBS (pH 7.4) for a total of 30 min, the silicone mask was removed from the slide with the mask holder submerged in 2 × PBS (pH 7.4), the slide was washed 3 × 2 min in 2 × PBS (pH 7.4), and spun dry on a Minicentrifuge. Slides were then scanned as described below. Sandwich Detection of TNF-R in Human Plasma. The experiments were performed as for the sandwich detection of pure analyte described above but with TNF-R at a final concentration of 330 pg/mL-100 ng/mL (20 pM-6 nM) in 1:100 diluted human plasma (obtained from Blodcentralen, Akademiska sjukhuset, Uppsala, Sweden) in 1 × PBS + Blocking Reagent for ELISA (Roche) and 0.05% casein, and with incubation for 1 h at room temperature instead of overnight. Detection of IgA in Normal Human Serum and in IgADeficient Serum Using Direct Labeling or Sandwich Detection. Thiol dextran slides with microarrays printed of the two Affibody dimers binding IgA and IgG: His6-(ZIgA)2-Cys and His6(ZIgG)2-Cys, respectively, were incubated with Cy5-labeled or unlabeled normal or IgA-deficient human serum37 for direct or sandwich detection of IgA, respectively. For the direct Journal of Proteome Research • Vol. 6, No. 1, 2007 173

research articles detection, normal and IgA-deficient human serum samples were buffer exchanged with Zeba spin columns (Pierce) to 0.1 M Na2CO3 pH 8.4 + 0.5 M NaCl and labeled with Cy5-dye on ice for 2 h according to manufacturer’s recommendations. Extensive dialysis against 1 × PBS pH 7.2 using Slide-A-Lyzer Dialysis Casettes (Pierce) was performed at 4 °C in the dark to remove unreacted dye. The labeled serum samples were stored at 4 °C until used. Labeled or unlabeled serum samples diluted to 1:1500 in 1 × PBS pH 7.2 were preincubated with a final concentration of 0.25% Polyvinylalcohol (Sigma) + 0.4% polyvinylpyrrolidone + 0.1% casein for 1 h at RT with occasional mixing. In parallel, microarrays were pre-blocked with 1 × PBS + 0.05% Tween 20 (PBST) 4 × 5 min, after which the preincubated samples (60 µL) were added to the arrays and incubated with agitation for 30 min at 4 °C in the dark, and again washed 4 × 5 min in PBST. For the direct-labeling experiments, the mask was removed and the slide was washed quickly in PBST before being blown dry with compressed air and scanned as described below. For the sandwich experiments, 60 µL of biotinylated goat anti-IgA antibody at a final concentration of 1 µg/mL in 1 × PBS + Blocking Reagent for ELISA (Roche) was incubated on the arrays for 45 min, washed 4 × 5 min with PBST, subsequently incubated with 60 µL 1 µg/mL Cy3-streptavidin 1 × PBS + Blocking Reagent for ELISA (Roche) for 30 min at 4 °C, and washed again 4 × 5 min PBST. The mask was removed and the slide was quickly washed in PBST before being blown dry with compressed air and scanned as described below. Microarray Slide Scanning and Evaluation. Slides were scanned with an Agilent (Agilent Technologies, Paramus, NJ) scanner using the green channel, except for the Cy5-labeled serum samples, which used the red channel. The 16-bit output TIFF images were imported into GenePix 5.1 (Axon Instruments, Union City, CA). For scanner signals above saturation (at a relative fluorescence intensity of 65,000), the fluorescence sensitivity setting was dimmed to 1/10 and the recorded values were subsequently multiplied by a factor of 10. The median local background was subtracted from the protein spot signals to give the relative fluorescent signal, and a mean was calculated from the triplicates of each concentration and protein. A new mean with interslide standard deviation was calculated from the two triplicates of two independent slides, except for the analysis of TNF-R in plasma in which the two triplicates were taken from separate arrays on the same slide and for the IgA normal and deficient serum samples, in which the median was calculated from the triplicate of one array. If one of the spots in a triplicate was damaged or missing, the mean of the two remaining spots was calculated instead. No leakage or cross-contamination was observed for any of the arrays, and all spots had similar shape and size, with a local decrease in intensity where the tip of the arrayer touched the surface during spotting. Signals were similar among array triplicates, with signal means in arrays made by different pins and on arrays on different slides contributing to the majority of the variation observed in the dilution series. The limit of detection was defined as the lowest concentration of target protein where the signal from the target-specific Affibody molecule was higher than the mean blank (the target-specific Affibody molecule incubated with buffer only) plus two standard deviations (95% confidence interval). For the plasma experiments with TNF-R, the blank was human plasma without added TNF-R. 174


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Figure 1. Schematic picture of mono- (A), di- (B), tri- (C), and tetrameric (D) Affibody molecules immobilized on a dextran matrix in an equal number of domains per spot. The multimeric Affibody molecules were used to investigate how binding ability is influenced by the degree of multimerization.

3. Results In the present study, multimeric Affibody molecules containing up to four identical domains were evaluated as capture agents in different protein microarray settings, using direct detection of fluorescent-labeled analytes or sandwich assays involving a secondary detection reagent. The different Affibody molecules were produced in E. coli as genetically linked headto-tail proteins, all containing a unique C-terminal cysteine residue, which allowed for the use of directed thiol-based immobilization chemistry, in addition to non-directed amine coupling. Detection of Labeled Target on Multimeric Affibody Protein Capture Microarray Slides. In a first set of experiments, Affibody mono-, di-, tri-, or tetramers were immobilized covalently via a unique C-terminal cysteine onto activated thiol dextran slides and analyte binding was evaluated by incubation with fluorescent-labeled target proteins. To allow for a direct evaluation of the different multimers, efforts were made to immobilize equal total numbers of Affibody domains, rather than equal numbers of Affibody molecules (Figure 1). The Affibody molecules were therefore spotted from solutions of the same concentrations (1 mg/mL) to yield the same amounts of protein on the chip surface. To verify this, the slides were stained with SYPRO Ruby Protein Blot Stain, a general protein stain, followed by scanning. The intensities of the spotted monomers or multimers of the same target specificity were approximately equal, and this indicates that the same amount of protein was immobilized. This suggests that the same numbers of domains were present for the different ligands of the same target specificity and that a comparison of analyte binding ability for the different Affibody domain presentation formats was possible. Furthermore, the comparison between the different multimers was performed with proteins spotted on the same microarray slides, to exclude possible effects caused by slide-to-slide variation due to age differences or other defects. In the experiment, Affibody molecules directed against IgA (one, two, or three domains) or insulin (one, two, three, or four domains) were used (Figure 2). The slides were incubated with dilution series of Cy3-labeled insulin (100 pg/mL-100 ng/mL) or IgA (100 pg/mL-100 ng/mL), and from triplicate spots on two slides, the means and standard deviations were calculated and plotted. All Affibody molecules bound specifically to their respective target analyte, with no cross-reactivity detected. Interestingly, the use of multimers resulted in increased signal intensities compared to the monomers, and the difference was


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Figure 2. (Left) Multimeric Affibody array of triplicates of mono- to trimeric ZIgA and mono- to tetrameric ZIns incubated with (A) 100 ng/mL Cy3-labeled human IgA or (B) 100 ng/mL Cy3-labeled human insulin. Each row of Affibody spots is separated by a row of buffer spots. (Right) Relative fluorescence intensities after incubation with dilution series of Cy3-labeled human IgA (100 pg/mL-100 ng/mL) and insulin (100 pg/mL-100 ng/mL) over multivalent Affibody molecules, containing one to four domains. The mean of three dilution series on triplicate spots on three slides is plotted with error bars of one standard deviation.

most pronounced between the monomers and the dimers. The signal intensities for the trimers were higher than for the corresponding dimers, but the use of a tetramer (for the insulinbinding Affibody molecule) did not increase the signal intensity further. These experiments were also performed on carboxymethyl dextran slides with similar results (data not shown). The results prompted us to further investigate the performance of multimeric Affibody molecules as capture agents in more complex microarray settings, in terms of sensitivity and cross-reactivity. For this experiment, six Affibody dimers directed against human IgG, human IgA, human IgE, insulin, TNF-R or Taq DNA polymerase, were covalently immobilized on thiol dextran slides and incubated with dilution series of Cy3-labeled target proteins (Figure 3). Also for these arrays, precautions were made to ensure that equal amounts of ligands (all dimeric) were immobilized. However, SYPRO Ruby staining intensities were slightly varying, and as a control, an SDS-PAGE gel loaded with equal amounts of the six dimers was also stained with SYPRO Ruby (data not shown). The signal intensities of the individual proteins on the stained gel and the microarray slide correlated well, and it was concluded that staining differences of the different proteins made up most of the variation observed for the stained arrays. Furthermore, a polyclonal goat anti-Affibody antibody was incubated on two arrays and detected with a secondary (R)-Phycoerythrin conjugated anti-goat antibody. This analysis confirmed that approximately equal amounts of all the Affibody dimers were immobilized, except for His6-(ZIgE)2-Cys and His6-(ZIns)2-Cys, which were immobilized at slightly lower levels. It should be noted that both of these Affibody molecules were printed in 1 × PBS pH 7.4 instead of 2 mM NaOAc pH 4.5, and the

difference in buffer seems to have led to less efficient immobilization. After analyte incubation and washing, a high selectivity in binding was observed for all included Affibody molecules, as only their specific target analytes were bound, with little or no cross-reactivity detected for the other proteins. The observed limits of detection (where the mean signal from dilution series on separate slides was higher than the buffer blank mean plus two standard deviations) were: 10 pg/mL (70 fM) for IgG, 100 pg/mL (600 fM) for IgA, 3.3 ng/mL (20 pM) for IgE, 330 pg/mL (60 pM) for insulin, 1 ng/mL (10 pM) for Taq DNA polymerase, and 330 pg/mL (20 pM) for TNF-R (Table 1). For IgA and Taq DNA polymerase, this was an improvement compared to the limits of detection observed for the microarrays produced with synthetic monomeric Affibody molecules in an earlier study, which were 3 pM for IgA and 30 pM for Taq DNA polymerase.33 Sandwich Detection of Unlabeled Target on Dimeric Affibody Protein Capture Microarray Slides. To investigate the performance of Affibody dimers as capture agents in the analysis of unlabeled analytes in samples of complex composition, different sandwich systems were tested. Here, Affibody molecules were used as capture agents and antibodies were used as secondary detection reagents. Four Affibody dimers directed against IgA, IgE, Taq DNA polymerase, and TNF-R were covalently immobilized on carboxymethyl dextran slides and incubated with dilution series of unlabeled IgA and TNFR, followed by incubation with a target-specific, biotinylated antibody and detected with a fluorescent-labeled antibody, for TNF-R, or Cy3-streptavidin, for IgA (Figure 4). The observed limit of detection (where the mean signal from dilution series on separate slides was higher than the buffer blank measureJournal of Proteome Research • Vol. 6, No. 1, 2007 175

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Figure 3. Relative fluorescence intensities after incubation with dilution series of Cy3-labeled human IgA (33 pg/mL-10 ng/mL), TNF-R (100 pg/mL-10 ng/mL), IgG (3.3 pg/mL-1 ng/mL), insulin (100 pg/mL-10 ng/mL), IgE (330 pg/mL-100 ng/mL), and Taq DNA polymerase (100 pg/mL-33 ng/mL) over arrays of dimeric Affibody molecules. The mean of two dilution series on triplicate spots on two slides is plotted with error bars of one standard deviation. Table 1. Limit of Detection of Fluorescently Labeled Target Proteins (Direct Detection) and Unlabeled Target Proteins in Pair with Antibodies (Sandwich Detection) target protein

direct detection

sandwich detection

IgG IgA IgE Insulin Taq DNA polymerase TNF-R

10 pg/mL (70 fM) 100 pg/mL (600 fM) 3.3 ng/mL (20 pM) 330 pg/mL (60 pM) 1 ng/mL (10 pM) 330 pg/mL (20 pM)

330 pg/mL (2 pM) 33 pg/mL (2 pM)

ment plus two standard deviations) was 33 pg/mL (2 pM) for detection of TNF-R in the sandwich format, which is lower than for the detection of directly fluorescent-labeled TNF-R (330 pg/ mL (20 pM)) (Table 1). For IgA, the limit of detection in the sandwich format was 330 pg/mL (2 pM) compared to 100 pg/ mL (600 fM) for directly fluorescent-labeled IgA. The biotinylated anti-TNF-R antibody was also evaluated for detection of TNF-R in combination with Cy3-streptavidin (data not shown), however, the combination with the anti-mouse antibody proved more sensitive. Similarly, the biotinylated antiIgA antibody was also evaluated for detection of IgA in combination with an anti-goat antibody labeled with R176


phycoerythrin, but this was not as sensitive as the combination with Cy3-labeled streptavidin (data not shown). A streptavidinphycobiliprotein conjugate (Sensilight PBXL-1), which contains supramolecular complexes of many fluorescent phycobiliproteins, was also evaluated instead of the other detection reagents, but, in our hands, was found not to be as sensitive as the anti-mouse antibody or Cy3-streptavidin (data not shown). Detection of Analytes in Human Serum or Plasma. Human serum and plasma represent complex protein mixtures of great interest for proteomics studies. To test the analysis of proteins in this type of samples, unlabeled TNF-R was spiked into human plasma followed by incubation on an Affibody capture array slide containing the four dimeric Affibody molecules directed against IgA, IgE, Taq DNA polymerase and TNF-R. Bound target molecules were detected in a sandwich format with the antibody directed against TNF-R, followed by the fluorescent-labeled goat anti-mouse antibody. A doseresponse curve was plotted (Figure 5) and the limit of detection for TNF-R in 1:100 diluted human plasma was 3.3 ng/mL (200 pM). The addition of blocking reagents to the human plasma was found crucial for decreasing the nonspecific signal to the

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Figure 4. Relative fluorescence intensities after incubation with dilution series of unlabeled IgA (33 pg/mL-10 ng/mL) and TNF-R (3.3 pg/mL-3.3 ng/mL) followed by the addition of biotinylated anti-IgA or anti-TNF-R target-specific antibodies and Cy3-labeled streptavidin for detection of IgA or an Alexa Fluor 555-labeled antibody for detection of TNF-R, over dimeric Affibody molecules. The mean of two dilution series on triplicate spots is plotted with error bars of one standard deviation.

4. Discussion

Figure 5. Relative fluorescence intensities after incubation with a dilution series of unlabeled TNF-R (330 pg/mL-100 ng/mL) in a background of 1:100 diluted human plasma, followed by an anti-TNF-R target-specific antibody and an Alexa Fluor 555labeled antibody, over dimeric Affibody molecules. The mean of two dilution series on triplicate spots is plotted with error bars of one standard deviation.

surface and producing measurable spots. This far, Superblock Dry Blend (Pierce) and Blocking Reagent for ELISA (Roche) produced the highest signals-to-background (data not shown). However, blocking was likely not optimal since the limit of detection had increased 2 orders of magnitude for the TNF-R detection in plasma compared to the detection of pure analyte. Further, unlabeled normal or IgA-deficient human serum was incubated on Affibody microarray slides containing the Affibody dimers directed against IgA and IgG, respectively. Bound analytes were detected with a biotinylated antibody directed against IgA, followed by Cy3-streptavidin. In the normal human serum containing IgA, a 100-fold stronger signal intensity was observed compared to the value obtained for the IgA-deficient serum, where the signal intensity was approximately equal to the background (Figure 6). For the directly Cy5labeled serum samples, IgA was detected only in the normal serum sample, not in the IgA-deficient serum samples. Also, for all three samples, IgG was detected at similar levels. For the directly labeled serum samples, extensive dialysis of the samples after labeling and preincubation with PVX were found to be very important for further reduction of the nonspecific binding to the slide surface and, hence, lower background. PVX treatment gave much better results compared to using Superblock (Pierce) and Blocking Reagent for ELISA (Roche), which had produced the highest signal-to-background for the earlier experiments.

In this study, head-to-tail multimers of Affibody molecules were investigated for use as randomly or site-specifically immobilized capture agents in protein microarray experiments. As shown for two Affibody molecules with different target specificities, the use of multimers significantly increased the obtained signal intensities, with the most pronounced difference observed between monomers and dimers, although inclusion of additional domains further increased the signals. The possible reasons for the observed advantageous effects from multimerization could be several and include parameters related to the immobilization procedure, solution accessibility, local ligand density and analyte characteristics. An effect from an improved accessibility to the outermost domains in the multimers, mediated by the inner domains via a spacer effect could be expected. This notion is supported from earlier work on monomeric Affibody capture agents, where a positive effect was seen from the introduction of a chemical linker between the array anchor point and the monomeric Affibody molecule.33 However, in the present study, no significant difference was observed between the results from the experiments performed on thiol dextran slides, where the Affibody molecules are sitespecifically immobilized through a C-terminal cysteine residue, and those performed on carboxymethyl dextran slides, where the Affibody molecules are randomly coupled through amino groups, which could have been expected if the spacer effect was the major contribution to the effect of multimerization. Alternatively, a multimeric ligand should confer a higher local concentration of analyte binding sites, promoting a local reassociation of analytes dissociating from its primary ligand binding site to a physically linked proximal domain. Depending on the target analyte quaternary structure (monomer, homodimer, homotrimer, etc.), co-operative binding (avidity) effects could have influence on the analyte-ligand interaction. A situation involving simultaneous binding to more than one of the binding domains in a single multimeric capture agent could possibly be envisioned for target proteins containing multiple binding epitopes, such as the symmetrical IgA molecule. Such avidity effects involving two separate neighboring ligands, rather than separate domains within a single ligand molecule should also be promoted by domain multimerization, owing an increased likelihood for the fulfillment of sterical constraints related to multiple-site analyte binding by immobilized ligands. Obviously, the reason for the observed beneficial effects from multimerization could also include combinations of the factors discussed above, and possibly be Journal of Proteome Research • Vol. 6, No. 1, 2007 177

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Figure 6. (Left column, Cy5-labeled serum) (Top) GenePix image of the signal intensities of Affibody spots after incubation with Cy5-labeled 1:1500 diluted normal serum (serum no 1) and two IgA-deficient serum samples (serum no. 2 and serum no. 3). (Bottom) The mean fluorescence intensities of the (ZIgA)2 and (ZIgG)2 triplicate spots shown above are plotted with standard bars of one standard deviation. (Right column, IgA sandwich detection) (Top) GenePix image of the signal intensities of Affibody spots after incubation with unlabeled 1:1500 diluted normal serum (serum no 1) and two IgA-deficient serum samples (serum no. 2 and serum no. 3). Detection was performed with biotinylated anti-IgA antibody and Cy3-streptavidin. (Bottom) The mean fluorescence intensities of the (ZIgA)2 and (ZIgG)2 triplicate spots shown above are plotted with standard bars of one standard deviation. All experiments were performed on the same slide and all spots were excised from the same array for each of the individual sera.

different for different target proteins. However, regardless of the underlying mechanism, the Affibody molecules evaluated in the study were more suitable for use as capture agents in protein microarrays as multimers than as monomers, and for the following experiments dimeric Affibody molecules were therefore employed. Detection assays using a sandwich format, where an unlabeled analyte is captured by a primary affinity reagent and detected by a secondary affinity reagent, are widely used methods for protein detection. The sandwich assay benefits from two recognition events, which can greatly increase the specificity of detection, and this feature is of particular importance for the analysis of proteins in complex sample mixtures. 178


Renberg et al.

Also, sandwich detection requires no labeling of the sample to be analyzed, which is often associated with a reduced binding activity, as the attachment of fluorophores on or in the proximity of the binding surface can disturb binding as well as folding of the labeled protein. For example, IgA and insulin were severely affected by Cy3 labeling in the present study. When studied by biosensor analysis, only approximately 40 and 20%, respectively, of the binding activities of IgA and insulin to ZIgA and ZIns were retained (data not shown). Furthermore, fluorescent labeling of proteins is likely to be highly variable, especially if a complex mixture such as human plasma or serum is labeled. In an earlier study by Andersson et al.,29 the advantage of using capture and detecting agents of different origins to reduce background were shown. Here, two dimeric Affibody molecules specific for TNF-R and IgA, respectively, were used as capture agents for sandwich detection in protein microarrays. Detection was performed with a biotinylated target-specific antibody, in combination with a fluorescentlabeled antibody or streptavidin. Again, good target specificity with no unspecific binding was observed, and the limit of detection was lowered ten times for TNF-R to 33 pg/mL (2 pM), compared to the use of a directly labeled analyte. A possible explanation for this effect is that the detector antibodies exhibit a higher fluorescence than directly labeled TNF-R, due to the incorporation of more fluorescent molecules. Another explanation is that for the sandwich assay no labeling of the target molecules, which is potentially destructive, has been performed. However, the limit of detection for IgA was slightly higher (0.33 ng/mL, 2 pM) compared to directly labeled IgA (0.1 ng/mL, 600 fM). The higher limit of detection for the sandwich detection could be explained by the relatively high background signal of the polyclonal antibody that was observed in the experiments, or possibly that this particular Affibody molecule and a fraction of the polyclonal detection antibody reagent have partly overlapping binding sites and thus compete for binding to IgA. It has previously been reported that signal amplification steps, for instance rolling circle amplification, can be used to increase the sensitivity of sandwich detection on microarrays.4 In the present study, an attempt to improve the sensitivity was made by using a streptavidin-fused phycoerithrin light harvesting complex, Sensilight PBXL-1, instead of the Alexa 555-labeled antibody or Cy3-labeled streptavidin, but the limit of detection or signal intensities were not improved for either IgA or TNFR. To challenge the selectivity of multimeric Affibody proteinbased microarrays, human plasma and serum, representing two highly complex and important biological samples for diagnostics and proteomics, were included for detection of TNF-R and IgA. A sandwich system could successfully be used for the detection of human TNF-R spiked into normal human plasma. Furthermore, normal human serum and a patient-derived IgAdeficient serum,37 respectively, were incubated on arrays and IgA was detected by the sandwich detection system. For the IgA-containing normal serum, the signal intensity was 100-fold stronger than that of the IgA-deficient serum, showing that this clinically relevant distinction could be clearly resolved. Provided that sufficient sensitivity and specificity can be obtained, direct fluorescent labeling and detection of complex samples is an attractive alternative to sandwich detection, as the need for a secondary affinity reagents is circumvented. Here, direct labeling of the normal human serum and IgAdeficient serum samples with Cy5 dye was performed and

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analyzed on microarrays with dimeric Affibody molecules. IgA was clearly detected in the normal IgA-containing serum samples, while no IgA was detected in the two IgA-deficient serum samples. Further, as expected, IgG was present in all serum samples and the signals were approximately equal, indicating that the labeling of the serum samples was reproducible. Thus, these experiments showed that multimeric Affibody molecules have high specificity for their respective targets and can be used as ligands for affinity capture in analyses of complex samples, such as human plasma or serum. Abbreviations: IgA, immunoglobulin A; IgE, immunoglobulin E; IgG, immunoglobulin G; TNF-R, tumor necrosis factor alpha; scFv, single chain variable fragment; Fab, fragment antigen binding; SPR, surface plasmon resonance; NHS, N-hydroxysuccinimide; EDC, 1-ethyl-3-(3-dimethylaminopropyl)-carbodiimide; MES, 2-morpholinoethanesulfonate; PVX, polyvinylalcohol/polyvinylpyrrolidone.

Acknowledgment. This study was supported by the EU project MolPAGE (Molecular Phenotyping to Accelerate Genomic Epidemiology). We thank Anna Sjo¨berg, Barbro Baastrup, Andreas Jonsson, and Malin Lindberg at Affibody AB for selection, cloning, expression and characterization of Affibody molecules. We are also grateful to Ing-Marie HoïdeńGuthenberg at Affibody AB for contributions to the sandwich assays, to Jochen Schwenk at KTH for valuable discussions on the analysis of plasma samples and blocking solutions, to Lennart Hammarstro¨m at Karolinska Institute for providing the normal and IgA-deficient human serum samples, and to Fredrik Ponteń at Uppsala University for providing the human plasma. Trademark: The phrase “affibody molecule” is used in this publication instead of “Affibody molecule”. Affibody is a trademark owned by Affibody AB. Affibody is a trademark registered in Sweden, EU, U.S.A., and under trademark application in Japan.

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