Detection of Antigens Using a Protein–DNA Chimera Developed by

May 8, 2012 - This indicates the generality of the A*-Zmab-based immuno-RCA assay that can be used in-sandwich ELISA format. Moreover, the enzymatic c...
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Detection of Antigens Using a Protein−DNA Chimera Developed by Enzymatic Covalent Bonding with phiX Gene A* Farhima Akter,† Masayasu Mie,† Sebastian Grimm,‡,§ Per-Åke Nygren,‡ and Eiry Kobatake*,† †

Department of Biological Information, Graduate School of Bioscience and Biotechnology, Tokyo Institute of Technology, 4259 Nagatsuta, Midori-ku, Yokohama 226-8501, Japan ‡ Division of Molecular Biotechnology, School of Biotechnology, Royal Institute of Technology (KTH), Roslagstullsbacken 21, 106 91 Stockholm, Sweden ABSTRACT: The chemical reactions used to make antibody−DNA conjugates in many immunoassays diminish antigen-binding activity and yield heterogeneous products. Here, we address these issues by developing an antibody-based rolling circle amplification (RCA) strategy using a fusion of φX174 gene A* protein and Zmab25 (A*-Zmab). The φX174 gene A* protein is an enzyme that can covalently link with DNA, while the Zmab25 protein moiety can bind to specific species of antibodies. The DNA in an A*-Zmab conjugate was attached to the A* protein at a site chosen to not interfere with protein function, as determined by enzyme-linked immunosorbent assay (ELISA) and gel mobility shift analysis. The novel A*Zmab-DNA conjugate retained its binding capabilities to a specific class of murine immunoglobulin γ1 (IgG1) but not to rabbit IgG. This indicates the generality of the A*-Zmab-based immuno-RCA assay that can be used in-sandwich ELISA format. Moreover, the enzymatic covalent method dramatically increased the yields of A*-Zmab-DNA conjugates up to 80% after a 15 min reaction. Finally, sensitive detection of human interferon-γ (IFN-γ) was achieved by immuno-RCA using our fusion protein in sandwich ELISA format. This new approach of the use of site-specific enzymatic DNA conjugation to proteins should be applicable to fabrication of novel immunoassays for biosensing.

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and yields heterogeneous products due to the uncontrollable and unpredictable attachment of DNA.12,14 To overcome common shortcomings of chemical conjugation or stv−biotin bridges, the present study describes a novel strategy for constructing protein−DNA conjugates by enzymatic means using a a fusion of φX174 gene A* protein and Zmab25 (A*-Zmab) fusion protein. The φX174 gene A* protein of coliphage φX174 can cleave the negatively supercoiled form of double-stranded φX174 DNA and become covalently bound to the 5′-phosphoryl group of the cleavage site via a phosphotyrosine link, forming a covalent protein− DNA complex.19 Therefore, one can imagine that A* could be used to construct fusion proteins for site-specific, covalent conjugation of DNA. However, no reports have yet described the preparation of protein−DNA conjugates by applying the catalytic activity of A*. On the other hand, the Zmab is an affibody molecule20 and has been isolated by ribosome display based on a small 58 amino acids three-helix bundle protein framework derived from staphylococcal protein A.21 In contrast to the widely used natural immunoglobulin (Ig) binding proteins (staphylococcal protein A or G), Zmab25 has very narrow binding spectra involving only murine immunoglobulin

uch effort has been devoted to the development of analytical techniques for ultrasensitive detection of protein biomarkers. Most of these techniques have relied on antibodies conjugated to enzymes (enzyme-linked immunosorbent assays, ELISA),1 isotopes (radioimmunoassay, RIA),2 or dyes (immunosensors).3 Although these techniques are powerful and accessible, later works revealed that greater sensitivity and specificity may be achieved using immunopolymerase chain reaction (IPCR)4−7 or immuno-rolling circle reaction (IRCA).8,9 Current research focuses on continuing to improve the performance and quality of these immunoassays.10−16 Common IPCR or IRCA formats may be derived from one or more general concepts such as the use of homotetrameric proteins (strept)avidin (stv) as a bridge to connect the biotinylated DNA template and the detection antibody. Although the stv−biotin complex has a high affinity, the tetrameric nature of stv makes it difficult to control the stoichiometry of the respective DNA−protein conjugates.6,17 In addition, steric obstacles due to the bulky nature of the stv connector protein and the requirement for multiple incubation, assembly, and wash steps reduce the sensitivity of IPCR.15 It has also been reported that the biotinylation of antibodies can affect their antigen binding affinity.18 An alternative strategy for constructing antibody−DNA conjugates is direct covalent linkage. However, the chemical means used to make antibody−DNA conjugates often weakens antigen binding activity © XXXX American Chemical Society

Received: March 13, 2012 Accepted: May 8, 2012

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γ1 (IgG1).21 Thus, an A*-Zmab construct has been used as a linker between antigen and primer DNA for rolling circle amplification (RCA)-based signal generation in a sandwich ELISA format (Figure 1). The sequence-specific covalent

QuikChange II Site-Directed Mutagenesis Kits (Agilent Technologies, Japan) because the mutation is necessary for overexpression of the A* protein.19 The plasmid that contains Y303H mutation was designated as pBS-A*-Y303H. The gene for phiX174 A* was amplified by polymerase chain reaction (PCR) from pBS-phiX A* Y303H using a pair of primer containing EcoRI and BamHI restriction sites, which were incorporated to facilitate the cloning of the segment in between the same restriction sites in pBluescript vector. The Zmab25 gene was also amplified from the plasmid pAY442-Zmab25 (constructed previously) with a set of primer containing BamHI and NotI and digested with the same enzyme and was inserted into the pBS- phiX A* Y303H vector; the resulting plasmid was named pBS-A*-Zmab. A*-Zmab fragment was digested from the pBluescript and inserted into the expression vector pET28 frame at EcoRI- NotI; the N-terminus of the plasmid carried His-Tag for efficient purification. Expression and Purification of Fusion Protein. The constructed pET28-NHis-A*-Zmab plasmid was transfected into E. coli BL21 (DE3) competent cells by heat shock. Transformed E. coli cells were added to Luria−Bertani (LB) medium in the presence of 50 μg/mL kanamycin and 0.5% glucose at 37 °C to an apparent optical density of 0.5 at 600 nm. Expression of A*-Zmab fusion protein was induced by the addition of 500 μM isopropyl-β-D-thiogalactopyranoside (IPTG), and the growth of the culture was continued overnight at 19 °C. Cells were harvested by centrifugation (18 000g) for 5 min and resuspended in 5 mL/g pellet of sucrose buffer (50 mM HEPES, 20% sucrose, 1 mM EDTA, pH 7.6) before repelleting by centrifugation (18 000g) for 5 min. The supernatant was discarded, and the pellet was resuspended in 5 mM MgSO4 for 10 min on ice. The cells were pelleted and were resuspended in cell lysis buffer (50 mM sodium phosphate, 300 mM NaCl, 10 mM MgCl2, 10% glycerol, 10% sucrose, 10 mM β-mercaptoethanol, 50 mM L-arginine hydrochloride, pH 8). Cell lysis was carried out by adding BugBuster (Novagen) with benzonase nuclease (Novagen) for 30 min and then sonicating on ice for 5 min. The supernatant was collected by centrifugation (25 000g) for 15 min and purified by His select TALON Metal Affinity Resins (Clontech). One milliliter of the gel containing Co2+ was packed into a Poly prep column (Bio-Rad), and the lysate sample (4 mL) was applied. After 1 h of incubation, the column was washed three times with four column volumes of wash buffer (50 mM sodium phosphate, 300 mM NaCl, 10% sucrose, pH 8) and followed by three times with four volumes of the same buffer including 5 and 10 mM imidazole each. His-A*Zmab fusion protein was eluted with 1 mL of extraction buffer (50 mM sodium phosphate, 300 mM NaCl, 10% sucrose, 10 mM β-mercaptoethanol, 200 mM imidazole, pH 7.4). The fusion protein was concentrated by the use of Amicon Ultra-0.5 mL 10K (MILLIPORE) and dialyzed against 150 mM NaCl, 50 mM Tris-HC1, pH 7.4, using Slide-A-lyzer dialysis cassette (PIERCE). All of the reactions listed above were performed at 4 °C unless otherwise mentioned. The purity of the protein was analyzed by 12% sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE), and the concentration was determined using a BCA assay kit (PIERCE). Characterization of the Fusion Protein. DNA Cleavage and Covalent Reactions by the A*-Zmab Fusion Protein. To monitor the covalent linkage of A*-Zmab fusion protein with DNA substrate, SDS-PAGE (10% acrylamide) was performed under denaturing conditions in the presence of β-mercaptoe-

Figure 1. Schematic representation of sandwich immuno-rolling circle amplification (IRCA) assay using recombinant protein of interest, A*Zmab. (Top Left) Antibody is captured on a solid surface. (Top Right) A reporter antibody binds to a test analyte that is captured on a solid surface by a capture antibody. (Bottom Left) A DNA oligonucleotide covalently linked with A*-Zmab binds to the detection antibody. (Bottom Right) The resulting complex is washed to remove excess reagents, and the DNA tag is amplified by RCA.

linkage can also site-specifically conjugate DNA to recombinant affinity proteins, yielding homogeneous reagents with 1:1 stoichiometry. Moreover, the fusion protein-based assay format described herein enhanced the signals of sandwich IRCA to detect human interferon-γ (IFN-γ), a cytokine related to many infectious disease and pathogen-directed responses of the human body.22

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MATERIALS AND METHODS Materials. Restriction enzymes, ligase, T4 DNA polymerase, exonuclease I, and exonuclease III were obtained from Takara Bio Inc. (Shiga, Japan). Plasmid pBluescriptSKII (+) was obtained from Stratagene (La Jolla, CA). Escherichia coli BL21 (DE3) and pET28 were purchased from Novagen (Madison, WI). Deoxynucleotide solution mix (dNTPs) and Phi29 DNA polymerase were from New England Biolabs (Ipswich, MA). Circligase ssDNA ligase II was purchased from Epicenter Biotechnologies (Madison, WI). All other chemicals were of analytical grade. Synthesized DNA fragments, the primer DNA (TCGACAACTTGATATTAATAACTTCCTctgtgcgccggtctctccca) modified with or without biotin at 3′ end were from FASMAC (Japan), and 5′-Phosphate-padlock probe 2 3 (cggcgcacagTTGAATTCGTCGTGACTGGGAAAACCCTGGGATCCTTtgggagagac-the ten 5′ and 3′ small letter terminal bases are complementary to the small letter sequences of primer DNA, respectively) were purchased from Operon (Tokyo, Japan). Construction of Plasmids. A pBS-phiX A* plasmid encoding phiX174 A* gene was constructed previously. At first, we did a point mutation at 303rd codon from TAC (Tyr) to CAC (His) Y303H as described in the instruction of B

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The unbound antibody was washed with HBS-T, and the excess sites on the plate were blocked with 2% BSA in HBS buffer for 1.5 h at 37 °C. The A*-Zmab fusion protein covalently linked with biotinylated DNA (500 nM) was added in each well and incubated for another 2 h at RT while shaking. The plate was washed in between each incubation step. The binding was detected as described above. Sandwich ELISA for IFN-γ. First, 10 μg/mL of capture antibody, rabbit anti-IFN-γ IgG, was adsorbed on the polystyrene plate overnight at 4 °C. The unbound Ab was aspirated and washed with HBS-T, and the free surface was blocked with 2% BSA in HBS buffer. Increasing concentrations of IFN-γ (PeproTech, USA) (10-fold) were added and incubated for 1.5 h at 37 °C. Negative control wells received HSA in HBS buffer. The IFN-γ was captured with 10 μg/mL of detection antibody and mouse anti-IFN-γ IgG1 and incubated for another 1.5 h at 37 °C. A*-Zmab fusion protein covalently linked with DNA (biotin modified, 500 nM) was added in each well for 2 h at RT while shaking. The plate was washed in between each incubation step. The binding was detected by incubating the plates with stv-HRP as described above. Circular Probe Generation. The circular probe was synthesized from the linear ssDNAs padlock probes through ligation catalyzed by the Circligase II ssDNA ligase. Circularization was performed at 60 °C for 120 min. Twenty microliters of the reaction mixture contained 10 pmol of linear padlock probe and 100 U Circligase II in the 1× reaction buffer containing 2.5 mM MnCl2 and 0.25 M Betaine, which was provided by the manufacturer. After deactivation of Circligase II by incubation at 80 °C for 10 min, the exonucleases 10 U Exo I and 90 U Exo III were added to digest the residual linear DNA at 37 °C for 45 min. The exonucleases were inactivated at 85 °C for 15 min. The product was purified by ethanol precipitation and verified by denatured PAGE gel electrophoresis and stored at −20 °C as a stock solution with an estimated concentration of 200 nM. Modeling of Immuno-Rolling Circle Amplification (IRCA). IRCA reactions were carried out using methods similar to conventional sandwich ELISA with slight modification. The A*-Zmab-DNA was used instead of biotinylated DNA. Instead of stv-HRP in ELISA, specimens were incubated with the RCA reaction mixture in 50 μL of reaction containing 200 μM of each dNTP, 500 μg/mL BSA, 20 nM circular probes, 0.4 units/ μL of phi29 DNA polymerase in the 1× reaction buffer provided by the manufacturer and continued for 3 h at 37 °C. The bound RCA products were detected by measuring the relative fluorescent intensity using CytoFluor Series 4000 (Fluorescence Multiwell plate reader, PerSeptive Biosystems) after SYBR gold staining.

thanol. The reaction was done in the presence of 5 mM dithiothreitol and 5 mM MgC12.19 Each reaction mixture contained varying concentrations of FITC conjugated DNA substrates and 4 μM of the A*-Zmab fusion protein in a total volume of 20 μL. To see the effect of reaction time for covalent coupling of the fusion protein with DNA, the same experiment was performed, and the reactions were incubated at 37 °C for 15, 30, 60, and 120 min and overnight; the reaction was terminated by the addition of gel electrophoresis loading buffer. Fluorescent imaging was performed on a FluoroImager 595 (Molecular Dynamics) for FITC conjugated DNA, and then, the gel was stained with Coomassee Brilliant Blue (CBB, Nacalai Tesque, Japan). The shifted band intensity was determined by ImageJ software. Antibody Binding Ability. Dot Blotting. Dot blotting was performed to check the binding activity of the fusion protein with different types of antibody. Ten microliters of 2 different concentrations of A*-Zmab fusion protein, protein G as a positive control, and human serum albumin (HSA, Sigma) as a negative control were applied to nitrocellulose membranes (Hybond-C, Amersham Biosciences). Two copies of membranes were prepared. Membranes were blocked with 0.25% Block Ace (DS pharma Biomedical, Japan) in HBS buffer (5 mM HEPES, 150 mM NaCl, 3.4 mM EDTA, pH 7.4) for 1 h followed by 3 washes with HBS-T (HBS with 0.05% Tween 20). One set of membranes was incubated with 1/500 diluted mouse monoclonal IgG1, and another set was incubated with 1/500 diluted rabbit polyclonal IgG in HBS buffer for 1 h followed by 4 washes with HBS-T. Then the membranes were incubated with alkaline phosphatase (ALP) labeled rabbit antimouse IgG1 (1/1000 diluted, Zymed Laboratories Inc., USA) and with goat antirabbit IgG (1/5000 diluted, Zymed Laboratories Inc., USA), respectively, for 1 h followed by 3 washes with HBS-T. The ALP signal was detected using ALP substrates (Sigma, Fast TR/Naphthol AS-MX). In a separate experiment, after A*-Zmab application, the membrane was directly incubated with the two different secondary antibodies used above. After 1 h of incubation, followed by 3 times washing, the signal was detected as described above. Enzyme-Linked Immunosorbent Assays (ELISA). To monitor the binding of Zmab fragment of the fusion protein with species specific antibody, 10 μg/mL of each mouse monoclonal anti-interferon-γ (IFN-γ) IgG1 (Mabtech, Inc. USA) and rabbit polyclonal IFN-γ IgG (U-Cytech biosciences) antibody was adsorbed in HBS buffer on a clear polystyrene plate (Costar #3361, Corning Incorporated, USA) overnight at 4 °C. The unbound Ab was aspirated and washed with HBS-T, and the excess sites on the plate were blocked with 2% bovine serum albumin (BSA) in HBS buffer for 1.5 h at 37 °C. Afterward, the wells were washed three times with HBS-T, and the antibody was captured with increasing concentrations of A*-Zmab fusion protein covalently linked with biotinylated DNA. The plate was incubated for 2 h at room temperature while shaking. Binding was detected by incubating the plate with streptavidin-horse radish peroxidase (stv-HRP; 1:10 000; Sigma) for 1 h at 37 °C. The plate was thoroughly washed three times, and the TMB peroxidase substrate (KPL, USA) was added and incubated for 5 min; the reaction was terminated with 1 N HCl. The signals were measured spectrophotometrically at a wavelength of 450 nm (Benchmark, Bio-Rad). Optimization of Mouse IgG1 Concentration. Two-fold serial dilution of mouse monoclonal anti-IFN-γ IgG1 in HBS buffer was adsorbed on the polystyrene plate overnight at 4 °C.

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RESULTS Expression and Purification of A*-Zmab Fusion Protein. A fusion protein, A*-Zmab, consisting of φX174 gene A* protein and an affibody molecule (Zmab25) derived from the immunoglobulin binding receptor staphylococcal protein A, was constructed for the purpose of antigen detection by a sandwich immuno-rolling circle amplification (IRCA) assay. The gene A* protein was fused to the N-terminus of the Zmab25 under control of the T7 promoter. To simplify the purification process utilizing one-step metal chelating affinity chromatography, a hexahistidine tag was introduced at the Nterminus of the fusion product (Figure 2A). The expression of A*-Zmab was induced by 500 μM IPTG at 19 °C overnight, C

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From Figure 3D, one can see that the covalent reaction was almost complete within 15 min at 37 °C, reaching a maximum level (approximately 85%) at 2 μM DNA in 120 min, thus indicating the potential of A* for the efficient preparation of protein−DNA conjugates. The reaction efficiency reached a plateau (about 80%) at DNA substrate concentrations above 2 μM. IgG Binding Ability of A*-Zmab Confirmed by Dot Blotting and ELISA. To monitor the binding of the Zmab moiety of the fusion protein with species-specific IgGs, dot blotting experiments were performed. Two different concentrations of A*-Zmab, a positive control (protein G), and a negative control (HSA) were applied to a nitrocellulose membrane, and binding was evaluated using two antibodies, a mouse monoclonal IgG1 and a rabbit polyclonal IgG. As shown in Figure 4A, the mouse monoclonal IgG1 exhibited stronger affinity to A*-Zmab compared to the rabbit polyclonal IgG. In addition, HSA gave no binding signal, even when added at up to a 2-fold excess concentration. In contrast, secondary antibodies (rabbit antimouse IgG1 and goat antirabbit IgG) did not show any binding to the A*-Zmab fusion protein (data not shown), suggesting that the Zmab moiety bound specifically to mouse monoclonal IgG1. Therefore, these antibody pairs could be useful in sandwich ELISAs. ELISA analysis was used to further assess the binding capability of A*-Zmab for different IgGs. Mouse monoclonal anti-IFN-γ IgG1 and rabbit polyclonal anti-IFN-γ IgG antibody were used as capture reagents on polystyrene plates. Serial dilutions of A*-Zmab-DNA modified with biotin were used to bind antibodies, and the signals were detected with HRPconjugated stv. As shown in Figure 4B, the signal for mouse IgG1 increased with increasing concentrations of A*-ZmabDNA in a dose-dependent manner up to 500 nM of A*-ZmabDNA, whereas rabbit IgG gave no significant binding signal. These data indicate that, when fused with the φX174 gene A*, the Zmab domain retained its biological functionalities in both ELISA and dot blotting assays. On the basis of these results, the 500 nM of A*-Zmab-DNA concentration was used for further studies. Similar experiments were performed with increasing concentrations of mouse monoclonal anti-IFN-γ IgG1 and a fixed concentration of A*-Zmab-DNA, to monitor the concentration dependence of antibody with A*-Zmab-DNA. Two-fold serial dilutions of mouse monoclonal IgG1 were coated on a microplate, and the signals were detected using HRP-stv via A*-Zmab-DNA. The peroxidase signal increased relative to the antibody concentration in a dose-dependent manner, indicating that A*-Zmab exhibited a concentrationdependent binding affinity for the mouse IgG1 antibody (Figure 4C). Detection of IFN-γ by Sandwich ELISA. Next, sandwich enzyme-linked immunosorbent assays were performed to investigate the feasibility of the use of this assay format and to confirm the utility of the A*-Zmab-DNA conjugate to detect immuno-proteins. IFN-γ was selected as the first test analyte due to its important role in pathogen-directed human responses and because of the requirement for high assay sensitivity. A polystyrene strip-well plate was first coated with rabbit antiIFN-γ IgG. IFN-γ was then added to bind the immobilized capture antibody. After this reaction, mouse anti-IFN-γ IgG1 was added to achieve sandwich-type bonding with the captured IFN-γ target. After the nonbound antibody was removed by washing with HBS-T buffer, the signal was detected as

Figure 2. Expression and purification of A*-Zmab fusion protein. (A) PhiX gene A* was fused to the Zmab25 under the control of the T7 promoter. A histidine tag sequence (His-tag) was used upstream of the fusion gene for purification. (B) The soluble fraction of the cell lysate containing the fusion protein was purified using TALON metalchelated affinity chromatography. Fifteen microliters of each fraction was resolved through 12% SDS-PAGE, followed by Coomassie brilliant blue staining. M, molecular weight marker; lane 1, soluble fraction of bacterial cell lysate; lane 2, insoluble fraction of bacterial cell lysate; lane 3, the purified A*-Zmab fusion protein after eluting with 200 mM imidazole. The predicted molecular mass is about 47 kDa.

and the expressed fusion protein was purified from the soluble fraction of the cell lysate by His select TALON metal-ion affinity chromatography. The results of 12% SDS-PAGE (Figure 2B) showed a single band with an apparent molecular mass of about 47 kDa in lanes containing the crude cell lysate and in the purified fraction. The fusion protein was purified to at least 90% purity, as indicated by SDS-PAGE. Characterization of the Fusion Protein. The activity of the purified A*-Zmab fusion protein was evaluated by its ability to covalently link to DNA (via the A* moiety) and to bind specific species of antibodies (via the Zmab affibody moiety). Covalent Bonding of A*-Zmab to DNA. To monitor the covalent linkage of DNA to the protein, a gel mobility shift assay was performed. The following concentrations of DNA were selected: 0.5, 1.0, 2.0, 4.0, 6.0, and 12.0 μM. Figure 3 shows that the 4 μM A*-Zmab fusion protein caused a shift in the mobility for a single protein−DNA conjugate compared to the free FITC-conjugated DNA tail (as judged by the presence of a single band on acrylamide gels by fluorescent imaging, Figure 3B) or compared to the free parental fusion protein (visualized by CBB staining, Figure 3C). The parental proteins did not form detectable amounts of this species (Figure 3B, lane 8) without any A* protein recognition sequence in the DNA, consistent with the idea that covalent linkage occurs between the A* protein and DNA. The shifted band intensity of the A*-Zmab-DNA conjugates increased with elevated DNA substrate concentration up to 2 μM. The effect of reaction time on the enzymatic covalent linkage of the A* fragment of the fusion protein was also evaluated. D

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Figure 3. Characterization of the fusion protein for covalent coupling by gel mobility shift assay. (A) Schematic representation of site-specific covalent bonding of A*-Zmab with DNA. (B) Fluor-image for protein−DNA conjugates. Increasing concentrations of FITC conjugated DNA substrates were reacted with A*-Zmab fusion protein, and 10% SDS-PAGE was performed to monitor the protein−DNA conjugates. Lanes 1 to 7 represent 0, 0.5, 1, 2, 4, 6, and 12 μM FITC conjugated DNA substrate; lane 8 represents 4 μM FITC conjugated DNA which does not have any recognition sequence for A* protein. Fluor-image for FITC showed the protein−DNA conjugates from the free nonreacted DNA. (C) CBB staining of the same gel for protein−DNA conjugates and nonreacted protein. The protein−DNA conjugates indicated the reduced electrophoretic mobility in the presence of DNA substrate as compared to the fusion protein alone. (D) The effect of reaction time for covalent coupling carried out at 37 °C.

described in the Materials and Methods section. Figure 5A shows the absorbance generated by different concentrations of the target IFN-γ. The absorbance produced by the target was the same as the blank signal when the target concentration was