Microfluidic Analysis of Antibody Specificity in a Compact Disk Format

Microfluidic Analysis of Antibody Specificity in a Compact Disk Format Cecilia Eriksson,† Charlotta Agaton,† Rikard Kånge,‡ Mårten Sundberg,† Peter Nilsson,† Bo Ek,‡ Mathias Uhle´ n,† Magnus Gustafsson,‡ and Sophia Hober*,† Royal Institute of Technology, AlbaNova University Center, Department of Biotechnology, SE-106 91 Stockholm, Sweden, and Gyros AB, Uppsala Science Park, SE-751 83 Uppsala, Sweden Received December 9, 2005

A new and flexible technology for high throughput analysis of antibody specificity and affinity is presented. The method is based on microfluidics and takes advantage of compact disks (CDs) in which the centrifugal force moves fluids through microstructures containing immobilized metal affinity chromatography columns. Analyses are performed as a sandwich assay, where antigen is captured to the column via a genetically attached His6-tag. The antibodies to be analyzed are applied onto the columns. Thereafter, fluorescently labeled secondary antibodies recognize the bound primary antibodies, and detection is carried out by laser-induced fluorescence. The CDs contain 104 microstructures enabling analysis of antibodies against more than 100 different proteins using a single CD. Importantly, through the three-dimensional visualization of the binding patterns in a column it is possible to separate high affinity from low affinity binding. The method presented here is shown to be very sensitive, flexible and reproducible. Keywords: microfluidics • miniaturization • compact disk • biochip • gyrolab • antibody specificity

1. Introduction When sequencing of the human genome had been performed1,2 we entered what often is described as the “postgenomic era”, an era which represents a shift from the study of individual genes and proteins to the parallel analysis of thousands of genes and proteins expressed under certain conditions and time points.3 One approach for proteomic analysis based on genomic information is high throughput generation of protein-specific affinity reagents, such as polyclonal antibodies, which can be used for a wide range of functional and biochemical studies.4-7 In order for any study to be reliable, the specificity of the affinity reagents used needs to be assessed, and one well-known problem is that affinity reagents are prone to cross-reactivity and therefore might give high background signals. Hence, polyclonal antibodies depend greatly on the purification to achieve monospecificity8,9 and validation of the achieved antibodies is of outermost importance. To quality ensure the specificity, different immunobased systems can be utilized. These systems should minimize sample consumption, since affinity reagents normally are expensive. Traditionally, the ELISA system has been used for this purpose. However, this method requires large sample volumes, tedious laboratory work and time-consuming incubation steps10 and is therefore not suitable for high-throughput projects. Hence, * To whom correspondence should be addressed. Tel: +46 8 5537 8330. Fax: +46 8 5537 8481. E-mail: [email protected]. † Royal Institute of Technology. ‡ Gyros AB.

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Published on Web 05/23/2006

significant efforts have been made in miniaturizing selectivity analyses into, for example, array-based formats.11-13 Protein arrays have the potential to be excellent tools for qualitative control assays in the evaluation of the specificity of antibodies and other binding ligands.14-16 However, protein arrays give limited information about the affinity and no information about differences between the antibodies in the same polyclonal sera. A strategy for minimizing sample consumption and increasing throughput, other than using protein arrays, is to use microfluidic technologies. The issues to be solved by microfluidic approaches largely differ and also the requirements in handling of the samples. Therefore, flexibility of the developed systems is essential. In addition, smooth and uniform liquid transportation is a challenge often complicated by heating from adjacent electrical components or pressure changes mediated by pumps. Through the design of compact disks (CDs) with integrated micro capillaries, fluid transportation can be accomplished by spinning the CD at an appropriate rate, thereby creating a sufficient g-force to transport the solutions through the columns that are enclosed in the CD microstructures.17 By careful design of the rotational speed and time, the flow rate can be very precisely defined. Furthermore, the flow is not disturbed by contiguous electrical components or pumps. Hence, many parallel samples can be simultaneously prepared in an identical way on the same disk.18,19 This technology can be used for a great variation of matrices and here we have used IMAC matrix to capture the antigens. After addition of the polyclonal antibodies as well as the covalently labeled secondary antibodies, fluorescence-based analysis is used for quality control. 10.1021/pr050447c CCC: $33.50

 2006 American Chemical Society

Microfluidic Analysis of Antibody Specificity

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In this paper, we describe a flexible system for simultaneous analysis of antibodies against 100 different antigens to investigate whether cross reactivity occurs to unrelated proteins or to the common fusion tag of the immunized antigens. For high throughput analyses it is essential to keep sample and time consumption at a minimum. To render the method even more effective and to increase throughput, we also show that it is possible to investigate antigen-antibody cross reactivity by pooling antibodies eight by eight.

2. Materials and Methods 2.1. Assay Preparations. 2.1.1. Production of Antigens and Polyclonal Antisera. To locate antigens containing suitable epitopes, blasting of protein fragments of 100-180 amino acids was performed against the peptide sequences of all human proteins in the Ensembl database.20 Transmembrane regions were avoided and the two fragments in each gene having the lowest sequence similarity scores were chosen.21 Subsequently, primers were designed and used in RT-PCR, resulting in DNA sequences encoding the selected antigens.21 Protein fragments and primers were designed using the software Bishop.22 Production of the antigens in E. coli, as well as immunization and antibody purification, was performed according to previously described methods.8,21 Briefly, the antigens were produced as fusion proteins to a common purification tag, His6ABP, and purified on IMAC matrix.21 The antibody purification was performed in a dual purification scheme, with an initial depletion step where antibodies directed toward the common His6ABP were removed, and a second step where specific antibodies were captured and purified.8 2.1.2. Gyrolab Workstation and CD Technology. A CD microlaboratory containing packed IMAC columns (∼15 nL) was used for selective capture of His6-tagged proteins. Within the plastic microfluidic CDs all compartments, channels, valves and solid phase columns are covered by a laminated lid and liquid flow is controlled by a combination of centrifugal force, hydrophobic barriers and channel geometry. The volume of liquids added to the structures is defined through channel geometry and hydrophobic barriers and is in this application set to 200 nL. Properly adjusted rotational speed allows for directed flow within the channels and compartments, and also constant flow rate over capturing columns. A user-defined method instructs the Gyrolab workstation to perform all steps of action required, such as addition of sample and reagents. The workstation controls the rotational speed of the CDs, handles all liquid transfers, and quantifies fluorescent-labeled proteins trapped on the IMAC-columns using a laser-induced fluorescence detector within the instrument. Intensities in 300 distinct spots recorded over a full column can be visualized using the software Gyrolab Viewer, which is helpful in distinguishing between different affinities. Up to 104 samples can be processed in parallel; such an analysis requires approximately 50 min, including all incubation, washing and detection steps as well as the transfer of raw data to the software. 2.1.3. Choice of Matrix. Three commercial IMAC matrices, Qiagen NTA-Ni (Nitrilotetraacetic acid (NTA)-silica, 16-24 µm particle size, Qiagen, Hilden Germany), Poros 20 MC (Polystyrene Di-vinyl Benzene, ∼20 µm particle size with rather high variability, Applied Biosystems, CA, US) and Ni-Sepharose (highly crossed-linked 6% agarose, ∼34 µm particle size, GE Healthcare, Uppsala, Sweden) were packed in individual CDs from approximately 2% (w/v) slurries. Qiagen NTA-Ni was silted up in 0.01% Tween20 and 0.1 M acetic acid, Poros 20 MC in

Figure 1. Column signal recorded by the laser-induced fluorescence detector is visualized. Liquid enters the column from the left and flows to the right, i.e., in parallel with the radius direction of the CD. The unit of measurement denoted Total Integrated Volume corresponds to the volume under the 3D curve.

40% EtOH and 0.1 M acetic acid and Ni-Sepharose in 40% EtOH and 0.1 M acetic acid prior to packing. Five proteins (Internal protein identities: P050, P054, P059, P061, and P076) were captured onto the different IMAC columns and detected using the corresponding antibodies recognized by the detecting antibodies Alexa Fluor 647 goat anti-rabbit IgG (Molecular Probes, Leida, NL). Column profiles were evaluated using Gyrolab Viewer. 2.2. Antibody Quality Assurance. 2.2.1. Analysis Using the Gyrolab Workstation. Prior to loading of His6-tagged proteins, the columns were reconditioned with a solution containing 50 mM Tris, 150 mM NaCl, 60 mM imidazole and 0.1% Tween20 at pH 8.0. Antigens were loaded on the column in a buffer containing 50 mM Tris, 6 M urea, 60 mM imidazole, 0.01% Tween20, 1 mg/mL casein, 17 mM NaH2PO4, 33 mM NaCl, 10 mM acetic acid and 23 mM NaAc at a pH of 8.0. Protein concentrations ranged between 0.2 and 2 mg/mL. The washing solution (50 mM Tris, 150 mM NaCl, 60 mM imidazole, 0.1% Tween20 and 1 mg/mL casein at pH 8.0), was used for dilution of primary and secondary antibodies as well as for washing the columns after the addition of sample. Primary antibodies were diluted in washing solution to a concentration of 1-1.5 µg/mL and 200 nL, i.e., 2 fmole, was added to each column. A total volume of 25 µL was needed to run the program. When antibodies were pooled the total concentration of primary rabbit antibodies was approximately 10 µg/mL. The detecting antibodies, Alexa Fluor 647 goat anti-rabbit IgG (Molecular Probes), were diluted to a concentration of 300 ng/mL. Here, 400 amole secondary antibodies were used per column. All dilutions were pipetted into a microtiterplate for subsequent automated addition to the CDs. Results were interpreted through the software Gyrolab Viewer and the two parameters Total Integrated Volume (TIV) of fluorescence (Figure 1) and the Sum of Intensity (SI) profiles (Figure 2) were used to illustrate antibody specificity. 2.2.2. Analysis Using Protein Arrays. The proteins were diluted to 40 µg/mL in 0.1 M urea and 1 × PBS (pH 7.4) and spotted and immobilized onto epoxy slides (Corning Life Sciences, NY, US) using a pin-and-ring arrayer (Affymetrix 427, Affymetrix, CA, US). The slides were washed in 1 × PBS (5 min) and the surface was then blocked (SuperBlock, Pierce, IL, US) Journal of Proteome Research • Vol. 5, No. 7, 2006 1569

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Figure 2. The curves in this figure show polyclonal antibodies Ab076 binding their corresponding antigen, P076, attached to three different IMAC matrices. Curves shapes are illustrated with the entity Sum of Intensity (SI), which is obtained through summarizing the intensities perpendicular to the column flow. The curves correspond to (A) a PDB matrix which resulted in sharp peaks and high signals, (B) a silica based NTA-Ni matrix which displayed sharper curves and comparable signal intensities, and (C) a cross-linked agarose based matrix which resulted in sharp curves with lower signals.

for 30 min. An adhesive 16-well silicone mask (Schleicher & Schuell, NH, US) was applied to the glass before the purified antibodies, 60 µL of a solution of approximately 50 ng/mL in 1 × PBST (1 × PBS, 0.1% Tween20, pH 7.4), were incubated on a shaker for 60 min. The slides were washed two times with PBST X and once with 1 × PBS for 10 min each. The secondary antibodies (Goat-anti rabbit Alexa 647, Molecular Probes) was diluted to 30 ng/mL in 1 × PBST and incubated for 60 min. After the same washing procedure as for the first incubation, the slides were spinned dry and scanned (G2565BA array scanner, Agilent, CA, US) and images were quantified using an image analysis software (GenePix 5.1, Axon Instruments, CA, US).

3. Results A novel high-throughput method for analysis of antibody specificity has been developed. The analyses are made in minute sample volumes taking advantage of a microfluidic system in a compact disk (CD) format. The CDs, manufactured through injection molding, contain 104 microstructures, each enclosing a 15 nL sized microcolumn. The liquid is moved through the capillaries by spinning the disks and thereby creating a g-force parallel to the radius direction which gives a well-defined flow velocity. Here, the system has been used for specificity analysis of polyclonal antibodies against 100 different antigens. Detection was made feasible by using fluorescently labeled secondary antibodies. Data achieved when analyzing the fluorescence can be displayed in two different ways. The Total Integrated Volume (TIV) corresponds to all fluorescence detected when scanning the three-dimensional column (Figure 1). When the data is presented as a two-dimensional picture, it shows the summarized intensities perpendicular to the column flow. Here this is denoted Sum of Intensity (SI), and examples of these curves are shown in Figure 2. In this project, all antigens were produced as fusion proteins to a His6ABP-tag. The His6-tag was utilized for antigen purification23 and the ABP-moiety was included to increase the immunoresponse when raising antibodies.24 To take advantage of the His6-tag, IMAC-matrix was chosen for a directed and efficient capture of the antigen to the CD microcolumns. Since CDs with IMAC matrices not are commercially available the performance of three different solid matrices was evaluated. Five antigens were bound to all of the different matrices. Thereafter, primary as well as labeled secondary antibodies 1570

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were loaded and the acquired fluorescence was measured. Analysis was performed using the software Gyrolab Viewer. An example from these experiments, where the antigen P076 and its interaction to antibodies Ab076 is analyzed, is shown in Figure 2. As can be seen the signal intensities and the peak profiles varied in size and shape between the different matrices. Highest signals were achieved by the Polystyrene Di-vinyl Benzene (PDB) matrix (Figure 2A), whereas the peak profiles were sharper using the silica and the cross-linked agarose based beads (Figure 2B and C, respectively). Since the cross-linked agarose particle shrinks when drying, automatic column volume measurement in the manufacturing process was complicated and long-term storage of the agarose-based matrix showed to be impossible since the dry matrix was at risk of escaping the columns. The silica based NTA-Ni matrix (Figure 2B) was chosen for further studies since it gave high signals, narrow peaks and reproducible and low background. Moreover, the possibility to store the CDs made the planning of the experiments easier. To minimize interactions of proteins without the His6-tag to the matrix, imidazole (which is a building block of histidine) was included in all buffers. The maximum allowed concentration of imidazole was investigated by elution of His6-tagged proteins from the IMAC columns. Three different matrices were tested and the results showed that His6ABP, used as test protein, was eluted at imidazole concentrations between 70 and 150 mM (data not shown). Hence, to ensure capture of the His6tagged proteins and to minimize the binding of other chelating groups to the matrix, the imidazole concentration was set to 60 mM in all washing solutions. Furthermore, to verify that binding to the matrix had not occurred, two antigen-free columns were included in every analysis. No substantial interaction between antibodies and matrix was detected for the antibodies tested, when imidazole was included in the solutions. Following optimization of buffer compositions and spinning profiles the specificity of several polyclonal antibodies was analyzed. Data from protein arrays with 100 different proteins spotted on glass surfaces (including the correct antigen) were compared with the data from the CD-system where the antigens were captured on microcolumns. In both methods, the common tag, His6ABP, was used as a negative control. Also, as earlier mentioned, to control the background caused by the matrix used in the CDs, two columns lacking His6-tagged antigen were included. When analyzing the specificity of, for example, antibodies Ab001 using protein arrays, a strong affinity to antigen P001 could be detected (Figure 3A). The data achieved show that antibodies directed toward the common tag (His6ABP) are completely depleted by the purification scheme used.8 Also a weak, but clear signal from P075 could be seen. For comparison, the specificity data from the CD-analysis is illustrated through bars representing the Total Integrated Volume (TIV) of fluorescence from each column (Figure 3B). All signals higher than the mean background level plus two times the standard deviation were regarded as positive. As can be seen, the CDanalysis using Ab001 shows the same strong interaction to P001 and a weaker interaction to P075. Comparison of the amino acid sequences of P001 and P075 reveals two identical amino acid sequences, that could be linear epitopes accounting for the detected high affinity binding of a subpopulation of Ab001 to P075 (Supporting Information, Figure 1). Interestingly, binding of Ab001 to two additional antigens (P009 and P071) is detected. The four interactions detected on CD can be


Figure 3. Comparison between specificity analysis using protein arrays and CDs. (A) Protein array analysis of antibodies Ab001 resulted in high signal intensity from the spot containing antigen P001 and lower intensity from the spot containing P075. (B) The same interactions plus two additional peaks were found with CD analysis. The nature of these interactions could be further investigated with the Gyrolab data. (C) The column profiles for Ab001 binding to P001 and P075 indicate strong, specific binding detected at the beginning of the column. The binding to P071 also seems specific although the signal intensity is weaker. Peaks with low intensity but narrow curve form, indicating specific interaction, can be explained by recognition of the antigen by a subset of the antibodies. The binding of Ab001 to P009 results in a low and broad peak, indicating binding with low affinity.

illustrated with the curves representing the SI of all four antigen-antibody-interactions (Figure 3C). Through their individual shapes, these curves illustrate the different interaction patterns between antigens and antibodies. For example, the column profiles depicting Ab001 binding to P001 and P075 both show high and narrow curve profiles representing specific, high affinity interactions. Similarly, a narrow peak is shown in the fluorescence profile of the column where the same antibodies, Ab001, interact with P071. However, this peak is smaller in size, probably due to that only a fraction of the polyclonal antibodies is able to bind to that specific antigen. The fourth curve representing the binding to P009 results in a very low but broad peak. Despite a TIV higher than the background, this curve represents a weak interaction. The binding patterns achieved

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Figure 4. Comparison between specificity analysis using protein arrays and CDs. (A) Protein arrays show clear interaction of Ab055 to its own antigen. However, no other binding is detected. (B) CD data show an additional interaction to antigen P095. (C) The CD column curves reveal that the binding to P095 is a low affinity interaction.

have shown large reproducibility demonstrating that the data are reliable. Taking into consideration that these interactions, clearly beyond the noise level, have not been found in protein array analyses, analysis on CD is more sensitive in terms of detection of additional binders (Figure 3). Another example showing that the results are similar but the sensitivity higher using CDs compared to protein arrays is shown in Figure 4. The polyclonal antibodies directed toward antigen P055 show specific binding to the expected fragment with both methods (Figure 4A and B), but when examining the TIV-data from the CD-analysis an additional interaction can be seen; to P095. However, when analyzing the shape of the curve a broad and low curve indicates that this interaction has very low affinity (Figure 4C). The results clearly demonstrate the difference in sensitivity between the two techniques, allowing for more accurate antibody specificity analyses using CDs. To further increase throughput, more than one polyclonal antibody were analyzed simultaneously. Hence, the antibodies were pooled together 8 × 8 before loading on the columns. Figure 5A illustrates the TIV-data from each antigen column when the eight polyclonal antibodies had been added. As can be seen in the figure, the pool of antibodies gave increased Journal of Proteome Research • Vol. 5, No. 7, 2006 1571

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higher than the mean background level plus two times the standard deviation were loaded onto a new CD and retested against each polyclonal antibody alone. When pooling different polyclonal antibodies, a decrease in fluorescent signal could be seen and thereby also a decrease of the signal-to-noise ratio. In Figure 5B, TIV values are shown from specificity analysis of the eight antibodies against all 11 antigens that showed elevated fluorescence levels in the first, pooled, analysis. These data show that all eight antibodies recognize their corresponding antigen with high affinity. Clearly, Ab050, Ab055, Ab061, and Ab083 show no cross reactivity to any of the 100 tested antigens. Again, all interactions can be further investigated and compared through their corresponding curve shapes (Figure 5C-F). For example polyclonal antibody Ab002 shows interactions with three of the antigens chosen for further analysis. From the curve shape it can be concluded that the antibodies have highest affinity to the expected antigen but also shows binding, although weaker, to P020 and P050 (Figure 5C). Noteworthy, all analyses performed with the antibodies denoted Ab054, raised against the antigen P054 also show specific binding to P062 (Figure 5B and F). This pattern is expected since the two antigens have a sequence of 64 amino acids in common. Moreover, two additional interactions can be detected with these antibodies, of which P069 has a rather sharp curve shape indicating high affinity for parts of the polyclonal antibodies. The interaction to P061 shows a very low and flat curve indicating a weak interaction. For the high affinity interactions, a great conformity can be seen when comparing the results from pooling of the antibodies with earlier data, both from CD and protein array-analyses. By this approach, all eight antibodies could be tested on two CDs instead of the eight CDs needed for analysis of one polyclonal antibody at a time. To further confirm the correctness of the assessed data, tissue Western blots were performed using the antibodies. The polyclonal antibodies shown to be cross-reactive in the CD analyses are showing similar behavior in the Western blot analyses (data not shown).

Discussion

Figure 5. Specificity analyses of eight pooled polyclonal rabbit antibodies raised against different proteins. (A) The four rightmost columns are used as controls; two columns with His6ABP and two blank columns where only washing solution has flown through. Despite that the concentration of antibodies is eight times higher, background signals are only increased two units in absolute values in the control columns. Eleven proteins are giving signals above the limit, the mean background level plus 2 × SD of the background signal. Green bars represent signals from the eight proteins to which the antibodies were raised and the yellow bar is the detected signal from a protein with a sequence of 64 amino acids in common with one of the target proteins. The red bars are positive signals from unrelated proteins. These proteins are loaded onto a second CD and the antibodies are reanalyzed one by one. (B) In the second step, when analyzing the antibodies separately, all antibodies recognized their target. Moreover, through examination of the curves (C-F) it can be stated that Ab002 and Ab054, i.e., two out of eight antibodies, show cross reactivity against unrelated proteins.

signals in 11 columns. To ensure that no antibody-antigen interaction was disregarded, all antigens causing TIV-values 1572


Here, a novel CD-based high throughput analysis system for specificity control of affinity reagents is presented. The method has been used for assessment of cross reactivity of affinity purified polyclonal antibodies and the obtained results clearly demonstrate the feasibility of this novel technology. The CDbased technology with integrated nanoliter-sized IMAC columns has shown high sensitivity and reproducibility. Antibody selectivity has been analyzed against 100 different antigens using one CD for each polyclonal antibody. For analysis of one antibody-antigen interaction, 2 fmole of the primary antibodies was needed. Achieved data corroborates with the protein arrays run in parallel as reference. Importantly, the sensitivity, i.e., the possibility to detect cross reactivity, is higher when using CD-structures than for classical protein arrays using glass slides. A possible reason for this could be that all antigens analyzed using the CD-system are attached to the surface by an interacting His6-tag, giving directed immobilization and high accessibility of the antigen. Antigens attached by directed immobilization through His6-tags to particles still have the possibility to display themselves efficiently to interaction partners, the antibodies. The antigens are added to the columns in denaturating conditions since all are purified using urea. Correct fold of all antigens might not be achieved before the antibodies are applied. The amount of correctly folded proteins

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is impossible to assess. However, many applications for the use of antibodies, display the proteins in denaturated or partly denaturated states. When spotting proteins on a glass surface, the epitopes recognized by the antibodies might be destroyed or hidden by adhesion to the surface. Furthermore, due to the use of columns with high density of metal ions, the protein is concentrated and thereby giving an improved sensitivity compared to protein arrays using a flat surface. When analyzing eight polyclonal antibodies simultaneously, it was noted that the signal intensity, and thereby also the signal/noise ratio, was decreased. Hence, weak interactions, such as that between the antibodies raised against P055 and the protein P095 (Figures 4 and 5), were harder to distinguish. This is of course a drawback for the pooling approach. However, the advantage of gain in time and expenses might outweigh the disadvantage of loss in performance. Importantly, in contrast to protein arrays on glass slides, data achieved from this system give the opportunity to achieve more detailed information about the interactions. The CD-system allows examination of all columns and their different binding patterns individually. High affinity binders can be separated from low affinity binders and the curve shape gives information about the strength of the interaction. Moreover, cross reactivity where only a subfraction of the polyclonal antibodies recognize the antigen can be assessed. This information could be used for further improvement of the polyclonal antibodies by depletion of the subpopulation that cross-reacts, for instance Ab054 with a subpopulation that binds to P062. Proteinantibody interactions with very low affinity also give a signal, which is important information for interpretation of data achieved when using the antibodies in further experiments. Another and important advantage of this system is the high flexibility regarding the design of the procedure, such as adding or exchanging the proteins or washing solutions or changing liquid flow. Here, it has been shown that quality control of antibodies can be performed successfully and with high sensitivity in a high-throughput manner using CDs with IMAC matrix in the microstructures. The His6-tag fused to the antigens enables directed immobilization of the antigens to the columns. Precise volume definitions are allowed by the microlab format on the CD thereby abolishing pipetting errors. In addition sample and manual labor time consumption is minimized. Importantly, the data handling system allowed further analysis of the signals and high affinity interactions were discriminated from the low affinity interactions. The results suggest that the method successfully also can be adapted to a wide range of systems by using different matrices, reagents and detection systems.

Acknowledgment. We thank Prof. J. Lundeberg for exquisite suggestions and valuable discussions. The authors are also very grateful to J. Steen, K. Larsson and P. Angleidou for technical assistance. This project was funded by the Knut and Alice Wallenberg Foundation and the EC project no. LSHG-CT 2004-512066 denoted MolPAGE. Supporting Information Available: Possible epitopes responsible for recognition of P075 using the polyclonal antibodies raised against P001, Ab001 (Figure S1. This material is available free of charge via the Internet at http://pubs.acs.org.

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