Antibody Specificity Profiling Using Protein Microarrays ... Key words Protein microarray, Monoclonal antibodies, Antibody specificity, Cross-reactivity.
Chapter 14 Antibody Specificity Profiling Using Protein Microarrays Pedro Ramos-Lo´pez, Jose´ Irizarry, Ignacio Pino, and Seth Blackshaw Abstract Antibodies are the most widely used reagent for isolation and detection of specific proteins. However, using antibodies that are not highly specific in these studies can generate inaccurate and misleading data. Protein microarrays offer a platform by which antibody cross-reactivity against a broad range of cellular antigens can be simultaneously and quantitatively profiled. This protocol describes in detail the process of array pretreatment, antibody binding, washing, scanning and quantitative analysis of antibody specificity. Key words Protein microarray, Monoclonal antibodies, Antibody specificity, Cross-reactivity
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Introduction Highly selective affinity reagents are essential for characterizing protein levels, localization, and function, and are also important clinical diagnostic tools and therapeutic agents. The most commonly used forms of protein affinity reagents are antibodies, both monoclonal and polyclonal. Although the majority of the human proteome is targeted by commercially available antibodies, it has become clear that the quality of these reagents leaves much to be desired [1, 2]. Many commercially available antibodies do not even recognize their intended targets, while many more cross-react with other, frequently unrelated proteins. The failure to replicate multiple high-profile biomedical studies has also been linked to problems with antibody quality. Clearly, a reliable supply of highly specific, well-characterized antibodies is an essential tool for biomedical research. Analysis of antibody specificity is challenging, and no consensus yet exists in the field as how best to do this. Validation using multiple different approaches (e.g., Western blotting, immunohistochemistry, and immunoprecipitation) has been proposed as one approach, but this runs into the fact that antibodies recognize a wide variety of conformational epitopes, and are often useful in only a limited number of assays [3]. Loss of signal following knockdown
Johan Rockberg and Johan Nilvebrant (eds.), Epitope Mapping Protocols, Methods in Molecular Biology, vol. 1785, https://doi.org/10.1007/978-1-4939-7841-0_14, © Springer Science+Business Media, LLC, part of Springer Nature 2018
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or knockout of the target gene is another criteria often used as a gold standard for antibody specificity. However, this only addresses antibody specificity in the cell type and developmental stages that are directly investigated, and does not exclude the possibility of cross-reactivity in other cell types. Ideally, it would be possible to screen antibodies against all possible antigens that they would encounter under experimental conditions. While this is not currently fully possible, the closest approximation is provided by analysis of antibody specificity using protein microarrays [4–6]. The larger and more comprehensive the protein microarray, the more accurate any antibody specificity profiles will be. The most comprehensive protein array currently available to the research community is the Human Proteome Microarray (HuProt™) version 3.1. This array contains nearly 20,000 unique affinity-purified, full-length human proteins. These proteins are expressed and purified under native conditions from Saccharomyces cerevisiae [7, 8], largely retain their biochemical activity and native conformation, and are suitable for screening antibodies against conformational epitopes (Fig. 1). The array can also be denatured to allow analysis of antibody specificity using linear epitopes. Here, we describe a protocol for the analysis of monoclonal antibody specificity using protein microarrays. This is written specifically for assays using HuProt™ microarrays, but can be readily adapted for any array. Starting from array pretreatment and antibody binding, through data acquisition and analysis, it details a procedure that allows antibody binding specificity to be quantitatively and comprehensively assessed.
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Materials Make all solutions with deionized water with a resistivity of 18 MΩ cm at 25 C. Store all reagents at room temperature (unless indicated otherwise).
2.1 Buffer Components
1. Tris-buffered saline 10 (TBS): 1.5 M NaCl, 0.1 M Tris–HCl, pH 7.5. For 1 L of 10 TBS stock buffer, dissolve 60.5 g Tris and 87.6 g NaCl in 800 mL of deionized water. Adjust pH to 7.5 and add ddH2O to 1 L. Store at room temperature. 2. Washing Buffer: Tris-buffered saline containing 0.1% Tween20 (TBS-T): Add 1.0 mL of Tween-20 to 100 mL of 10 TBS solution. Make up to 1 L with ddH2O and stir until homogeneous. Store at room temperature. 3. Blocking buffer: 5% BSA/TBS-T. Weigh 5 g of Bovine Serum Albumin (IgG-Free, Protease-Free; Jackson ImmunoResearch Laboratories). Make up to 100 mL with TBS-T. Store at 4 C.
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Fig. 1 Example of a highly specific mAb analyzed using HuProt™ microarrays. (a) Cy5 signal reveals selective binding to the intended target, which is printed as a pair of adjacent, duplicate spots (yellow box). In the control row, Cy5-labeled BSA and mouse IgG serve as positive controls that allow ready alignment of each block of printed proteins (blue boxes). (b) The same block stained with Alexa 555-coupled anti-GST, which detects all purified human proteins on the array
4. Denaturation Buffer: 9 M Urea, 5 mM DTT. Weigh 54.05 g of Urea and 80 mg of Dithiothreitol (DTT). Make up to 100 mL with deionized water. 5. Secondary Antibodies: Make a 1 mg/mL stock solution of the Alexa-647 Goat Anti-Mouse IgG (Jackson ImmunoResearch) and the Alexa-555 Goat Anti-Rabbit IgG (Invitrogen) following the manufacturer’s directions. 6. GenePix® 4000B Microarray Scanner (Molecular Devices).
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Methods
3.1 Microarray Binding
Store protein microarrays inside closed plastic holders at 80 C, or on dry ice, until the blocking step. Wear gloves at all times. For HuProt™ microarrays, the active surface is the side with the barcode. Do not let liquid condense onto the microarray surface
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before use. Please avoid touching or scratching the surface at any point during assay, it may result in loss of proteins and introduce background signal. Since all the human proteins on the HuProt™ are native, it is important to mention that if a WB-grade antibody is being screened, a HuProt™ denaturation step is recommended after blocking the arrays. 1. Add 3.0 mL of Blocking Buffer to each compartment of the four-well plates. 2. Gently use a fine-nosed tweezers to remove one HuProt™ microarray from the slide holder that is resting in dry ice. 3. Submerge the microarray, active surface up, in a compartment of the four-well plate containing Blocking Buffer and let the HuProt™ microarray incubate at room temperature for 1.5–2 h on an orbital shaker at 40 rpm. For analysis of WB-grade antibodies an extra step is highly recommended at this point (see Note 1). 4. If the source of the primary antibody is hybridoma supernatant, make a 1:12 dilution in Blocking Buffer, and dilute to a final volume of 3 mL. Store in ice until use (see Note 2). 5. If the primary antibody is purified, dilute it to a final concentration of 0.001 mg/mL in 3 mL of Blocking Buffer. Store in ice until use. 6. If using HuProt™ microarrays, add rabbit anti-Glutathione-STransferase Antibody (Millipore, AB3282) into the diluted primary antibody to a final concentration of 1:10,000 (see Note 3). 7. Carefully use aspiration to remove the Blocking Buffer from the four-well plates that contain the immersed the microarray. 8. Add the 3 mL mixture of primary antibodies starting from the frosted or barcoded area of the slide to completely cover the entire active surface of the array. 9. Incubate with gentle shaking on an orbital shaker at room temperature for 1 h at 40 rpm. 10. Rinse each slide briefly with 5 mL of TBS-T added from the barcoded area, and completely remove the buffer after each wash by aspiration. Repeat for a total of three quick washes. 11. Add 5 mL of TBS-T and let it incubate at room temperature for 10 min with gentle shaking. 12. Remove the Washing Buffer by aspiration and repeat for a total of three washes. 13. Add the Alexa-647 Goat Anti-Mouse IgG (and the Alexa-555 Goat Anti-Rabbit IgG, if anti-GST has been applied) at a final
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concentration of 1:1000 in total volume of 3 mL of Blocking Buffer for each slide. 14. Add 3 mL diluted secondary antibodies into each compartment of the four-well plate containing the microarrays. 15. Cover the four-well plate with aluminum foil and incubate at room temperature for 1 h with gentle shaking (see Note 4). 16. Rinse each slide briefly with 5 mL of TBS-T added from the barcoded area and completely remove the buffer after each wash by aspiration. Repeat for a total of three quick washes. 17. Add 5 mL of TBS-T and let it incubate at room temperature for 10 min with gentle shaking. The four-well plate should be covered with foil at all times. 18. Remove the Washing Buffer by aspiration and repeat for a total of three washes. 19. Briefly rinse the microarray with 5 mL of ddH2O. 20. Place a clean room wipe or paper towels on the bottom of a black microscope slide box. 21. Remove the microarray from the four-well plate and tap the edge of the slide lightly on the paper towel to remove excess fluid. 22. Carefully slot the microarrays into the black microscope slide box by placing the microarrays perpendicular to the paper towel lining the base of the box (see Note 5). 23. To remove excess fluid, spin the microscope slide box or the 50 mL conical tube in a centrifuge at 800 rpm for 3 min. Spinning at higher speeds may break the microarrays. 24. Carefully remove and discard the paper towel from the microscope slide box. 25. The microarray should be scanned immediately or stored overnight at 4 C in a black microscope box for the next day. 26. Turn on the GenePix® 4000B scanner and let it warm for at least 15 min. 27. Place the microarray with the active surface facing down into the scanner and initiate the GenePix® software. 28. Make a preview image and set the photomultiplier tube (PMT) gain values to each channel based on the excitation of the secondary antibodies fluorescence. 29. Make the real scan based on the pre-selected PMT gain values. 30. After the microarray is scanned, place the Gal File and make the alignment of each spot (see Note 6). 31. Generate the GPR (GenePix® Results) file from the software to conduct Data Analysis.
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3.2 Data Analysis (First Described in [7])
1. Filter controls and spot sets without significant signal (>1.5 Foreground/Background ratio). 2. Calculate the average signal of each spot set. 3. Calculate the average signal of all spot sets. 4. Calculate the standard deviation of the signal of all spot sets. 5. Calculate the Z-Score for each spot set: z¼ (α αavg)/αstd 6. Rank all spot sets by Z-Score. 7. Calculate the S-Score for each spot set: s¼ z1 z2 8. To ensure low cross-reactivity and a high quality reagent, check the top result is your expected target and the S-Score is higher than 2.5 (see Note 7).
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Notes 1. Carefully use aspiration to remove the Blocking Buffer from the four-well plates that contain the immersed the microarray. Immediately, add 3 mL of the Denaturation Buffer from the barcode area and let it incubate without shaking for 20 min at room temperature. After the incubation, aspirate the Denaturation Buffer and proceed to rinse each slide briefly with 5 mL of TBS-T added from the barcoded area, and completely remove the buffer after each wash by aspiration. Repeat for a total of three quick washes. At this time, the HuProt™ is ready for incubation with the primary antibody. 2. This dilution yields, on average, a final concentration of 20 ng/ μL of Blocking Buffer. A dilution series may be tested if desired. If antibody concentration is limiting, the antibody may be diluted 1:12 in Blocking Buffer, and a HybriSlip™ Hybridization Cover (Grace Bio-Labs) used minimize the evaporation. Also a humidification chamber is recommended to avoid dryness. 3. Since all human proteins on the HuProt™ have a GST tag, the use of an anti-GST antibody is highly recommended to ensure that the expected target was successfully printed on the microarray, in case the primary antibody does not bind its intended target. 4. After the secondary antibody is added, store the microarrays in the dark. For all incubation and washing steps after it, cover the four-well plate containing HuProt™ microarrays with aluminum foil to minimize light exposure, which can quench the fluorescence. 5. Drying can be done using a plastic 50 mL conical tube prepared with paper towel at the bottom. Place a single microarray
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lengthwise into the tube. The tube must be covered at all times with aluminum foil. 6. The HuProt™ v3.0 is divided into 24 blocks that contains both the full-length human proteins and a series of controls. The Gal File is a grid-based file that represents the HuProt™ printing pattern and gives the protein’s identity across the microarray slide. It is crucial to do a good alignment of each spot in order to get reliable results. To facilitate analysis, align each block using the IgG controls strategically printed on the last row of each block (Fig. 1). These controls will light up if the secondary antibodies were properly added into the assay. Use the “feature finding” tool on the GenePix® software to get a tight alignment on each spot and also obtain a better signal value for the data analysis. 7. If the top result is not your expected target, or if the S-Score is lower than 2.5, this indicates a low-quality reagent with crossreactivity issues.
Acknowledgments This work was supported by NIH grant U54HG006434. References 1. Bradbury A, Pluckthun A (2015) Reproducibility: standardize antibodies used in research. Nature 518(7537):27–29 2. Baker M (2015) Reproducibility crisis: blame it on the antibodies. Nature 521(7552):274–276 3. Bordeaux J et al (2010) Antibody validation. BioTechniques 48(3):197–209 4. Zhu H, Snyder M (2003) Protein chip technology. Curr Opin Chem Biol 7(1):55–63 5. Sjoberg R et al (2016) Exploration of highdensity protein microarrays for antibody validation and autoimmunity profiling. New Biotechnol 33(5 Pt A):582–592
6. Zandian A et al (2017) Whole-proteome peptide microarrays for profiling autoantibody repertoires within multiple sclerosis and narcolepsy. J Proteome Res 16(3):1300–1314 7. Jeong JS et al (2012) Rapid identification of monospecific monoclonal antibodies using a human proteome microarray. Mol Cell Proteomics 11(6):O111.016253 8. Zhu H et al (2001) Global analysis of protein activities using proteome chips. Science 293 (5537):2101–2105
