Introduction

Immune response biomarker profiling using the ProtoArray® human protein microarray provides a rapid, simple method for examining changes in patient immune responses to over 9,000 targets with low volumes of unpurified clinical samples such as sera.

ProtoArray®human protein microarrays are compatible with many commercial microarray scanners and the data can be analyzed using our free ProtoArray® Prospector Software.

The first step in the immune response biomarker profiling workflow is to determine the two patient groups you wish to study. Examples include pre- and post- drug treatment and healthy versus diseased patients. Once the patients have been identified, samples need to be collected. The collected samples are then diluted in buffer and incubated onto the array. This is then followed by incubation of a fluorescently conjugated secondary antibody. The probed arrays are then scanned and the resulting data is compared using ProtoArray® Prospector or your software of choice.

The following protocols were developed for the profiling of human serum samples and can be readily adapted to other clinical samples containing antibodies.
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Materials

  • ProtoArray® Human Protein Microarray v5.0
  • Human serum or plasma sample
  • Blocking Buffer and Washing Buffer
  • 10X Synthetic Block (Invitrogen)
  • Alexa Fluor® 647 Goat Anti-Human IgG (Invitrogen)
  • Clean, covered 4-chamber incubation tray, chilled on ice
  • Forceps and deionized water
  • Shaker (capable of circular shaking at 50 rpm, place the shaker at 4oC)
  • Microarray slide holder and centrifuge equipped with a plate holder (Optional)
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Ordering Information

Catalog # Name Size List Price (USD) Qty
A21445 Alexa Fluor® 647 Goat Anti-Human IgG (H+L) 0.5 mL 149.00

Protocol

1.  Preparing buffers

  1. Prepare 100 mL of the blocking buffer by mixing 5 mL of 1 M HEPES, pH 7.5, 4 mL of 5 M NaCl, 800 l of 10% Triton® X-100, 50 mL of 50% Glycerol, 610 mg of Reduced glutathione, 10 mL of 10X Synthetic Block, and sufficient deionized water for a final volume of 100 mL.
  2. Mix the reagents, adjust the pH of the buffer to 7.5 with NaOH and add 100 L of 1 M DTT prior to use.
  3. Prepare 1,000 mL of washing buffer by mixing 100 ml of 10X PBS, 10 ml of 10% Tween 20, 100 mL of 10X Synthetic Block, and sufficient deionized water for a final volume of 1,000 mL.
  4. Mix reagents and cool to 4°C.
  5. Use the buffers immediately and store any remaining buffer at 4°C for <24 hours.


2.  Before Starting

  1. Before starting the probing procedure, make sure you have all items on hand especially buffers and an incubation tray.
  2. Dilute serum or plasma sample 1:500 in Washing Buffer and store at 4°C or on ice.
  3. Make sure the buffers are cold and stored on ice until use. Place an incubation tray on ice to chill until use.


3.  Blocking Step


  1. Place the mailer containing the ProtoArray® Human Protein Microarray v5.0 at 4°C upon removal from storage at –20°C and equilibrate the mailer at 4°C for at least 15 minutes prior to use.
  2. Place ProtoArray® Human Protein Microarrays with the barcode facing up in the bottom of a 4-chamber incubation tray.
  3. Using a sterile pipette, add 5 mL Blocking Buffer into each chamber. Avoid pipetting buffer directly onto the array surface.
  4. Incubate the tray for 1 hour at 4°C on a shaker set at 50 rpm (circular shaking).
  5. After incubation, aspirate Blocking Buffer by vacuum or with a pipette. Tilt the tray so that any remaining buffer accumulates at the end of the tray. Aspirate the accumulated buffer.

  6. Important: Do not position the tip or aspirate from the microarray surface as this can cause scratches. Immediately proceed to adding the next solution to prevent any part of the array surface from drying which may produce high or uneven background.

  7. Proceed immediately to Probing the Array.
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4.  Probing the array

  1. Add 5 mL Washing Buffer at the end of the 4-chamber incubation tray without touching the array surface. Incubate the tray for 5 minutes at 4°C on a shaker set at 50 rpm (circular shaking).
  2. Aspirate the buffer using vacuum or pipette as described above (Step 3.5).
  3. Add 5 mL serum or plasma diluted (1:500) in Washing Buffer at the end of the 4-chamber incubation tray without touching the array surface. Allow the sample to flow across the array surface. Avoid pipetting sample directly onto the array surface.
  4. Incubate the tray for 90 minutes at 4°C on a shaker set at 50 rpm (circular shaking).
  5. Aspirate the sample using vacuum or pipette as described above (Step 3.5).
  6. Wash each array with 5 mL Washing Buffer with gentle shaking on a shaker set at 50 rpm for 5 minutes at room temperature. Aspirate the Washing Buffer as described above page (Step 3.5).
  7. Repeat Step 4.6 four more times using fresh Washing Buffer each time to obtain a total of 5 wash steps.
  8. During the wash steps, mix 2.5 L Alexa Fluor® 647 goat anti-human IgG antibody with 5 mL Washing Buffer per array to obtain a final antibody concentration of 1 g/mL. Store on ice until use. Optional: add Alexa Fluor®®Prospector software (see ProtoArray® Prospector manual for details).
  9. Add 5 mL Alexa Fluor® 647 antibody solution from Step 4.8 to the incubation tray at the end of the tray without touching the array surface. Allow the solution to flow across the array surface. Avoid pipetting solution directly onto the array surface.
  10. Incubate the tray for 90 minutes at 4°C on a shaker set at 50 rpm (circular shaking).
  11. Aspirate the antibody solution as described above (Step 3.5).
  12. Wash each array with 5 mL Washing Buffer with gentle shaking on a shaker set at 50 rpm for 5 minutes at room temperature. Aspirate the Washing Buffer as described above (Step 3.5).
  13. Repeat Step 4.12 four more times using fresh Washing Buffer each time to obtain a total of 5 wash steps.
  14. Proceed immediately to Drying the Array.

647-labeled anti-V5 antibody diluted in Washing Buffer to 0.1 g/mL. Signals from the V5 Control Protein gradient printed in each subarray can be used for sample-independent (external) normalization of the IRBP data using the ProtoArray

5.  Drying the array

  1. To remove the array from the 4-chamber incubation tray, insert the tip of forceps into the end near the barcode and gently pry the array upward. Using a gloved hand, pick up the microarray by holding the array by its edges.
  2. Place the array in a slide holder (or a sterile 50 mL conical tube, if you do not have a slide holder). Ensure the array is properly placed and is secure in the holder to prevent any damage to the array during centrifugation. Briefly dip the slide holder containing the arrays into room temperature distilled water three times to remove salts. If you are not using a slide holder, dip the array into a 50 mL conical tube filled with room temperature distilled water three times.
  3. Centrifuge the array in the slide holder or 50 mL conical tube at 200 × g for 1 minute in a centrifuge (equipped with a plate rotor, if you are using the slide holder) at room temperature. Verify the array is completely dry.
  4. After drying, store the arrays vertically or horizontally in a slide box protected from light. Avoid prolonged exposure to light. To obtain the best results, scan the array within 24 hours of probing.

Proceed to Scanning and Data Analysis.


6.  Scanning the Array

  1. Insert array into a compatible fluorescence microarray scanner. The scanner must fit a standard 1” by 3” microscope slide, have 10 M resolution, have filters compatible with your probe’s dye, and have the emitter and detector on the same face of the slide.
  2. Adjust scanner settings. The PMT setting for the scanner needs to be adjusted so that the pixels in the feature with the highest concentration of IgG in the control gradient are slightly below saturation.
  3. Preview the microarray and adjust settings, if needed.
  4. Scan the microarray.
  5. Save image data.
  6. Export and analyze results.


7.  Data Acquisition and Analysis

  1. To acquire data from the scanned image, use the barcode on the array to download the .GAL file from Central at  ProtoArray®www.invitrogen.com.
  2. Use the .GAL file and suitable microarray data acquisition software to acquire pixel intensity values for all features on the array.
  3. Analyze data with ProtoArray® Prospector to determine significant signals with the controls and your protein probe.

Note:   Set the Application in ProtoArray® Prospector to Immune Response Profiling for serum samples, or to Immune Response Profiling with Plasma for plasma samples.

After data analysis, ProtoArray®Prospector presents a summary of the analyzed data in a table format (see ProtoArray®Prospector manual for details). The proteins that score as positive in the experiment are proteins that satisfy the basic program options. We recommend that candidate biomarkers be validated in a follow-on experiment using ProtoArray® or other methods. There are several appropriate assay formats including ELISA, Luminex, and immunoblotting.

The following control features can be observed after probing a ProtoArray® Protein Microarray:

  • Alexa Fluor® Ab signal: This is an antibody labeled with Alexa Fluor® 647. The fluorescent antibody signals indicate that the array has been properly scanned, and are used as reference spots to orient the microarray and help assign spot identities.
  • Human IgG Signal: A protein gradient of purified human IgG is printed on each subarray and serves as a positive control when anti-human IgG is used for detection. The Human IgG signals are used to verify proper probing and detection reagents.
  • Anti-human IgG Signal: A protein gradient of goat anti-human IgG is printed on each subarray. The IgG from human serum binds to the anti-human IgG on the array and is used to verify proper probing and detection reagents.
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Troubleshooting

Weak or no signal observed after probing with serum sample can be caused by: Low serum concentration during probingIncorrect probing procedureIncorrect scanning or imaging of the arrayDecreased stringency during washing
High background on the array can be caused by: Improper blocking of the arrayImproper washing of the arrayAllowing the array to dry during probingArray not dried properly before scanningHigh serum concentration during probingSecondary antibody cross-reactivity
Uneven background: Uneven blocking or washing of the arrayImproper washing of the arrayPortions of array have driedImproper array handlingSerum sample or detection reagents  
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References

  1. Marina et al. (2010) Serologic Markers of Effective Tumor Immunity against Chronic Lymphocytic Leukemia Include Nonmutated B-Cell Antigens. Cancer Res 70:1344-1355.
  2. Wadia PP et al. (2010) Antibodies specifically target AML antigen NuSAP1 after allogeneic bone marrow transplantation. Blood 115:2077-2087
  3. Biernacki MA et al. (2010) Efficacious immune therapy in chronic myelogenous leukemia (CML) recognizes antigens that are expressed on CML progenitor cells. Cancer Res 70:906–915.
  4. Babel I et al. (2009) Identification of tumor-associated autoantigens for the diagnosis of colorectal cancer in serum using high density protein microarrays. Mol Cell Proteomics 8:2382–2395.
  5. Sutherland SM et al. (2009) Protein microarrays identify antibodies to protein kinase C that are associated with a greater risk of allograft loss in pediatric renal transplant recipients. Kidney Int 76:1277–1283.
  6. Li L et al. (2009) Identifying compartment-specific non-HLA targets after renal transplantation by integrating transcriptome and “antibodyome” measures. Proc Natl Acad Sci U S A 106:4148–4153.
  7. Auger I et al. (2009) New autoantigens in rheumatoid arthritis: screening 8268 protein arrays with RA patients’ sera. Ann Rheum Dis 68:591–594.
  8. Gunawardana CG et al. (2009) Identifying novel autoantibody signatures in ovarian cancer using high-density protein microarrays. Clin Biochem 42:426–429.
  9. Gnjatic S et al. (2009) Seromic analysisa of antibody responses in non-small cell lung cancer patients and healthy donors using conformational protein arrays. J Immunol Methods 341:50–58.
  10. Roche S et al. (2008) Autoantibody profiling on high-density protein microarrays for biomarker discovery in the cerebrospinal fluid. J Immunol Methods 338: 75–78.
  11. Hudson ME et al. (2007) Identification of differentially expressed proteins in ovarian cancer using high-density protein microarrays. Proc Natl Acad Sci U S A 104:17494–17499.
  12. Lalive PH et al. (2006) Identification of new serum autoantibodies in neuromyelitis optica using protein microarrays. Neurology 67:176–177.
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