Viral Antigen Microarray Assay to Detect Antibody Isotypes in Serum Against Viral Antigen

1. Produce influenza antigen microarrays

1. Print antigens onto microarray slides
   1. Reconstitute each lyophilized antigen to a concentration of 0.1 mg/mL in phosphate-buffered saline (PBS) with 0.001% Tween-20 (T-PBS). Transfer 10 μL of each reconstituted antigen to individual wells of an untreated 384-well flat-bottom plate.
   2. Print antigens using a microarray printer with low-volume microarray spotting pins that aspirate antigen into the sample channel and deposit via direct contact and capillary action onto 16-pad nitrocellulose-coated glass slides.
      1. Program the printing software (e.g., Gridder) with the source plate configuration and printing parameters.
         1. Use the pull-down menu to select the name of the plate type that will be used with this printing method. For this study, use an untreated 384-well flat-bottom plate.
         2. Select the text box next to Number of Plates and type the number of sample plates that will be used in this printing protocol. For this study, use 1 plate.
         3. Select a pin configuration to use with this method. For this study, use an 8-pin configuration.
         4. Ensure the origin offsets are the distances (in the X- and Y-directions) between the slide origin (which is calibrated in the Administrative section) and the location where the printing pins will start printing on the slides.
         5. Using the Array Design tab, define the size and shape of the arrays (dot spacing and the number of dots per subarray). For this study, print 324 spots (180 μm diameter with 300 μm spacing) onto 16-pad slides in an 18×18 format using 8 pins.
         6. Select the parameters for how the printing pins pick up and dispense samples. For this study, each pin aspirates 250 nL of antigen solution and prints 1 nL onto each of 40 spots (total of 20 slides for all 8 pins)
         7. Configure the pin cleaning protocol and blotting protocol. For this study, each pin prints antigen is dipped in sterile ddH₂O in sonicated wash container and then aspirates the next antigen.
         8. Define the sequence in which the sample blocks are printed onto the slides. The array software will construct an annotated grid index (.gal) file to describe the arrangement of antigens within each microarray.
            NOTE: The top row is used for fiducials (with fluorescence at all wavelengths used in imaging, e.g., a mix of Qdot 585 nm streptavidin conjugate and Qdot 800 nm streptavidin conjugate in this study) to orient grids during imaging. For long printing runs, antigens in the source plate can be periodically re-suspended by pipetting up and down and then centrifuging, or a new source plate can be prepared and used.
   3. Place un-probed microarray slides in a lightproof box and keep them in a desiccator cabinet at room temperature for long-term storage.
      NOTE: The protocol may be paused indefinitely at this point.
2. Perform quality control check (requires poly-histidine tags)
   1. Attach the slide to probing chambers and rehydrate with a blocking buffer as described in steps 2.1.1-2.1.2 and shown in Figure 1.
   2. Dilute the mouse monoclonal poly-His antibody 1:100 in filtered 1x blocking buffer.
      NOTE: If non-purified protein antigens (e.g., expressed in in vitro transcription and translation system) are directly used to print microarrays, add components of the protein expression system (e.g., E. coli lysate) in a ratio of 1:10 with the blocking buffer used for serum dilution in order to block any antibodies directed against these components.
   3. Add 100 μL of diluted poly-His antibody to each slide chamber containing the array pad after aspiration and incubate for 2 h at room temperature or overnight at 4 °C on a shaker. Wash 3x with T-TBS buffer as described in step 2.2.1. Dilute biotin-conjugated goat anti-mouse IgG secondary antibody 1:200 in blocking buffer, add 100 μL per well after aspiration, and incubate for 1 h at room temperature on a shaker. Wash 3x with T-TBS buffer.
   4. Dilute Qdot 585 nm streptavidin conjugate to 4 nM in blocking buffer, add 100 μL per well after aspiration, and incubate for 1 h at room temperature on the shaker. Wash 3x with T-TBS buffer, and then once with TBS buffer (without Tween).
   5. Dissemble and quantify slides as described in steps 2.2.5 – 3.1.2.
      NOTE: Protein antigens must contain His10 tags to use this quality control protocol. Alternatively, if a different tag is included, a quality control check can be performed with an antibody or ligand for that tag.

2. Probe sera for influenza antibodies using microarrays

1. Incubate sera on microarrays for antibody binding.
   1. Attach microarray slides to chambers using clips and place them in frames as shown in Figure 2.
      NOTE: Always avoid touching the microarray pad with hands and instruments. Ensure that slides are oriented with the pad side up and the small notch in the upper right corner.
   2. Rehydrate microarray slides with 100 μL per well of filtered 1x blocking buffer, and dilute serum 1:100 in 100 μL of blocking buffer (can use untreated 96-well plates or 2 mL tubes). Incubate both rehydrated microarray slides in covered frames and diluted sera separately for 30 minutes at room temperature on an orbital shaker at 100-250 rpm. Perform all subsequent incubation steps similarly on the shaker.
      NOTE: Sera should be aliquoted and frozen at -80 °C for long-term storage to minimize freeze-thaw cycles and should be vortexed to mix and centrifuged to remove particulates prior to use. Observe slide chambers carefully during and after this step to detect any leakage that requires re-assembling slide chambers.
   3. Using pipette tips connected to a vacuum line with a secondary collection flask, carefully aspirate the blocking buffer from the corner of each chamber without touching the pads. Perform all subsequent aspiration steps similarly. Add diluted sera to pads quickly after aspiration in order to not allow pads to dry.
   4. Place covered frames in trays inside a secondary container surrounded by moist paper towels and sealed to prevent evaporation. Incubate overnight at 4 °C on a rocking shaker (alternatively, can incubate for 2 h at room temperature on an orbital shaker at 100-250 rpm).
2. Label bound serum antibodies with quantum-dot-conjugated secondary antibodies
   1. Aspirate sera from chambers carefully as described above, add 100 μL per well of  T-TBS buffer (20 mM Tris-HCl, 150 mM NaCl, 0.05% Tween-20 in ddH₂O adjusted to pH 7.5 and filtered, can be obtained commercially), and incubate for 5 min on an orbital shaker at 100-250 rpm. Repeat this wash step a total of 3x (all subsequent wash steps are performed similarly).
   2. Prepare a mixture of secondary antibodies diluted to 1 μM in blocking buffer and mix thoroughly by pipetting prior to and during use to maintain homogeneity.
      NOTE: To maintain assay reproducibility, the same batch of each secondary antibody should be used for all probing experiments for which quantitative comparison of data across experiments is planned. The specific concentration of secondary antibody may need to be varied depending on the affinity; follow the manufacturer's protocols whenever available.
   3. Aspirate buffer from chambers after final wash, add 100 μL per well of secondary antibody mixture, and incubate for 2 h at room temperature on a shaker.
   4. Aspirate secondary antibody mixture wash 3x with T-TBS buffer, and then wash once with TBS buffer (without Tween).
   5. Dissemble microarray slides from chambers carefully to avoid touching pads, rinse gently with filtered ddH₂O, and dry by placing them in 50 mL tubes and centrifuging at 500 x g for 10 min.
   6. Place probed microarray slides in a light-proof box and keep them in a desiccator cabinet at room temperature for long-term storage.
      NOTE: The protocol may be paused for up to 1 week at this point.

3. Quantify antibody binding to antigens within microarray

1. Visualize microarray slides and quantify spot fluorescence intensity to measure antibody binding.
   1. Acquire images of microarray slides using the portable imager with built-in software.
      1. In the Configure Imager tab, select the proper slide configuration. For this study, use 16-pad slides.
      2. In the Image Control tab, select the proper fluorescent channel, and adjust the gain, exposure time, and acquisition time depending on the reactivity of the sera to obtain optimal images. For this study, the fluorescent channels for IgA and IgG were 585 nm and 800 nm respectively, and imaging settings were gain of 50, exposure time of 500 ms, and acquisition time of 1 s.
      3. Click on Capture to start the process of acquiring the image.
         NOTE: Microarray slides can be re-imaged at multiple settings without degradation of signals as long as they are stored dark and dry. Other imaging systems can be used if compatible with the slides.
2. Detect array spots using grids oriented based on the fiducial markers and measure spot intensity as the median of pixel intensity minus background measured around spots. Perform this quantification algorithm in batch using the built-in imager software, which utilizes the .gal file constructed in step 1.1.2 to connect spot intensities to individual antigens on each microarray.
   1. In the File Info panel, upload the .gal file by selecting from its folder on the computer, and specify the folder where the analysis output files are to be saved in the Analysis Options section.
   2. In the Image Control tab, open one of the acquired images to be quantified and select the Auto button in the upper right corner.
   3. In the Array Analysis section in the lower right corner, create a fiducial template as instructed by the software.
   4. Click on the Batch Analysis, select the folder that contains the images to be quantified, and select the fiducial template that was created in the previous step. The software analyzes each image and quantifies the spot intensity.
      NOTE: This step will generate a .csv file containing spot intensities quantifying antibodies within each serum specimen that bind to each individual antigen on the microarray that can subsequently be manipulated in spreadsheet manipulation or analysis software.
3. Analyze raw data to compare antibody binding across antigens and across serum specimens. For this study, IgA and IgG antibodies measured as 585 nm and 800 nm fluorescent spot intensities were compared across all antigens between 2 independent runs of the experiment using different slides on different days, and correlation analysis was performed to measure assay reproducibility.
   NOTE: For non-purified proteins printed as expression mixture, data analysis should begin with background subtraction of no DNA control.