The authors would like to acknowledge Prof. Don Milton (Institute of Applied Public Health, University of Maryland, College Park, MD, USA) for collecting the human sera under University of Maryland IRB protocol #313842 funded by DARPA N66001-18-2-4015 P00001. The authors would also like to acknowledge Prof. Florian Krammer (Icahn School of Medicine, Mt. Sinai, NY, USA) for providing trimerized HA and NA antigens funded by ODNI IARPA DJF-15-1200-K-0001725. S. Khan is partially supported by the National Center for Research Resources and the National Center for Advancing Translational Sciences, National Institutes of Health, through grant KL2 TR001416. The content is solely the responsibility of the authors and does not necessarily represent the official views of the NIH.

All human sera are handled and disposed according to approved institutional protocols for biosafety with use of protective personal equipment. All laboratory personnel participating in this protocol have received training in biosafety and research ethics.
1. Produce influenza antigen microarrays
<ol>
	<li>Design and obtain antigen set for microarray
	<ol>
		<li>Obtain expressed and purified protein antigens as lyophilized powder.</li>
	</ol>
	</li>
	<li>Print antigens onto microarray slides
	<ol>
		<li>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 &#956;L of each reconstituted antigen to individual wells of an untreated 384-well flat-bottom plate.</li>
		<li>Print antigens using a microarray printer (the microarray printer used in this study is no longer commercially available, see Discussion) 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.
		<ol>
			<li>Program the printing software (e.g., Gridder) with the source plate configuration and printing parameters.
			<ol>
				<li>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.</li>
				<li>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.</li>
				<li>Select a pin configuration to use with this method. For this study, use an 8-pin configuration.</li>
				<li>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.</li>
				<li>Using the Array Design tab, define the size and shape of the arrays (dot spacing and number of dots per subarray). For this study, print 324 spots (180 &#956;m diameter with 300 &#956;m spacing) onto 16-pad slides in an 18x18 format using 8 pins.</li>
				<li>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)</li>
				<li>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 next antigen.</li>
				<li>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., 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 source plate can be periodically re-suspended by pipetting up and down and then centrifuging, or new source plate can be prepared and used.</li>
			</ol>
			</li>
		</ol>
		</li>
		<li>Place un-probed microarray slides in a lightproof box and keep in a desiccator cabinet at room temperature for long-term storage. 
		NOTE: The protocol may be paused indefinitely at this point.</li>
	</ol>
	</li>
	<li>Perform quality control check (requires poly-histidine tags)
	<ol>
		<li>Attach the slide to probing chambers and rehydrate with blocking buffer as described in step 2.1.1-2.1.2 and shown in Figure 2.</li>
		<li>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.</li>
		<li>Add 100 &#956;L of diluted poly-His antibody to each slide chamber containing array pad after aspiration and incubate for 2 h at room temperature or overnight at 4 &#176;C on 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 &#956;L per well after aspiration, and incubate for 1 h at room temperature on shaker. Wash 3x with T-TBS buffer.</li>
		<li>Dilute Qdot 585 nm streptavidin conjugate to 4 nM in blocking buffer, add 100 &#956;L per well after aspiration, and incubate for 1 h at room temperature on shaker. Wash 3x with T-TBS buffer, and then once with TBS buffer (without Tween).</li>
		<li>Dissemble and quantify slides as described in step 2.2.5 &#8211; 3.1.2. 
		NOTE: Protein antigens must contain His10 tags to use this quality control protocol. Alternatively, if a different tag is included, quality control check can be performed with antibody or ligand for that tag.</li>
	</ol>
	</li>
</ol>
2. Probe sera for influenza antibodies using microarrays
<ol>
	<li>Incubate sera on microarrays for antibody binding
	<ol>
		<li>Attach microarray slides to chambers using clips and place in frames as shown in Figure 3. 
		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 upper right corner.</li>
		<li>Rehydrate microarray slides with 100 &#956;L per well of filtered 1x blocking buffer, and dilute serum 1:100 in 100 &#956;L 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 orbital shaker at 100-250 rpm. Perform all subsequent incubation steps similarly on the shaker. 
		NOTE: Sera should be aliquoted and frozen at -80 &#176;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.</li>
		<li>Using pipette tips connected to a vacuum line with secondary collection flask, carefully aspirate blocking buffer from corner of each chamber without touching pads. Perform all subsequent aspiration steps similarly. Add diluted sera to pads quickly after aspiration in order to not allow pads to dry.</li>
		<li>Place covered frames in trays inside a secondary container surrounded by moist paper towels and sealed to prevent evaporation. Incubate overnight at 4 &#176;C on rocking shaker (alternatively, can incubate for 2 h at room temperature on orbital shaker at 100-250 rpm).</li>
	</ol>
	</li>
	<li>Label bound serum antibodies with quantum-dot-conjugated secondary antibodies
	<ol>
		<li>Aspirate sera from chambers carefully as described above, add 100 &#956;L 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 orbital shaker at 100-250 rpm. Repeat this wash step a total of 3x (all subsequent wash steps are performed similarly).</li>
		<li>Prepare mixture of secondary antibodies diluted to 1 &#956;M 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 manufacturer&#8217;s protocols whenever available.</li>
		<li>Aspirate buffer from chambers after final wash, add 100 &#956;L per well of secondary antibody mixture, and incubate for 2 h at room temperature on shaker.</li>
		<li>Aspirate secondary antibody mixture wash 3x with T-TBS buffer, and then wash once with TBS buffer (without Tween).</li>
		<li>Dissemble microarray slides from chambers carefully to avoid touching pads, rinse gently with filtered ddH2O, and dry by placing in 50 mL tubes and centrifuging at 500 x g for 10 min.</li>
		<li>Place probed microarray slides in a lightproof box and keep 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.</li>
	</ol>
	</li>
</ol>
3. Quantify antibody binding to antigens within microarray
<ol>
	<li>Visualize microarray slides and quantify spot fluorescence intensity to measure antibody binding
	<ol>
		<li>Acquire images of microarray slides using the portable imager with built-in software.
		<ol>
			<li>In the Configure Imager tab, select the proper slide configuration. For this study, use 16-pad slides.</li>
			<li>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.</li>
			<li>Click on Capture to start the process of acquiring the image. 
			NOTE: Microarray slides can be re-imaged at multiple settings without degradation of signal as long as they are stored dark and dry. Other imaging systems can be used if compatible with the slides.</li>
		</ol>
		</li>
		<li>Detect array spots using grids oriented based on the fiducial markers and measure spot intensity as 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 1.2.2 to connect spot intensities to individual antigens on each microarray.
		<ol>
			<li>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.</li>
			<li>In the Image Control tab, open one of the acquired images to be quantified and select the Auto button in the upper right corner.</li>
			<li>In the Array Analysis section in the lower right corner, create a fiducial template as instructed by the software.</li>
			<li>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.</li>
		</ol>
		</li>
		<li>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 a no DNA control.</li>
	</ol>
	</li>
</ol>

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The authors have no disclosures.

The influenza antigen microarray protocol described here is adaptable to any project that requires analyzing antibody responses to many antigens. The microarray platform can be used with any desired set of protein antigens expressed in any system that can achieve 0.1 mg/mL or higher yield with or without purification as previously described12. If non-purified protein antigens (e.g., expressed in in vitro transcription and translation system) are directly used to print microarrays, components of protein expression system (e.g., E. coli lysate) should be added 1:10 to blocking buffer used for dilution of sera and quality control antibodies in order to block any antibodies directed against these components. Slide configurations are available with a lower number of larger pads on each slide to accommodate a higher number of antigens per array. For this study, we used a GeneMachines OmniGrid 100 microarray printer that utilizes pins that directly contact the nitrocellulose surface to deposit spots on the array. While this microarray printer is no longer commercially available, other commercially available microarray printers can be used in this protocol but may require custom pins (either contact or non-contact) and software and should have sufficient spot resolution and compatibility with slides and imager. Depending on reactivity of sera, dilutions from 1:50 to 1:400 can be used. Serum-free buffer can be used as a negative control, while monoclonal antibodies known to bind to antigen can be used as a positive control.
Users should be aware of a few troubleshooting issues. The purpose of the quality control check using antibodies to the His tag is to check for any antigens for which printing was not successful. Any spots that consistently give low signal in quality control check of multiple arrays likely represent insufficient antigen printed. Possible reasons include aggregation or precipitation or antigen in source plate or poor contact between printing pin and microarray slide due to variable thickness of nitrocellulose pad. At this point, we do not perform assay normalization based on quantitative results of the quality control check, given that the detected binding of anti-His antibodies can be influenced by availability of this tag, which depends on the 3-dimensional conformation specific to each antigen.
The influenza antigen microarray provides several advantages over traditional methods such as ELISA and is complementary to functional assays such as HAI and MN. The 16-pad protein microarray is a sample-sparing technology with capacity to measure antibodies of multiple isotypes simultaneously against approximately 300 antigens from 1 &#181;L of serum. The number of antigens can be increased to the thousands by decreasing the number of pads per slides. The multiplexed assay also spares personnel time and consumable resources, given that hundreds of sera can be probed for antibodies in 2 days, and all materials other than slides and reagents are re-usable so do not generate large amounts of plastic waste.
While microarray printers may not be widely distributed, microarray slides can be printed at a centralized location and then transported to the end user for probing. The only equipment required for probing is the low-cost and portable imager. The goal of disseminating this protocol is to make utilization of this technique more widespread.
The main limitations of the influenza antigen microarray are the inability to characterize the function and kinetics of the detected antibodies. The microarray is detecting a polyclonal set of binding antibodies for each antigen. These antibodies may or may not be functional in neutralizing virus in HAI and MN assays. However, HAI and MN assays require live virus culture with associated need for specialized facilities with high-level biosafety cabinets to test for antibodies against avian subtypes of influenza, whereas the protein microarray does not involve live virus components so can be utilized in any basic laboratory. With respect to binding kinetics, a single dilution of serum probed with an array containing a single concentration of each antigen yields a single data point, which represents a composite of quantity and affinity summated over all antibodies that bind to the antigen. To fully resolve the antigen-antibody binding kinetics, multiple antigen concentrations and/or serial dilutions of sera are required.
Despite these limitations, the influenza antigen microarray is a useful tool to characterize breadth of influenza antibodies across the antigenic landscape that can complement functional assays that are more limited in throughput and availability.

The influenza virus is responsible for a loss of 20 million life-years annually by death or disability, including 1% of all deaths worldwide each year, with disproportionate impacts on the elderly and populations in the tropics and developing world1,2,3. In addition to the disease burden of seasonal epidemics, the emergence of novel influenza strains via genetic re-assortment either naturally in common hosts or artificially for bioterrorism could lead to worldwide pandemics with rapid spread and high lethality4,5. While numerous influenza vaccines are currently available, their effectiveness is limited by subtype specificity6, creating the need to develop universal influenza vaccines that confer long-lasting immunity against multiple virus strains7.
A key challenge to the development of universal influenza vaccines is high antigenic diversity across strains. The antigenic specificity of current vaccines combined with antigenic variation of circulating viruses creates a mismatch between vaccine strains and circulating strains. This confers an evolutionary advantage favoring further genetic drift away from vaccine strains during an epidemic, limiting vaccine efficacy often to less than 50%8,9. An additional source of antigenic mismatch is egg-adaptive viral mutations generated during vaccine manufacture, which lead to antibodies that bind poorly to circulating viruses10,11.
Overcoming this challenge of high antigenic diversity will require novel research tools to characterize the breadth of pre-existing and elicited immune responses across clinically relevant antigenic variants in serum and mucosal specimens. Currently available methods, including hemagglutination inhibition (HAI), microneutralization (MN), and traditional ELISA, are limited to detecting antibodies against a single virus strain at a time, so their use for detection of multiple antibody isotypes against multiple virus strains quickly exhausts available clinical specimen and laboratory resources. Furthermore, HAI and MN require live virus culture that is only available in specialized laboratories.
Protein microarrays, potentially consisting of up to thousands of antigens printed onto nitrocellulose-coated slides as shown in Figure 1, can fill this need12. These microarrays can be produced and probed in a high throughput manner while consuming small quantities of clinical specimen to determine quantitative antibody isotype/subtype levels against each individual antigen on the array. This approach to antigen discovery has been applied to diagnostic and vaccine development against multiple infectious pathogens13. To date, we have produced protein microarrays for over 35 pathogens including over 60,000 total expressed proteins and used them to probe over 30,000 human sera from infected and control individuals. A recently developed portable imaging platform for microarray slides has made this methodology more accessible to the end user14.
Building on extensive previous work by multiple contributors in the field15,16,17,18,19, an influenza protein microarray was recently developed that contains over 250 purified hemagglutinin (HA) antigenic variants with representation of all 18 subtypes12,20. Using this methodology, a natural influenza infection was demonstrated to generate broadly reactive IgG and IgA antibodies against phylogenetically related HA subtypes, while an intramuscular influenza vaccination generated only subtype-specific IgG antibodies21. However, adding an adjuvant that activates toll-like receptors to influenza vaccines was shown to broaden the elicited IgG antibody response across HA subtypes in animal studies22.
This microarray is currently being used to probe sera collected from a prospective cohort study of college students who were followed for influenza infection. Here, the methodology of the influenza antigen microarray with demonstration of assay reproducibility to detect subtype-specific and cross-reactive antibodies in a subset of specimens from this study is reported.

<table>
	<tbody>
		<tr>
			<td>16-pad nitrocellulose-coated glass slides</td>
			<td>Grace Bio Labs</td>
			<td>305016</td>
			<td></td>
		</tr>
		<tr>
			<td>1x GVS FAST blocking buffer</td>
			<td>Fischer Scientific</td>
			<td>10485356</td>
			<td></td>
		</tr>
		<tr>
			<td>ArrayCam portable imager</td>
			<td>Grace Bio Labs</td>
			<td>400S</td>
			<td>Other imaging devices can be used to visualize slides if capable of achieving the resolution of the microarray spots and the excitation and emission wavelengths of the quantum dots.</td>
		</tr>
		<tr>
			<td>Biotin-conjugated goat anti-mouse-IgG antibody</td>
			<td>Thermo Fischer</td>
			<td>31800</td>
			<td></td>
		</tr>
		<tr>
			<td>HiBase 384-well plate</td>
			<td>Greiner Bio-One</td>
			<td>T-3037-11</td>
			<td></td>
		</tr>
		<tr>
			<td>Microarray pins</td>
			<td>ArrayIt</td>
			<td>GMP2</td>
			<td>Each different microarray printer may require its own custom microarray pins.</td>
		</tr>
		<tr>
			<td>Mouse monoclonal poly-His antibody</td>
			<td>Sigma-Aldrich</td>
			<td>H1029</td>
			<td></td>
		</tr>
		<tr>
			<td>OmniGrid 100 microarray printer</td>
			<td>GeneMachines</td>
			<td></td>
			<td>The version of the microarray printer used in this work is no longer commercially available, but the updated similar equipment is the OmniGrid Accent microarray printer from Digilab (Hopkinton, MA), and the same protocol can be carried out with most commercially available microarray printers.</td>
		</tr>
		<tr>
			<td>ProPlate slide chambers</td>
			<td>Grace Bio Labs</td>
			<td>246890</td>
			<td></td>
		</tr>
		<tr>
			<td>ProPlate slide clips</td>
			<td>Grace Bio Labs</td>
			<td>204838</td>
			<td></td>
		</tr>
		<tr>
			<td>ProPlate slide frames</td>
			<td>Grace Bio Labs</td>
			<td>246879</td>
			<td></td>
		</tr>
		<tr>
			<td>Quantum dot 585 nm conjugated goat anti-human-IgA antibody</td>
			<td>Grace Bio Labs</td>
			<td>110620</td>
			<td></td>
		</tr>
		<tr>
			<td>Quantum dot 585 nm streptavidin conjugate</td>
			<td>Thermo Fischer</td>
			<td>Q10111MP</td>
			<td></td>
		</tr>
		<tr>
			<td>Quantum dot 800 nm conjugated goat anti-human-IgG antibody</td>
			<td>Grace Bio Labs</td>
			<td>110610</td>
			<td></td>
		</tr>
	</tbody>
</table>

use of an influenza antigen microarray to measure the breadth of serum antibodies across virus subtypes

The influenza virus remains a significant cause of mortality worldwide due to the limited effectiveness of currently available vaccines. A key challenge to the development of universal influenza vaccines is high antigenic diversity resulting from antigenic drift. Overcoming this challenge requires novel research tools to measure the breadth of serum antibodies directed against many virus strains across different antigenic subtypes. Here, we present a protocol for analyzing the breadth of serum antibodies against diverse influenza virus strains using a protein microarray of influenza antigens.
This influenza antigen microarray is constructed by printing purified hemagglutinin and neuraminidase antigens onto a nitrocellulose-coated membrane using a microarray printer. Human sera are incubated on the microarray to bind antibodies against the influenza antigens. Quantum-dot-conjugated secondary antibodies are used to simultaneously detect IgG and IgA antibodies binding to each antigen on the microarray. Quantitative antibody binding is measured as fluorescence intensity using a portable imager. Representative results are shown to demonstrate assay reproducibility in measuring subtype-specific and cross-reactive influenza antibodies in human sera.
Compared to traditional methods such as ELISA, the influenza antigen microarray provides a high throughput multiplexed approach capable of testing hundreds of sera for multiple antibody isotypes against hundreds of antigens in a short time frame, and thus has applications in sero-surveillance and vaccine development. A limitation is the inability to distinguish binding antibodies from neutralizing antibodies.

As a demonstration of the protocol, baseline sera were assayed from 16 individuals within a prospective cohort study of college students followed for influenza infection on the influenza antigen microarray. To demonstrate assay reproducibility, these specimens were probed twice, on different slides and different days.
For this study, purified influenza antigens containing His10 tags were obtained from commercial vendor (see Table of Materials) and collaborators. These antigens include 251 total HA antigens, with 63 globular head domains (HA1) and 186 full-length proteins (HA0), including 96 monomeric HA0 proteins and 90 trimerized HA0 proteins containing fused trimerization (&#34;foldon&#34;) domain23. A full list of antigens and controls used in this study is included as a Supplementary File. For this study, secondary antibodies used were goat anti-human IgG conjugated to quantum dot emitting at 800 nm (GAH-IgG-Q800) and goat anti-human IgA conjugated to quantum dot emitting at 585 nm (GAH-IgA-Q585) for multiplex detection of IgG and IgA antibodies as shown in Figure 3. IgG and IgA antibody binding to different subtypes and molecular forms of influenza HA antigens were compared for sera obtained from the clinical cohort described above.
The resulting heat map is shown in Figure 4 with graphical representation in Figure 5. In these figures, only the clinically relevant subtypes with high representation on the array are labeled to save space, with &#34;+&#34; denoting all remaining subtypes of higher number, and minor or less well represented subtypes included as ordered (e.g., H2 in between H1 and H3). A full list of strains and subtypes on the array is included as Supplementary File. These data demonstrate that antibodies to the head group of HA are subtype-specific with expected high quantity of antibodies to clinically prevalent strains (H1N1, H3N2, and B) and low quantity of antibodies to other strains. However, antibodies to the whole HA, which includes the stalk domain, are more cross-reactive across subtypes, and this effect appears to be augmented when the whole HA is trimerized. This result is not unexpected, as the whole HA includes the stem region, which is highly conserved across subtypes. Therefore, antibodies to whole HA molecules from non-clinical subtypes (e.g., H5 and H7) likely represent anti-stem antibodies originally elicited against clinical subtypes (e.g., H1 and H3) that are cross-reacting with the stem regions of the other HA subtypes on the array. This point illustrates the importance of including both head HA and whole molecule HA on the array to distinguish between antibodies to the head and the stem regions which show different reactivity profiles.
The assay demonstrates good reproducibility across probing runs. The second run does show slightly lower IgG antibodies across all strains, although the pattern between the strains is consistent. This across-the-board slight decrease is likely due to batch-to-batch variability in the secondary antibody, which was changed between runs for IgG but not for IgA. Thus, as noted in the protocol, it is recommended to use the same batch of each secondary antibody for any experiments between which quantitative comparison is planned. If different batches of antibody are necessary due to a high number of samples to be tested, we recommended including shared samples between experimental runs with different antibody batches to allow for quantitative comparison with correction.
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/59973/59973fig1.jpg" /> 
Figure 1: Schematic of protein microarray. Each slide contains multiple pads each with a single array, which consists of hundreds of antigens printed onto spots arranged in a grid, with each spot containing one antigen adsorbed onto the 3-dimensional topography of the nitrocellulose surface to which antibodies from serum are bound. <a href="https://www.jove.com/files/ftp_upload/59973/59973fig1large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 2" class="xfigimg" src="/files/ftp_upload/59973/59973fig2.jpg" /> 
Figure 2: Schematic of influenza antigen microarray printing and probing protocol. From left to right, microarray is printed using onto nitrocellulose-coated slides, which are used to probe sera for IgG and IgA antibodies using quantum-dot-conjugated secondary antibodies, with slides imaged using a portable imager, and results analyzed to generate a heat map. <a href="https://www.jove.com/files/ftp_upload/59973/59973fig2large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 3" class="xfigimg" src="/files/ftp_upload/59973/59973fig3.jpg" /> 
Figure 3: Procedure for attaching probing chamber to microarray slide. From A to F, the probing chamber is placed on top of slide in correct orientation, attached to the slide using horizontal clips on the sides, and placed in the probing tray. <a href="https://www.jove.com/files/ftp_upload/59973/59973fig3large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 4" class="xfigimg" src="/files/ftp_upload/59973/59973fig4.jpg" /> 
Figure 4: Representative results of influenza antigen microarray. Heat maps represent antigen-specific antibody responses, with each row representing a single antigen arranged by molecule, subtype, and strain, and each column representing a probing run of a single specimen, arranged by antibody isotype and run (A, white = 0, black = 20000, red = 40000 fluorescence intensity). The antigen subtypes including all hemagglutinin subtypes from 1 to 18 and all neuraminidase subtypes from 1 to 10 are arranged vertically and labeled on the left. A comparison of the fluorescence intensity between two runs demonstrates good assay reproducibility by linear regression for IgA (B) and IgG(C). <a href="https://www.jove.com/files/ftp_upload/59973/59973fig4large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 5" class="xfigimg" src="/files/ftp_upload/59973/59973fig5.jpg" /> 
Figure 5: Breadth of serum antibodies measured on influenza antigen microarray. Serum IgA (A) and IgG (B) are grouped by HA and NA molecular forms and subtypes to demonstrate high specificity of HA head group antibodies for clinical subtypes and high cross-reactivity of whole HA and trimerized whole HA antibodies with inclusion of stalk region. <a href="https://www.jove.com/files/ftp_upload/59973/59973fig5large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
Supplementary File: List of antigens on influenza antigen microarray. Content is shown for all 324 spots on the array, including blanks, fiducials, controls (human IgG and IgA and anti-human IgG and IgA at 0.1 mg/mL and 0.3 mg/mL), and antigens with information on source, molecular form, subtype, and strain. Abbreviations are as follows: for source, Sino = Sino Biological Inc., FKL = Florian Krammer Laboratory; for molecular form, HA1 = head HA, HA0 = whole HA, HA2 = stalk HA, NP = nucleoprotein. For antigens sourced from Sino Biological Inc., catalog numbers are shown; for antigens sourced from Krammer Laboratory, antigen IDs are listed. <a href="https://www.jove.com/files/ftp_upload/59973/Supplementary_File.xlsm">Please click here to download this file.</a>

Watch this Scientific Journal Video about Use of an Influenza Antigen Microarray to Measure the Breadth of Serum Antibodies Across Virus Subtypes at JoVE.com

Use of an Influenza Antigen Microarray to Measure the Breadth of Serum Antibodies Across Virus Subtypes

We present a protocol for using a protein microarray constructed by printing influenza antigens onto nitrocellulose-coated slides to simultaneously probe serum for multiple antibody isotypes against over 250 antigens from different virus strains, thus allowing measurement of the breadth of serum antibodies across virus subtypes.

The influenza virus remains a significant cause of mortality worldwide due to the limited effectiveness of currently available vaccines. A key challenge ...

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8.0K Views.  University of California Irvine Health. We present a protocol for using a protein microarray constructed by printing influenza antigens onto nitrocellulose-coated slides to simultaneously probe serum for multiple antibody isotypes against over 250 antigens from different virus strains, thus allowing measurement of the breadth of serum antibodies across virus subtypes.

Video: Use of an Influenza Antigen Microarray to Measure the Breadth of Serum Antibodies Across Virus Subtypes

Generation of Recombinant Influenza Virus from Plasmid DNA

Efforts by a number of influenza research groups have been pivotal in the development and improvement of influenza A virus reverse genetics. Originally established in 1999 1,2 plasmid-based reverse genetic techniques to generate recombinant viruses have revolutionized the influenza research field because specific questions have been answered by genetically engineered, infectious, recombinant influenza viruses. Such studies include virus replication, function of viral proteins, the contribution of specific mutations in viral proteins in viral replication and/or pathogenesis and, also, viral vectors using recombinant influenza viruses expressing foreign proteins 3.

Rescue of influenza A viruses from plasmid DNA is a basic and essential experimental technique that allows influenza researchers to generate recombinant viruses to study multiple aspects in the biology of influenza virus, and to be used as potential vectors or vaccines.

Efforts by a number of influenza research groups have been pivotal in the development and improvement of influenza A virus reverse genetics. Originally ...

Using Bioluminescent Imaging to Investigate Synergism Between Streptococcus pneumoniae and Influenza A Virus in Infant Mice

During the 1918 influenza virus pandemic, which killed approximately 50 million people worldwide, the majority of fatalities were not the result of infection with influenza virus alone. Instead, most individuals are thought to have succumbed to a secondary bacterial infection, predominately caused by the bacterium Streptococcus pneumoniae (the pneumococcus). The synergistic relationship between infections caused by influenza virus and the pneumococcus has subsequently been observed during the 1957 Asian influenza virus pandemic, as well as during seasonal outbreaks of the virus (reviewed in 1, 2). Here, we describe a protocol used to investigate the mechanism(s) that may be involved in increased morbidity as a result of concurrent influenza A virus and S. pneumoniae infection. We have developed an infant murine model to reliably and reproducibly demonstrate the effects of influenza virus infection of mice colonised with S. pneumoniae. Using this protocol, we have provided the first insight into the kinetics of pneumococcal transmission between co-housed, neonatal mice using in vivo imaging 3.

Using Bioluminescent Imaging to Investigate Synergism Between Streptococcus pneumoniae and Influenza A Virus in Infant Mice

A concurrent infection with influenza A virus is one of the factors implicated in the induction of invasive pneumococcal disease during asymptomatic Streptococcus pneumoniae carriage. Here we describe a mixed infection method using infant mice to investigate the synergism between these two respiratory pathogens.

During the 1918 influenza virus pandemic, which killed approximately 50 million people worldwide, the majority of fatalities were not the result of ...

ampliPHOX Colorimetric Detection on a DNA Microarray for Influenza

DNA microarrays have emerged as a powerful tool for pathogen detection.1-5 For instance, many examples of the ability to type and subtype influenza virus have been demonstrated.6-11 The identification and subtyping of influenza on DNA microarrays has applications in both public health and the clinic for early detection, rapid intervention, and minimizing the impact of an influenza pandemic. Traditional fluorescence is currently the most commonly used microarray detection method. However, as microarray technology progresses towards clinical use,1 replacing expensive instrumentation with low cost detection technology exhibiting similar performance characteristics to fluorescence will make microarray assays more attractive and cost-effective.
The ampliPHOX colorimetric detection technology is intended for research applications, and has a limit of detection within one order of magnitude of traditional fluorescence11, with a main advantage being an approximate ten-fold lower instrument cost compared to the confocal microarray scanners required for fluorescence microarray detection. Another advantage is the compact size of the instrument which allows for portability and flexibility, unlike traditional fluorescence instruments. Because the polymerization technology is not as inherently linear as fluorescence detection, however, it is best suited for lower density microarray applications in which a yes/no answer for the presence of a certain sequence is desired, such as for pathogen detection arrays. Currently the maximum spot density compatible with ampliPHOX detection is &tilde;1800 spots/array. Because of the spot density limitations, higher density microarrays are not suitable for ampliPHOX detection.
Here, we present ampliPHOX colorimetric detection technology as a method of signal amplification on a low density microarray developed for the detection and characterization of influenza viruses (FluChip). Although this protocol uses the FluChip (a DNA microarray) as one specific application of ampliPHOX detection, any microarray incorporating biotinylated target can be labeled and detected in a similar manner. The microarray design and biotinylation of the target to be captured are the responsibility of the user. Once the biotinylated target has been captured on the array, ampliPHOX detection can be performed by first tagging the array with a streptavidin-label conjugate (ampliTAG). Upon light exposure using the ampliPHOX Reader instrument, polymerization of a monomer solution (ampliPHY) occurs only in regions containing ampliTAG-labeled targets. The polymer formed can be subsequently stained with a non-toxic solution to improve visual contrast, followed by imaging and analysis using a simple software package (ampliVIEW). The entire FluChip assay from un-extracted sample to result can be performed in about 6 hours, and the ampliPHOX detection steps described above can be completed in about 30 min.

ampliPHOX colorimetric detection technology is presented as an inexpensive alternative to fluorescence detection for microarrays. Based on photopolymerization, ampliPHOX produces solid polymer spots visible to the naked eye in just a few minutes. Results are then imaged and automatically interpreted with a simple yet powerful software package.

DNA microarrays have emerged as a powerful tool for pathogen detection.1-5  For instance, many examples of the ability to type and subtype influenza virus ...

Rapid Diagnosis of Avian Influenza Virus in Wild Birds: Use of a Portable rRT-PCR and Freeze-dried Reagents in the Field

Wild birds have been implicated in the spread of highly pathogenic avian influenza (HPAI) of the H5N1 subtype, prompting surveillance along migratory flyways. Sampling of wild birds for avian influenza virus (AIV) is often conducted in remote regions, but results are often delayed because of the need to transport samples to a laboratory equipped for molecular testing. Real-time reverse transcriptase polymerase chain reaction (rRT-PCR) is a molecular technique that offers one of the most accurate and sensitive methods for diagnosis of AIV. The previously strict lab protocols needed for rRT-PCR are now being adapted for the field. Development of freeze-dried (lyophilized) reagents that do not require cold chain, with sensitivity at the level of wet reagents has brought on-site remote testing to a practical goal.
Here we present a method for the rapid diagnosis of AIV in wild birds using an rRT-PCR unit (Ruggedized Advanced Pathogen Identification Device or RAPID, Idaho Technologies, Salt Lake City, UT) that employs lyophilized reagents (Influenza A Target 1 Taqman; ASAY-ASY-0109, Idaho Technologies). The reagents contain all of the necessary components for testing at appropriate concentrations in a single tube: primers, probes, enzymes, buffers and internal positive controls, eliminating errors associated with improper storage or handling of wet reagents. The portable unit performs a screen for Influenza A by targeting the matrix gene and yields results in 2-3 hours. Genetic subtyping is also possible with H5 and H7 primer sets that target the hemagglutinin gene.
The system is suitable for use on cloacal and oropharyngeal samples collected from wild birds, as demonstrated here on the migratory shorebird species, the western sandpiper (Calidrus mauri) captured in Northern California. Animal handling followed protocols approved by the Animal Care and Use Committee of the U.S. Geological Survey Western Ecological Research Center and permits of the U.S. Geological Survey Bird Banding Laboratory. The primary advantage of this technique is to expedite diagnosis of wild birds, increasing the chances of containing an outbreak in a remote location. On-site diagnosis would also prove useful for identifying and studying infected individuals in wild populations. The opportunity to collect information on host biology (immunological and physiological response to infection) and spatial ecology (migratory performance of infected birds) will provide insights into the extent to which wild birds can act as vectors for AIV over long distances.

This study describes diagnosis of avian influenza in wild birds using a portable rRT-PCR system. The method takes advantage of freeze-dried reagents to screen wild birds in a non-laboratory setting, typical of an outbreak scenario. Use of molecular tools provides accurate and sensitive alternatives for rapid diagnosis.

Wild birds have been implicated in the spread of highly pathogenic avian influenza (HPAI) of the H5N1 subtype, prompting surveillance along migratory ...

High-throughput Detection Method for Influenza Virus

Influenza virus is a respiratory pathogen that causes a high degree of morbidity and mortality every year in multiple parts of the world. Therefore, precise diagnosis of the infecting strain and rapid high-throughput screening of vast numbers of clinical samples is paramount to control the spread of pandemic infections. Current clinical diagnoses of influenza infections are based on serologic testing, polymerase chain reaction, direct specimen immunofluorescence and cell culture 1,2.
Here, we report the development of a novel diagnostic technique used to detect live influenza viruses. We used the mouse-adapted human A/PR/8/34 (PR8, H1N1) virus 3 to test the efficacy of this technique using MDCK cells 4. MDCK cells (104 or 5 x 103 per well) were cultured in 96- or 384-well plates, infected with PR8 and viral proteins were detected using anti-M2 followed by an IR dye-conjugated secondary antibody. M2 5 and hemagglutinin 1 are two major marker proteins used in many different diagnostic assays. Employing IR-dye-conjugated secondary antibodies minimized the autofluorescence associated with other fluorescent dyes. The use of anti-M2 antibody allowed us to use the antigen-specific fluorescence intensity as a direct metric of viral quantity. To enumerate the fluorescence intensity, we used the LI-COR Odyssey-based IR scanner. This system uses two channel laser-based IR detections to identify fluorophores and differentiate them from background noise. The first channel excites at 680 nm and emits at 700 nm to help quantify the background. The second channel detects fluorophores that excite at 780 nm and emit at 800 nm. Scanning of PR8-infected MDCK cells in the IR scanner indicated a viral titer-dependent bright fluorescence. A positive correlation of fluorescence intensity to virus titer starting from 102-105 PFU could be consistently observed. Minimal but detectable positivity consistently seen with 102-103 PFU PR8 viral titers demonstrated the high sensitivity of the near-IR dyes. The signal-to-noise ratio was determined by comparing the mock-infected or isotype antibody-treated MDCK cells.
Using the fluorescence intensities from 96- or 384-well plate formats, we constructed standard titration curves. In these calculations, the first variable is the viral titer while the second variable is the fluorescence intensity. Therefore, we used the exponential distribution to generate a curve-fit to determine the polynomial relationship between the viral titers and fluorescence intensities. Collectively, we conclude that IR dye-based protein detection system can help diagnose infecting viral strains and precisely enumerate the titer of the infecting pathogens.

This method describes the use of Infrared dye based imaging system for detection of H1N1 in bronchioalveolar lavage (BAL) fluid of infected mice at a high sensitivity. This methodology can be performed in a 96- or 384-well plate, requires &lt;10 &mu;l volume of test material and has the potential for concurrent screening of multiple pathogens.

Influenza virus is a respiratory pathogen that causes a high degree of morbidity and mortality every year in multiple parts of the world. Therefore, ...

Affinity Purification of Influenza Virus Ribonucleoprotein Complexes from the Chromatin of Infected Cells

Like all negative-strand RNA viruses, the genome of influenza viruses is packaged in the form of viral ribonucleoprotein complexes (vRNP), in which the single-stranded genome is encapsidated by the nucleoprotein (NP), and associated with the trimeric polymerase complex consisting of the PA, PB1, and PB2 subunits. However, in contrast to most RNA viruses, influenza viruses perform viral RNA synthesis in the nuclei of infected cells. Interestingly, viral mRNA synthesis uses cellular pre-mRNAs as primers, and it has been proposed that this process takes place on chromatin1. Interactions between the viral polymerase and the host RNA polymerase II, as well as between NP and host nucleosomes have also been characterized1,2.
Recently, the generation of recombinant influenza viruses encoding a One-Strep-Tag genetically fused to the C-terminus of the PB2 subunit of the viral polymerase (rWSN-PB2-Strep3) has been described. These recombinant viruses allow the purification of PB2-containing complexes, including vRNPs, from infected cells. To obtain purified vRNPs, cell cultures are infected, and vRNPs are affinity purified from lysates derived from these cells. However, the lysis procedures used to date have been based on one-step detergent lysis, which, despite the presence of a general nuclease, often extract chromatin-bound material only inefficiently. 
Our preliminary work suggested that a large portion of nuclear vRNPs were not extracted during traditional cell lysis, and therefore could not be affinity purified. To increase this extraction efficiency, and to separate chromatin-bound from non-chromatin-bound nuclear vRNPs, we adapted a step-wise subcellular extraction protocol to influenza virus-infected cells. Briefly, this procedure first separates the nuclei from the cell and then extracts soluble nuclear proteins (here termed the "nucleoplasmic" fraction). The remaining insoluble nuclear material is then digested with Benzonase, an unspecific DNA/RNA nuclease, followed by two salt extraction steps: first using 150 mM NaCl (termed "ch150"), then 500 mM NaCl ("ch500") (Fig. 1). These salt extraction steps were chosen based on our observation that 500 mM NaCl was sufficient to solubilize over 85% of nuclear vRNPs yet still allow binding of tagged vRNPs to the affinity matrix.
After subcellular fractionation of infected cells, it is possible to affinity purify PB2-tagged vRNPs from each individual fraction and analyze their protein and RNA components using Western Blot and primer extension, respectively. Recently, we utilized this method to discover that vRNP export complexes form during late points after infection on the chromatin fraction extracted with 500 mM NaCl (ch500)3.

Influenza viruses replicate their RNA genome in association with host-cell chromatin. Here, we present a method to purify intact viral ribonucleoprotein complexes from the chromatin of infected cells. Purified viral complexes can be analyzed by both Western blot and primer extension of protein and RNA content, respectively.

Like all negative-strand RNA viruses, the genome of influenza viruses is packaged in the form of viral ribonucleoprotein complexes (vRNP), in which the ...

Influenza Virus Propagation in Embryonated Chicken Eggs

Influenza infection is associated with about 36,000 deaths and more than 200,000 hospitalizations every year in the United States. The continuous emergence of new influenza virus strains due to mutation and re-assortment complicates the control of the virus and necessitates the permanent development of novel drugs and vaccines. The laboratory-based study of influenza requires a reliable and cost-effective method for the propagation of the virus. Here, a comprehensive protocol is provided for influenza A virus propagation in fertile chicken eggs, which consistently yields high titer viral stocks. In brief, serum pathogen-free (SPF) fertilized chicken eggs are incubated at 37 &#176;C and 55-60% humidity for 10 &#8211; 11 days. Over this period, embryo development can be easily monitored using an egg candler. Virus inoculation is carried out by injection of virus stock into the allantoic cavity using a needle. After 2 days of incubation at 37 &#176;C, the eggs are chilled for at least 4 hr at 4 &#176;C. The eggshell above the air sac and the chorioallantoic membrane are then carefully opened, and the allantoic fluid containing the virus is harvested. The fluid is cleared from debris by centrifugation, aliquoted and transferred to -80 &#176;C for long-term storage. The large amount (5-10 ml of virus-containing fluid per egg) and high virus titer which is usually achieved with this protocol has made the usage of eggs for virus preparation our favorable method, in particular for in vitro studies which require large quantities of virus in which high dosages of the same virus stock are needed.

Fertile chicken eggs are widely used to produce large amounts of human influenza A virus as they provide a convenient and cost-effective system to prove high yields of virus.

Influenza infection is associated with about 36,000 deaths and more than 200,000 hospitalizations every year in the United States. The continuous ...

Co-immunoprecipitation of the Mouse Mx1 Protein with the Influenza A Virus Nucleoprotein

Studying the interaction between proteins is key in understanding their function(s). A very powerful method that is frequently used to study interactions of proteins with other macromolecules in a complex sample is called co-immunoprecipitation. The described co-immunoprecipitation protocol allows to demonstrate and further investigate the interaction between the antiviral myxovirus resistance protein 1 (Mx1) and one of its viral targets, the influenza A virus nucleoprotein (NP). The protocol starts with transfected mammalian cells, but it is also possible to use influenza A virus infected cells as starting material. After cell lysis, the viral NP protein is pulled-down with a specific antibody and the resulting immune-complexes are precipitated with protein G beads. The successful pull-down of NP and the co-immunoprecipitation of the antiviral Mx1 protein are subsequently revealed by western blotting. A prerequisite for successful co-immunoprecipitation of Mx1 with NP is the presence of N-ethylmaleimide (NEM) in the cell lysis buffer. NEM alkylates free thiol groups. Presumably this reaction stabilizes the weak and/or transient NP&#8211;Mx1 interaction by preserving a specific conformation of Mx1, its viral target or an unknown third component. An important limitation of co-immunoprecipitation experiments is the inadvertent pull-down of contaminating proteins, caused by nonspecific binding of proteins to the protein G beads or antibodies. Therefore, it is very important to include control settings to exclude false positive results. The described co-immunoprecipitation protocol can be used to study the interaction of Mx proteins from different vertebrate species with viral proteins, any pair of proteins, or of a protein with other macromolecules. The beneficial role of NEM to stabilize weak and/or transient interactions needs to be tested for each interaction pair individually.

This co-immunoprecipitation protocol allows to study the interaction between the influenza A virus nucleoprotein and the antiviral Mx1 protein in human cells. The protocol emphasizes the importance of N-ethylmaleimide for successful co-immunoprecipitation of Mx1 and influenza A virus nucleoprotein.

Studying the interaction between proteins is key in understanding their function(s). A very powerful method that is frequently used to study interactions ...

Nasal Wipes for Influenza A Virus Detection and Isolation from Swine

Surveillance for influenza A viruses in swine is critical to human and animal health because influenza A virus rapidly evolves in swine populations and new strains are continually emerging. Swine are able to be infected by diverse lineages of influenza A virus making them important hosts for the emergence and maintenance of novel influenza A virus strains. Sampling pigs in diverse settings such as commercial swine farms, agricultural fairs, and live animal markets is important to provide a comprehensive view of currently circulating IAV strains. The current gold-standard ante-mortem sampling technique (i.e. collection of nasal swabs) is labor intensive because it requires physical restraint of the pigs. Nasal wipes involve rubbing a piece of fabric across the snout of the pig with minimal to no restraint of the animal. The nasal wipe procedure is simple to perform and does not require personnel with professional veterinary or animal handling training. While slightly less sensitive than nasal swabs, virus detection and isolation rates are adequate to make nasal wipes a viable alternative for sampling individual pigs when low stress sampling methods are required. The proceeding protocol outlines the steps needed to collect a viable nasal wipe from an individual pig.

The authors present a protocol to collect swine nasal wipes to detect and isolate influenza A viruses.

Surveillance for influenza A viruses in swine is critical to human and animal health because influenza A virus rapidly evolves in swine populations and ...

A Miniaturized Glycan Microarray Assay for Assessing Avidity and Specificity of Influenza A Virus Hemagglutinins

Influenza A virus (IAV) hemagglutinins recognize sialic acids on the cell surface as functional receptors to gain entry into cells. Wild waterfowl are the natural reservoir for IAV, but IAV can cross the species barrier to poultry, swine, horses and humans. Avian viruses recognize sialic acid attached to a penultimate galactose by a &#945;2-3 linkage (avian-type receptors) whereas human viruses preferentially recognize sialic acid with a &#945;2-6 linkage (human-type receptors). To monitor if avian viruses are adapting to human type receptors, several methods can be used. Glycan microarrays with diverse libraries of synthetic sialosides are increasingly used to evaluate receptor specificity. However, this technique is not used for measuring avidities. Measurement of avidity is typically achieved by evaluating the binding of serially diluted hemagglutinin or virus to glycans adsorbed to conventional polypropylene 96-well plates. In this assay, glycans with &#945;2-3 or &#945;2-6 sialic acids are coupled to biotin and adsorbed to streptavidin plates, or are coupled to polyacrylamide (PAA) which directly adsorb to the plastic. We have significantly miniaturized this assay by directly printing PAA-linked sialosides and their non PAA-linked counterparts on micro-well glass slides. This set-up, with 48 arrays on a single slide, enables simultaneous assays of 6 glycan binding proteins at 8 dilutions, interrogating 6 different glycans, including two non-sialylated controls. This is equivalent to 18x 96-well plates in the traditional plate assay. The glycan array format decreases consumption of compounds and biologicals and thus greatly enhances efficiency.

Using a printed glycan microarray strategy, a conventional 96-well plate assay was miniaturized for analysis of influenza A virus hemagglutinin avidity and specificity for sialic acid containing receptors.

Influenza A virus (IAV) hemagglutinins recognize sialic acids on the cell surface as functional receptors to gain entry into cells. Wild waterfowl are the ...