This work was supported in part by the Scripps Microarray Core Facility, and a contract from the Centers for Disease Control (J.C.P.). RPdV is a recipient of a Rubicon and VENI grant from the Netherlands Organization for Scientific Research (NWO). The Consortium for Functional Glycomics (http://www.functionalglycomics.org/) funded by NIGMS grant GM62116 (J.C.P.) provided several glycans used in this study. This is publication 29113 from The Scripps Research Institute.

NOTE: All steps are carried out at room temperature unless otherwise indicated.&#160;
1. Array Construction
<ol>
	<li>Glycan Preparation and Plate Setup
	<ol>
		<li>Prepare a stock of printing buffer. First make 500 ml of a 150 mM stock solution of dibasic sodium phosphate. Also make 50 ml of a 150 mM stock solution of monobasic sodium phosphate solution. In a flask, using a magnetic stir bar and pH meter, slowly titrate the dibasic sodium phosphate solution with the monobasic solution until a pH of 8.5 is reach...

<ol>
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Assessing IAV receptor specificity is an important step in analyzing pandemic potential of avian viruses. Sialic acid recognition by the virus is linked to several biological properties such as binding to and release from the cell. Knowledge of which amino-acid mutations are necessary for avian viruses to achieve &#945;2-6 binding and cross the species barrier enables pandemic preparedness. Several assays are used to determine receptor specificity; however, all have their drawbacks, including only measuring avidity and n...

Wild waterfowl are the natural reservoir for IAV, but IAV is able to cross the species barrier to poultry and mammals, including, humans. Avian IAVs recognize &#945;2-3 linked sialic acids (avian-type receptors), whereas human viruses bind &#945;2-6 linked sialic acids (human-type receptors). To be able to efficiently replicate and transmit between humans an avian IAV needs to bind to human-type receptors 1.
IAVs are divided based on serology that characterizes the antigenicity of their hemagglutinin (HA) and neuraminidase (NA) envelope glycoproteins. HA binds to sialic acids, whereas NA is the receptor-destroying enzyme at the other end of the viral lifecycle and cleaves sialic acid 2. All human infecting viruses, including H1N1, H2N2 and H3N2, have an avian origin 3. Over the last two of decades several avian to human crossovers have occurred, with H5N1, H7N7, and H7N9 being the most well-known; however, other subtypes have infected humans more sporadically (H6N1, H7N1, H7N2, H9N2, H10N7, H10N8) 4. Fortunately, it seems that none of these viruses have been able to fully adapt to human-type &#945;2-6-linked sialic acid receptors 5-8. Adaptation of avian or other zoonotic viruses to accommodate replication and transmission in human hosts could have a devastating effect on human health. Therefore, prior knowledge of how these viruses are evolving to bind human-type receptors will help world-wide surveillance of emerging influenza viruses.
Determination of receptor preference was first elucidated using erythrocytes of different species and remains a favored assay among flu researchers 9-12. The demonstration that avian viruses recognize &#945;2-3 sialic acid and human viruses &#945;2-6 linked sialic acid was originally based on an assay using hemagglutination of erythrocytes enzymatically engineered to contain each of the linkages 13,14. Although the readout is hemagglutination, a standard assay for virologists, the underlying glycan structures are not defined, only the terminal linkage. Additionally, the limited availability of the sialyltransferases, used to re-sialylate the cells, have limited the use of this assay 15-18. Subsequently, other methods of determining receptor-binding preferences were introduced using sialylated glycan structures linked to poly-acrylamide (PAA) or poly-glutamate (PGA) structures in plate-based assays 19,20. Several variations are possible in coating either the glycans or viruses to microtiter plates, each of which results in a robust, reliable and very sensitive ELISA-type assay 21-23. Alternatively, biotin-linked glycans can replace PAA/PGA and can be conjugated to streptavidin-coated plates 2,24. Although some specific sera may be required, ELISAs are standard and several glycans linked to PAA are readily commercially and non-commercially available (Consortium for Functional Glycomics (http://www.functionalglycomics.org)).
Glycan microarray technologies have emerged as an invaluable tool to determine receptor specificity, as multiple different glycans are spotted, and binding to a wide array of different structures can be assessed within a single assay 25-29. The binding of IAV to these structures provides a better understanding of the glycan structures that IAV preferentially recognizes 30-33. Glycan microarrays require small amounts of sample volume to perform a binding assay and only uses minute amounts of glycan per spot (2 nl). However, these arrays are typically used only to evaluate specificity of glycans receptors. Analysis of multiple viruses, or hemagglutinin proteins, at multiple concentration ranges can be prohibitive due to the number of slides required. Furthermore, to date, no relative avidity assay has been developed using glycan array techniques.
To combine the low sample requirement afforded by glycan microarray techniques and the sensitivity of PAA-linked glycans in ELISA-based assays, we sought to develop a multi-well glycan array that would allow for high-throughput analysis with similar or better resolution as compared to the traditional ELISA-based assay. Simultaneously, we wanted to minimize the amount of biologicals and reporter chemicals consumed. The ultimate result is a miniaturized avidity assay, specifically developed for monitoring IAV specificity and is equally applicable to assess other glycan binding proteins. Using a glass slide separated into 48 micro-wells by a Teflon mask, 6 different glycans are spotted in 6 replicates per well. The microarray platform affords the same trends in receptor binding seen in the macro ELISA format with several advantages. These include (I) printing of compound in 6 replicates, using minimal sample, versus the coating of several rows in a plate, using 100 &#181;l per well; (II) multiple different compounds analyzed simultaneously in a single well, including controls; (III) a massive decrease in incubation volume and; (IV) a larger dynamic range using a fluorescent readout. A single slide can be calculated to be equivalent to 18x 96-well plates.
With the following protocol, any lab capable of fabricating and analyzing spotted microarrays should be able to manufacture this miniaturized ELISA format.

<table>
	<tbody>
		<tr>
			<td>NEXTERION&#174; Slide H MPX-48&#160;</td>
			<td>Schott</td>
			<td>1091525</td>
			<td>Microwell slides</td>
		</tr>
		<tr>
			<td>ProScanArray Plus&#160;</td>
			<td>PerkinElmer</td>
			<td>discontinued</td>
			<td>confocal microarray scanner</td>
		</tr>
		<tr>
			<td>Innoscan 1100AL Scanner/Mapix Software</td>
			<td>Innopsys</td>
			<td>-</td>
			<td>confocal microarray scanner</td>
		</tr>
		<tr>
			<td>MicroGrid II&#160;</td>
			<td>Digilab</td>
			<td>-</td>
			<td>microaray printer</td>
		</tr>
		<tr>
			<td>SNA</td>
			<td>Vector Labs</td>
			<td>B-1305&#160;</td>
			<td>Plant Lectin</td>
		</tr>
		<tr>
			<td>ECA</td>
			<td>Vector Labs</td>
			<td>B-1145&#160;</td>
			<td>Plant Lectin</td>
		</tr>
		<tr>
			<td>Anti-Strep Antibody</td>
			<td>IBA</td>
			<td>2-1509-001&#160;</td>
			<td>Anitbody for HA binding</td>
		</tr>
		<tr>
			<td>Anti-Mouse Alexa-647</td>
			<td>Life</td>
			<td>A-21235</td>
			<td>Anitbody for HA binding</td>
		</tr>
		<tr>
			<td>Tween-20</td>
			<td>Sigma</td>
			<td>P2287&#160;</td>
			<td>detergent</td>
		</tr>
		<tr>
			<td>di-basic Sodium Phosphate</td>
			<td>Sigma</td>
			<td>255793</td>
			<td>printing buffer component</td>
		</tr>
		<tr>
			<td>mono-basic Sodium phosphate</td>
			<td>Sigma</td>
			<td>229903&#160;</td>
			<td>printing buffer component</td>
		</tr>
		<tr>
			<td>poly-l-lysine solution</td>
			<td>Sigma</td>
			<td>P8920&#160;</td>
			<td>pre-spotting slide component</td>
		</tr>
		<tr>
			<td>sodium hydroxide</td>
			<td>Sigma</td>
			<td>221465</td>
			<td>pre-spotting slide component</td>
		</tr>
		<tr>
			<td>ethanol</td>
			<td>Sigma</td>
			<td>493546</td>
			<td>pre-spotting slide component</td>
		</tr>
		<tr>
			<td>phosphate buffered saline</td>
			<td>Corning</td>
			<td>46-013-CM</td>
			<td>incubation/washing buffer</td>
		</tr>
		<tr>
			<td>SMP4B pins</td>
			<td>Telechem</td>
			<td>SMP4B</td>
			<td>printing pin</td>
		</tr>
		<tr>
			<td>Compressed Nitrogen (Grade5)</td>
			<td>Praxair</td>
			<td>UN1066</td>
			<td>general dusting/drying tool</td>
		</tr>
		<tr>
			<td>Boric Acid</td>
			<td>Sigma</td>
			<td>B6768-500G</td>
			<td>Slide blocking reagent</td>
		</tr>
		<tr>
			<td>ethanolamine</td>
			<td>Sigma</td>
			<td>E9508-500ML</td>
			<td>Slide blocking reagent</td>
		</tr>
		<tr>
			<td>Atto 488</td>
			<td>AttoTec</td>
			<td>AD 488-91</td>
			<td>Gridmarker on array</td>
		</tr>
		<tr>
			<td>PAA-LNLN</td>
			<td>Consortium for Functional Glycomics</td>
			<td>PA368</td>
			<td>Spotted glycans</td>
		</tr>
		<tr>
			<td>PAA-3SLNLN</td>
			<td>Consortium for Functional Glycomics</td>
			<td>PA362</td>
			<td>Spotted glycans</td>
		</tr>
		<tr>
			<td>PAA-6SLNLN</td>
			<td>Consortium for Functional Glycomics</td>
			<td>PA343</td>
			<td>Spotted glycans</td>
		</tr>
		<tr>
			<td>LNLN</td>
			<td>Consortium for Functional Glycomics</td>
			<td>Te98</td>
			<td>Spotted glycans</td>
		</tr>
		<tr>
			<td>3SLNLN</td>
			<td>Consortium for Functional Glycomics</td>
			<td>Te175</td>
			<td>Spotted glycans</td>
		</tr>
		<tr>
			<td>6SLNLN</td>
			<td>Consortium for Functional Glycomics</td>
			<td>Te176</td>
			<td>Spotted glycans</td>
		</tr>
		<tr>
			<td>384-well microtiter plate</td>
			<td>Matrix TechCorp</td>
			<td>4361</td>
			<td>Printing plate</td>
		</tr>
		<tr>
			<td>VWR lab marker</td>
			<td>VWR</td>
			<td>52877-150</td>
			<td>Slide Numbering</td>
		</tr>
		<tr>
			<td>Wheaton slide staining dish</td>
			<td>Sigma</td>
			<td>Z103969-1EA</td>
			<td>Blocking and Drying</td>
		</tr>
	</tbody>
</table>

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.

Printing, Scanning &#38; Data analyses
To ensure proper printing, it is vital to have the correct alignment of the spotted grid within the teflon mask, which delineates each array on the slide. During printing, due to the nature of the teflon coating, spots cannot be seen by the naked eye on the MPX slides. Attention is paid then to the appearance of the pre-spots on the poly-L-Lysine coated slides. Directly after printing, each...

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

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 ...

The overall goal of this miniaturized glycan array is to analyze the specificity and avidity of influenza A virus hemagglutinins. This assay can help to answer key questions regarding influenza A virus receptor binding and avidity. The main advantage to this technique is that within a single assay we can address specificity as well as relative binding avidity, normally not achieved within typical glycan array and plate-based assays.This protocol begins with preparations of the glycan samples. The base solution for the glycans is a stock printing buffer which, when made, should be slowly titrated to a pH of 8.5 and then given an addition of surfactant. Using the prepared printing buffer, dilute the PAA-conjugated glycans to 100 micrograms per milliliter.Then, dilute the monovalent glycans to 100 micromolar also in the printing buffer. Finally, make working stocks of the amine-functionalized dyes at one micromolar. Now load ten microliters of each glycan sample into a well of a 384-well microtiter plate.This is enough solution to print five slides. Also transfer ten microliters of the dye marker to one well of the printing plate. Prepare to print the arrays by first positioning the arrayer pins and printing head according to the layout.In this case, one pin can print all the samples. The arrays of glycans are printed in blocks of six surrounded by an empty spot on all sides. Put on gloves to handle the slides.Unpack them and blow any debris off their surfaces with ultra high purity nitrogen gas. Next, position the slides coated-side up on the arrayer stage. Then, program the arrayer.In this case, program 27 spots per visit to the source plate. Three pre spots placed on the non-array area of a poly-L-lysine coated slide are used to remove excess liquid at the tip of the pin. The other 24 spots per pin visit are used to place six spots in four replicate arrays.Details are provided in the text protocol. Now, load the multi-well plates into the printer and start printing the arrays. While printing, make sure that the humidity stays between 55 and 65%Pay attention to the appearance of the pre spots on the poly-L-lysine coated slides.After printing, visually inspect each slide. Check for correct alignment of the spots within the teflon borders, which is vital, and check for the correct number of spotted features. To help homogenize the attachment of the dots, put the printed slides in a 100%humidity chamber with their printed side up immediately after they are made.Use wet paper towels to line a container and a makeshift rack. Wrap the chamber in plastic to trap in the moisture. After half an hour, remove the slides.Then, number the slides with a solvent-resistant marker and store them in a desiccation box overnight. The next day, prepare a fresh stock of blocking buffer with vigorous stirring. Adjust the pH of 9.2 using concentrated sodium hydroxide.Then, incubate the slides in the buffer for an hour. To remove the block, rinse the slides in double-distilled water. Then, transfer the slides to glass staining holders and load the holders into a swinging plate holder to spin the slides dry at 10 G's for five minutes.Once dry, if needed store the slides at minus 20 degrees celsius in sealed plastic bags. In preparation, make another humidification chamber as before. To detect the immobilized glycans, prepare SNA and ECA biotinylated lectins at 10 micrograms per milliliter in probing buffer, and add two micrograms per milliliter of streptavidin 555 to each dilution.To stain with the hemagglutinins, make a 20 microgram per milliliter solution of pre complex antibody in blocking buffer at a four-to-two-to-one molar ratio as described in the text protocol. Transfer 20 microliters of the prepared solutions into wells of a 384-well plate. Next, serially dilute the lectins and hemagglutinins one-to-one in their appropriate buffers.Make eight dilutions, filling each well with 10 microliters of sample and 10 microliters of buffer. Now, incubate the plate on ice for 30 minutes. Now add eight microliters of each dilution to one of the 48 microwells on a printed slide.Then, incubate the prepared slide in the humidification chamber for 90 minutes. After 90 minutes, wash the slide while holding it from the edge;dip it in PBS-T four times, then dip it in PBS four times and finally, dip it in deionized water four times. Now, spin-dry the slide as before.After spin-drying, immediately scan the slide. Be sure to carefully load the slide into the scanner in the correct orientation. Using a low laser power of five milliwatts iteratively scan the slides, gradually increasing the gain from 25 to 75%While optimizing the scan settings keep in mind that the fluorophores will bleach out with repeated scans.Analyze the saved image for binding signals using the associated software. An array list can be loaded which has the spotting pattern. Overlay it on the scan to help navigate the placement of the grid markers.Then, use the software to analyze the spot intensities and save the results as a tab delimited text file to export into a spreadsheet or other statistical package. Analyze the data by calculating the mean signal intensity minus the background. Take an average of four of the six replicate spots for each sample, leaving out the highest signal and the lowest signal.Then, produce a line graph of the average mean signal minus the background against the eight protein concentrations. Smooth the resulting line using nonlinear regression. The PAA array was used to assess receptor binding specificity of influenza A virus hemagglutinins.The array included non-sialylated controls in the same microwells. Plant lectins with known specificities for the printed compounds were used as positive controls. The ECA lectin bound to terminal galactose beta 1-4 linked to anacetylglucosamine and did not bind to the same compound if the terminal galactose was capped with sialic acid.To test alpha 2-6 linked sialic acid, the SNA lectin was used. As expected, SNA only bound to alpha 2-6 linked sialic acids, but with notable differences in affinity for PAA linked versus non-PAA linked dye lacNAc repeats. Next, an H5 hemagglutinin derived from a Vietnam/1203/04 virus which only recognizes alpha 2-3 linked sialic acids was tested.The binding profile of the H5 hemagglutinin showed the expected specificity with a higher avidity for the PAA linked structures. Finally, the H1 hemagglutinin from a human seasonal H1N1 strain was tested. As expected, this hemagglutinin only bound to alpha 2-6 linked sialic acid-containing structures.Once mastered, this technique can reveal the binding specificities and relative avidities of influenza viruses within a single assay. This technique will greatly enhance the efficiency of influenza binding assays as well as reducing the consumption of precious biologicals.

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Immunology and Infection

7.8K 조회수.  The Scripps Research Institute. The overall goal of this miniaturized glycan array is to analyze the specificity and avidity of influenza A virus hemagglutinins. This assay can help to answer key questions regarding influenza A virus receptor binding and avidity. The main advantage to this technique is that within a single assay we can address specificity as well as relative binding avidity, normally not achieved within typical glycan array and plate-based assays.This protocol begins with preparations of the glycan s...