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

Summary

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.

Abstract

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 α2-3 linkage (avian-type receptors) whereas human viruses preferentially recognize sialic acid with a α2-6 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 α2-3 or α2-6 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.

Introduction

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 α2-3 linked sialic acids (avian-type receptors), whereas human viruses bind α2-6 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 o....

Protocol

NOTE: All steps are carried out at room temperature unless otherwise indicated. 

1. Array Construction

1. Glycan Preparation and Plate Setup
   1. 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 reached. Add Tween-20 to 0.005%.
   2. Prepare the glycan samples. Dilute PAA conjugated glycans, (diLacNAc (Galβ1-4GlcNAcβ1-3Galβ1-4Gl....

Results

Printing, Scanning & 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.......

Discussion

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 α2-6 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.......

Materials

List of materials used in this article

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