Introduction

Influenza viruses attach to target cells via multivalent interactions of the viral envelope protein hemagglutinin (HA) with sialyloligo-saccharide moieties of cellular glycoconjugates traditionally called viral receptors. The interactions of the virus with cellular receptors and extracellular inhibitors in the target tissues determine viral host range, tissue tropism, and pathogenicity (for reviews, see refs. 1-5).

Yoshihiro Kawaoka and Gabriele Neumann (eds.), Influenza Virus: Methods and Protocols,

Methods in Molecular Biology, vol. 865, DOI 10.1007/978-1-61779-621-0_5, © Springer Science+Business Media, LLC 2012

First assays of influenza virus receptor-binding specificity were based on either virus-mediated agglutination of erythrocytes from different species or hemagglutination inhibition by natural and chemically modified glycoprotein inhibitors (for review, see ref. 6). A major advance was associated with utilization of specific sialyltransferases by Paulson and colleagues to generate resialylated erythrocytes with defined structure of terminal sialic acid motifs (7). Studies using this assay for the first time demonstrated that receptor specificity of influenza virus depends on the viral host species as avian and equine viruses preferentially bound to Neu5Aca2-3Gal-terminated receptors (avian-type receptors), whereas swine and human viruses preferentially bound to the alternative (humantype) terminal receptor moiety Neu5Aca2-6Gal (reviewed in ref. 8). Another useful assay type took advantage of the fact that glycolip-ids, unlike sialylglycoproteins, contain only one oligosaccharide chain per molecule and can be purified to homogeneity. Furthermore, the hydrophobic ceramide component easily anchors glycolipids in various assay media for binding studies. Several different assay formats have been developed to characterize influenza virus binding to gangliosides (sialic acid-containing glycolipids) immobilized on solid phase (for reviews, see refs. 2, 3). The most recent new assay platform, glycan microarrays, comprises a library of structurally defined sugars printed on glass or cellulose slides (9, 10). The capacity of the arrays to provide highly detailed profiles of influenza virus binding to sialyloligosaccharides has been demonstrated (11-13). This powerful assay can greatly facilitate studies on the fine receptor specificity of avian and mammalian influenza viruses. However, the assay is relatively expensive and technically demanding, making its routine use in general virological laboratories impossible.

The solid-phase enzyme-linked assays analogous to sandwich ELISA were developed in the early 1990s (14, 15). These assays were easy to perform, sensitive, quantitative, and allowed comparison of large panels of viruses for their binding to sialylglycoproteins and to low molecular mass sialic acid compounds, such as free Neu5Ac, 3'-sialyllactose and 6'-sialyl-N-acetyllactosamine (16-22). Binding experiments using structurally defined monovalent sialosides provided valuable information about HA-receptor interactions, but could not account for the effects of presentation of the sialyloligosaccharide moiety in the context of the receptor macromolecule as a whole and for polyvalency of virus-receptor interactions. In addition, these experiments require large amounts of expensive sialosides because of the low binding affinity of the virus for monovalent receptors. These pitfalls were overcome with the development by Nicolai Bovin and colleagues of synthetic sialylglycopolymers (SGPs), monospecific macromolecular probes which comprised multiple copies of sialyloligosaccharide moieties attached to soluble hydrophilic polymeric carrier (23, 24).

Analyses of the virus binding to monovalent sialosides and to corresponding SGPs have been proven to be useful for characterizing the receptor-binding properties of influenza viruses from different hosts (25-27). In particular, by comparing the viral binding to a panel of SGPs that harbored the same Neu5Aca2-3Gal motif in a context ofdifferent oligosaccharide core sequences, Gambaryan, Bovin, and colleagues were able to specify the recognition of the inner parts of sialyloligosaccharide sequences by viruses from different avian species (28-31). Unexpectedly, these studies revealed significant distinctions between duck, gull, and chicken viruses raising an intriguing question about the role of these distinctions in the interspecies transmission of avian viruses.

The protocols described in this chapter are based on our long-term working experience with the solid-phase enzyme-linked receptor-binding assays. Two assay types are described, a direct binding assay and a competitive fetuin-binding inhibition (FBI) assay. At the first stage of each assay, the virus is adsorbed in the wells of 96-well ELISA plates coated with sialylglycoprotein fetuin. In the direct binding assay (Subheading 3.2), the immobilized virus is allowed to interact with dilutions of labeled macromolecular receptor analogues. Two types of such analogues are described: monospecific synthetic biotinylated SGPs 3'SL-PAA-biot and 6'SLN-PAA-biot (Subheading 3.2.1) and monospecific peroxidise-labeled preparations of fetuin, 3-Fet-HRP, and 6-Fet-HRP (Subheading 3.2.2). After non-bound material is removed by washing, the binding of labeled receptor analogues is evaluated using standard colori-metric assay of peroxidise activity. The binding data are converted into Scatchard plots, and association constants of virus-analogue complexes are determined from these plots (Subheading 3.2.1.1). In the FBI assay (Subheading 3.3), the virus is incubated with a mixture of unlabeled receptor analogue and standard preparation of Fet-HRP; the association constant of the analogue is calculated based on the level of its competition with Fet-HRP (Subheading 3.3.2.1).

2. Materials

2.1. Preparation and Optimization of Reagents

2.1.1. Viruses

Suspensions of influenza A and B viruses propagated in either embryonated hen's eggs or cell culture (see Chapter 2 in this book) with hemagglutination titer higher than 32 are required (see Notes 1 and 2). Viruses in allantoic fluid (AF) or culture fluid (CF) can be stored at 4°C for up to 4 weeks. It is recommended to add protease inhibitors (e.g., Complete Mini, Roche) and 0.02% sodium azide. For longer storage, either keep in aliquots at -20°C or prepare partially purified virus concentrates and store in 50% glycerol at low temperature as described in Subheading 3.1.1.

2.1.2. Fetuln-Coated 96-Well Plates

2.1.3. DeslalylatedBSA (BSA-NA) (see Note 3)

2.1.4. Standard Fet-HRP and Hlgh-Avldlty Fet-HRP

2.1.5. Monospecific 3-Fet-HRP and 6-Fet-HRP

2.1.6. Determination of Working Dilutions of Fet-HRP Preparations

1. Flat-bottom 96-well ELISA plates.

2. 5% solution of fetuin (Sigma, Cat No F3004) in water. Store in aliquots at -20°C.

3. Phosphate-buffered saline (PBS), 0.02 M, pH 7.2-7.4.

4. 8-12-channel pipette or 8-channel dispenser (0.2 ml).

1. BSA powder or solution, for example, Sigma, Cat. No. A3059 or A0336, respectively.

3. Neuraminidase from Vibrio cholerae (Sigma, Cat. No. N7885).

1. Fetuin from fetal calf serum (Sigma, Cat. No. F3004).

2. 1 and 0.1 M sodium carbonate buffers, pH 9.3.

3. Horseradish peroxidase, lyophilized powder, absorbency ratio (RZ) not lower than 3.

4. Bidistilled water (see Note 4).

5. Sodium periodate (NaIO4), reagent grade.

6. Sephadex G-25 column, 5 ml, equilibrated with water.

7. Sodium borohydride (NaBH4), reagent grade.

9. Glycerol, reagent grade.

11. Sephacryl S-200 column, 60 ml, equilibrated with 0.1 M Tris-HCL buffer, pH 7.2.

1. Items 3-10 described in Subheading 2.1.4.

2. 0.1 M sodium carbonate buffer, pH 9.3.

3. Asialofetuin from fetal calf serum (Sigma, Cat. No. A1908).

4. Slide-A-Lyzer Dialysis Cassettes, 10,000 MWCO (Pierce) or dialysis tubing (10-30 kDa).

6. 1 M solution of MgCl2 in water.

7. CMP-sialic acid (Calbiochem, Cat. No. 233264). Prepare 4 mM solution in water, store in aliquots at -20°C.

8. Alfa-2,6-(N)-sialyltransferase from rat liver (WAKO Chemicals).

9. Rat recombinant alfa-2,3-(N)-sialyltransferase (Merck).

1. Stock solutions ofFet-HRP, high-avidity Fet-HRP (ha-Fet-HRP), 3-Fet-HRP, or 6-Fet-HRP (see Subheadings 3.1.4 and 3.1.5).

2. Human influenza virus with good binding to fetuin, for example, A/X31 (H3N2), A/Chile/1/83 (H1N1), or A/Taiwan/1/86 (H1N1).

2..1.7. Determination of Working Dilutions of Viruses for Adsorption

2.1.8. Solutions Used in Receptor-Binding Assays

3. Avian influenza virus, for example, A/duck/Ukraine/1/63 (H3N8) or A/duck/Czechoslovakia/56 (H4N6).

4. Fetuin-coated plates.

5. Assay solutions TBS, BS,WS, RS, SS, and stop solution (see Subheading 2.1.8)

6. 8-12-channel pipette or 8-channel dispenser (0.2 ml).

7. 8-Position multiwell plate washer/dispenser manifold (Sigma, Cat. No. M2656).

8. Microplate reader (450 nm).

2. Non-purified virus suspensions or partially purified viruses (see Subheadings 2.1.1 and 3.1.1).

3. Fetuin-coated plates.

4. Assay solutions TBS, BS, WS, RS, SS, and stop solution (see Subheading 2.1.8).

5. 8-12-channel pipette or 8-channel dispenser (0.2 ml).

6. 8-Position multiwell plate washer/dispenser manifold (Sigma, Cat No M2656).

7. Microplate reader (450 nm).

1. Tris-buffered saline (TBS): 0.02 M, pH 7.2-7.4 (diluent for virus suspensions).

3. Stock solution of neuraminidase inhibitor, either oseltamivir carboxylate (Roche) or zanamivir (GlaxoSmithKline) (see Note 5). Prepare 1 mM stock solution in water, store in aliquots at -20°C.

4. 1% (w/v) stock solution of 3,3',5,5'-tetramethylbenzidine (TMB). Dissolve TMB in dimethyl sulfoxide (reagent grade), store in aliquots at -20°C.

5. Washing solution (WS): 0.01% tween-80 in PBS. Cool to 4°C before use.

6. Blocking solution (BS): 0.1% solution of BSA-NA in PBS. Prepare from 5% stock solution (see Subheading 3.1.3).

7. Reaction solution (RS): PBS containing 0.02% tween-80, 0.1% BSA-NA (see Subheading 3.1.3), and 1 mM neuraminidase inhibitor. Store at 4°C for up to 5 days.

8. Substrate solution (SS). Add 0.1 ml of1% stock solution of TMB to 10 ml of 0.05 M sodium acetate buffer pH 5.5. Add 10 ml of 30% hydrogen peroxide (Sigma, Cat. No. H0904) and mix.

2.2. Direct Binding Assays

Prepare SS immediately before use, do not store. Any commercial peroxidase substrate can be used as an alternative to SS described here.

1. Items 1-7 described in Subheading 2.1.7 are common for both assay variants.

2. Materials specific for each of the two assay variants are described below (Subheadings 2.2.1 and 2.2.2).

2.2.1. Binding to

Biotinylated

Sialylglycopolymers

1. Biotinylated SGPs 3' SL-PAA-biot and 6 ' SLN-PAA-biot (Lectinity Holding, Inc., Moscow, Russia) (24). These SGPs contain 20 mol% of 3' -sialyllactose (Neu5Aca2-3Gaipi-4Glc) and 6 ' -sialyl-N-acetyllactosamine (Neu5Aca2-6Galpi-4GlcNAc), respectively, and 5 mol% of biotin attached to poly[N-(2-hydroxyethyl)acrylamide] backbone. SGPs are available with average molecular mass of either 30 or 1,500 kDa. The 30 kDa SGPs are suitable for a majority of applications; however, they bind weakly if at all to many non-egg-adapted H3N2 human viruses isolated after 1992 (32); for these viruses as well as for other low-avidity viruses, high-molecular mass SGPs (1,500 kDa) should be used.

Prepare 0.1 mM stock solutions (with respect to sialic acid) by dissolving SGPs in bidistilled water; store in aliquots at -20°C.

2. Peroxidase-labeled streptavidin, for example, Pierce, Cat. No. 21126.

2.2.2. Binding to 3-Fet-HRP and 6-Fet-HRP

2.3. Fetuin-Binding 1. Items 1-7 listed in Subheading 2.1.7.

Inhbition Assay 2. Receptor analogues. A variety of natural and synthetic sialic acid-containing compounds can be used depending on availability and goals of the study. Below, we list some of the commercially available compounds of defined chemical structure with references to reports describing their use for characterization of viral receptor-binding specificity in FBI assay.

(a) Monovalent compounds (contain one sialic acid residue per molecule) (14, 15, 18-22, 25, 26).

• 3 ' sialyllactose, sodium salt (3 'SL, Neu5Aca2-3Galp1-4Glc) (Sigma, Cat. No. A8681).

• 6' -sialyl-N-acetyllactosamine, sodium salt (6'SLN, Neu5Aca2-3Galb1-4GlcNAc) (Sigma Cat. No. 37966).

Prepare 40 mM stock solutions of 3' SL and 6 ' SLN in water, store in aliquots at -20°C.

• N-acetylneuraminic acid (a,bNeu5Ac) (Sigma, Cat. No. A0812). Prepare 800 mM stock solution, which will contain 40 mM of binding-competent a-anomeric form (aNeu5Ac) (19). Dissolve 250 mg of a,bNeu5Ac in 0.75 ml of1 M solution of NaOH, adjust to neutral pH with 10 M NaOH, adjust volume to 1 ml with water. Store in aliquots at -20°C.

Non-labeled poly[N-(2-hydroxyethyl)acrylamide]-based sialylglycopolymers are produced by Lectinity Holding, Inc., Moscow, Russia (23, 24). Standard SGPs contain 20 mol% of a sialyloligosaccharide moiety (~98% purity) linked to the 30 kDa polymeric carrier. Several sialyloli-gosaccharides that were particularly useful in the studies on species-specific distinctions in receptor specificity of avian influenza viruses (14, 29-31, 34) are listed below.

Neu5Aca2-3Galp 1-4GlcNAcb

3 'SLN

Neu5Aca2-3Galp 1-4(6-HSO 3)GlcNAcb

Su-3'SLN

Neu5Aca2-3Galp 1-4(Fuca1-3)GlcNAcb

SLex

Neu5Aca2-3Galp 1-4(Fuca1-3)-(6-HSO3)GlcNAcb

Su-SLex

Neu5Aca2-3Galp 1-3GlcNAcb

3Lec

Neu5Aca2-3Galp 1-3GalNAca

3TF

Neu5Aca2-3Galp 1-3(Fuca1-4)GlcNAcb

SLea

Neu5Aca2-6Galpi-4GlcNAcp

3 'SLN

Prepare 1 mM stock solutions with respect to sialic acid by dissolving SGPs in bidistilled water; store in aliquots at -20°C.

3. Methods

3.1. Preparation and Optimization of Reagents

3.1.1. Viruses

Avian viruses and most egg-adapted mammalian viruses can be used without purification.

Many non-egg-adapted human and swine viruses and some laboratory-derived receptor-binding mutants do not bind efficiently to fetuin-coated plates from allantoic or cultural fluid. To solve this problem, such viruses must be partially purified from egg- or cell-derived protein and concentrated as described below.

1. Clarify allantoic or culture fluid by low-speed centrifugation (5,000 xg, 20 min).

2. Pellet the virus by high-speed centrifugation (120,000 xg, 1 h).

3. Resuspend the virus pellet thoroughly in 50% glycerol-0.1 M Tris-HCL (pH 7.2) at 1% of the original volume. Remove remaining cellular debris by centrifugation for 5 min at 1,000 x g.

4. Prepare aliquots. Store one aliquot for routine work at -20°C and store the rest at -80°C.

3.1.2. Fetuin-Coated 96-Well Plates

3.1.4. Synthesis of Peroxldase-Labeled Fetuin (Fet-HRP)

1. To coat 25 plates, prepare 510 ml of a 10 mg/ml working solution of fetuin in PBS.

2. Add 0.2 ml of fetuin solution to each well, cover plates, and incubate at 4°C overnight.

3. Flick plate content into a waste container. Rinse and flick the plates three times with 0.35-0.4 ml of tap water and once with distilled or deionized water. Strike the plates sharply onto paper towel to remove water droplets.

4. Let plates air-dry for at least 3 h at room temperature in a clean bench area.

5. Store in a moisture-proof bag at room temperature away from light and heat for up to several months.

1. Prepare 50 ml of 5% solution of BSA in PBS, add 1 ml PEN-STREP and adjust pH to 7.3-7.5.

2. Add 1 unit of Vibrio cholerae neuraminidase and incubate for 24 h at 37°C.

3. Incubate for at least 24 h at 60°C to inactivate the neuraminidase.

Synthesis of the four following preparations is described.

1. Standard Fet-HRP is routinely used for the FBI assay (Subheading 3.3) with the majority of avian and mammalian influenza viruses.

2. High-avidity Fet-HRP which is prepared from heat-aggregated fetuin is routinely used in all assay variants for determination of working dilutions of viruses for adsorption (see Subheading 3.1.7). High-avidity Fet-HRP can also be used for direct binding assay and FBI with viruses that show insufficient avidity for the standard Fet-HRP.

3-4. Monospecific Neu5Aca2-3Gal- and Neu5Aca2-6Gal-contai-ning peroxidase-labeled fetuin preparations (3-Fet-HRP and 6-Fet-HRP) are primarily used for differentiation between virus preference for the type of Neu5Ac-Gal linkage in the direct binding assay (Subheading 3.2.2).

3.1.4.1. Standard Fet-HRP 1. Dissolve 2 mg of fetuin in 0.6 ml of 0.1 M sodium carbonate buffer, pH 9.3.

2. Dissolve 4 mg of HRP in 0.9 ml of bidistilled water.

3. Add 0.1 ml of freshly prepared 0.2 M NaIO4 solution in water to the solution of HRP. Close the tube, mix, and incubate for 20 min in the dark at room temperature.

4. Desalt reaction mixture by filtration through 5-ml Sephadex G-25 column, elute with water. Collect major colored fraction (1.2-1.4 ml) containing oxidized HRP.

5. Add collected solution of oxidized HRP to the solution of fetuin. Mix, incubate for 4 h in the dark at room temperature.

6. Transfer reaction mixture to a 10-ml tube with cap to avoid potential losses due to formation of foam at the next steps. Add 0.1 ml of freshly prepared 5 mg/ml solution of NaBH4 in water, mix, and incubate for 30 min on ice. Add 0.2 ml of another freshly prepared solution of NaBH4, incubate for 30 min on ice.

7. Slowly add 1/5 volume of 1 M Tris-HCL pH 6 on ice. Caution: this will destroy residual NaBH4 and may result in foaming. Leave overnight at 4°C to ensure complete hydrolysis of NaBH4 before the next step.

8. Separate Fet-HRP from nonconjugated peroxidase by chromatography on Sephacryl S-200 column in 0.1 M Tris buffer pH 7.0. Collect and combine major HRP-containing fractions eluted in the void volume of the column. Add equal volume of glycerol, mix, and aliquot. Store one aliquot for routine work at -20°C and store the rest at -80°C.

1. Dissolve 15 mg of fetuin in 0.3 ml water (5% solution) in a 0.4-ml capped tube. Incubate for 4 h at 90°C, vortex tube each 30 min (4ee Note 6). Heating leads to aggregation of fetuin and increases avidity of its binding to influenza viruses.

2. Dissolve 15 mg HRP in 1 ml water in a 2-ml tube.

3. Add 0.1 ml of freshly prepared 0.4 M NaIO4 solution in water to the solution of HRP. Mix and incubate for 20 min in the dark at room temperature.

4. Desalt reaction mixture by filtration through 5-ml Sephadex G-25 column, elute with water. Collect major colored fraction (1.2-1.5 ml) containing oxidized HRP.

5. Combine solution of oxidized HRP with solution of heat-aggregated fetuin, add 0.1 ml of 1 M carbonate buffer, pH 9.3. Mix, incubate for 4 h at room temperature in the dark.

6. Follow the instructions for the steps 6-8 in Subheading 3.1.4.1.

3.1.4.2. High-Avidity Fet-HRP

3.1.5. Monospecific 1. MonospecificNeu5Aca2-3Gal- andNeu5Aca2-6Gal-containing

3-Fet-HRPand6-Fet-HRP peroxidase-labeled fetuin preparations 3-Fet-HRP and

6-Fet-HRP are primarily used for differentiation between virus preference for the type of Neu5Ac-Gal linkage in the direct binding assay (Subheading 3.2.2). Dissolve 15 mg of asialofe-tuin in 0.3 ml of 0.1 M sodium carbonate buffer, pH 9.3 in a 2-ml tube.

2. Dissolve 15 mg HRP in 1 ml of water.

3. Add 0.1 ml of freshly prepared 0.4 M NaIO4 solution in water to the solution of HRP. Mix and incubate for 20 min in the dark at room temperature.

4. Desalt reaction mixture by filtration through a 5-ml Sephadex G-25 column, elute with water. Collect major colored fraction (1.2-1.4 ml) containing oxidized HRP.

5. Add solution of oxidized HRP to the solution of asialofetuin. Mix, incubate for 4 h at room temperature in the dark.

6. Follow the instructions for the steps 6-7 in Subheading 3.1.4.1.

7. Dialyze HRP-labeled asialofetuin against PBS, pH 7.0 at 4°C.

8. Collect dialyzed product. Add MgCl2 to 2 mM and CMP-Neu5Ac to 1.5 mM.

9. Divide the mixture into two aliquots (about 1.6 ml each) for the separate preparation of 3-Fet-HRP and 6-Fet-HRP. Add 2,3-(N)-sialyltransferase to one aliquot to a final concentration 20 mU/ml. Add 2,6-sialyltransferase to another aliquot to 80 mU/ml. Incubate for 16 h at 37°C (see Note 7).

10. Dialyze 3-Fet-HRP and 6-Fet-HRP against 0.1 M Tris-HCL buffer, pH 7.2 at 4°C.

11. Collect dialyzed products. Add equal volume of glycerol, mix, and aliquot. Store one aliquot of each preparation for routine work at -20°C and store the rest at -80°C.

3.1.6. Determination of 1. Dilute virus suspensions in TBS to hemagglutination titer 128.

WMmg DMions of 2. Dispense 0.05 ml of diluted human and avian viruses into wells

Fet-HRp preparations of a fetuin-coated plate. Use two replicate 12-well columns for each virus and preparation of Fet-HRP. Use two replicate 12-well columns for the negative control (0.05 ml of TBS).

4. Empty wells by vacuum suction using 8-positon manifold. Wash the plate three times with 0.2 ml of PBS.

5. Add 0.2 ml of the blocking solution (BS). Incubate for 1 h at room temperature or leave under BS at 4°C overnight.

6. Prepare 12 serial twofold dilutions of Fet-HRP stock in RS starting from 1/100.

7. Remove blocking solution; wash plate twice with 0.2 ml of ice-cold WS. Add 0.05 ml of each Fet-HRP dilution to duplicate virus-coated and non-coated wells (see Note 9).

9. Wash five times with 0.2 ml of ice-cold WS (see Note 10).

10. Add 0.1 ml of freshly prepared substrate solution; incubate for 30 min at room temperature away from direct light.

11. Stop the reaction by adding 0.05 ml of 3% H2SO4.

12. Read plate on a microplate reader at 450 nm and transfer the absorbency data from the reader into Microsoft Excel or another spreadsheet program.

13. Prepare plots of absorbency (A450) versus Fet-HRP dilution in semilogarithmic scale. As illustrated in Fig. 1, avian viruses typically show saturated binding curves with the plateau at low Fet-HRP dilutions. Binding plots of human influenza viruses often lack a clear-cut plateau because these viruses have low avidity for fetuin. The optimal working dilutions of the FetHRP depend on specific application and on viruses included in the study as outlined below.

- For virus detection (Subheading 3.1.7) use the highest dilution of Fet-HRP that still shows high values of A450 with both avian and human virus.

- For the direct binding assay with all preparations of FetHRP and for titration of Fet-HRP and ha-Fet-HRP in the binding inhibition assay (Subheading 3.3.1) use dilutions that cover the range of absorbencies from 0.1 to the beginning of the plateau.

Dilution of Fet-HRP

Fig. 1. Determination of working dilutions of Fet-HRP preparations (Subheading 3.1.6). Serial twofold dilutions of Fet-HRP were incubated in the wells of 96-well plate coated with viruses A/X31 (H3N2) (open circles) and A/Duck/Ukraine/1/63 (H3N8) (filled diamonds). Wells without virus were used for a control of nonspecific binding of Fet-HRP to the plate (plus symbols).

Dilution of Fet-HRP

Fig. 1. Determination of working dilutions of Fet-HRP preparations (Subheading 3.1.6). Serial twofold dilutions of Fet-HRP were incubated in the wells of 96-well plate coated with viruses A/X31 (H3N2) (open circles) and A/Duck/Ukraine/1/63 (H3N8) (filled diamonds). Wells without virus were used for a control of nonspecific binding of Fet-HRP to the plate (plus symbols).

- In any assay, avoid using dilutions of Fet-HRP that produce significant nonspecific binding (A450 > 0.2) in the control virus-non-coated wells.

3.1.7. Determination of Working Dilutions of Viruses for Adsorption

3.2. Direct Binding Assays

3.2.1. Binding to

Biotinylated

Sialylglycopolymers

1. Prepare eight serial twofold dilutions ofviruses in TBS (0.05 ml each) in eight consecutive wells of fetuin-coated plate (two replicate rows for each virus). For non-concentrated allantoic or culture fluid, start from undiluted material; for concentrated viruses, start from 1/50 dilution. Use four to eight wells of a plate for a negative control (0.05 ml TBS).

2. Incubate overnight at 4°C (see Note 11).

3. Empty wells by vacuum suction using 8 position manifold. Wash the plate three times with 0.2 ml of PBS.

4. Add 0.2 ml of blocking solution. Incubate for 1 h at room temperature or leave under BS overnight at 4°C.

5. Remove blocking solution; wash twice with 0.2 ml of WS.

6. Add 0.05 ml of working dilution of ha-Fet-HRP in RS as determined in Subheading 3.1.6.

7. Follow the instructions for the steps 8-12 in Subheading 3.1.6.

8. Prepare plots of absorbency (A450) versus virus dilution (Fig. 2). For routine work, choose maximal dilution of the virus that still saturates the plate (end of absorbency plateau, see Fig. 2). If plateau is not reached, choose the dilution corresponding to A450 in the range from 0.4 to 2.5 (optimally 1.5-2).

This assay was initially introduced to study phenotypic differences between mammalian H1, H2, and H3 viruses and their avian precursors (26). Afterwards, the assay was used for a number of different applications, among them, comparison of the receptor-binding phenotypes of human and swine influenza viruses isolated in cell culture and eggs (27, 32), analysis of effects of antibody-escape mutations on receptor specificity of H9N2 virus ( 35), detection of receptor specificity changes during adaptive evolution of the influenza virus in the mouse lung (36). The assay is particularly useful for characterization of non-egg-adapted human and swine influenza viruses, many of which do not bind to fetuin.

1. Determine optimal dilutions of viruses to be compared as described in Subheading 3.1.7.

2. Dispense 0.05 ml of chosen virus dilutions in TBS into the wells of two replicate fetuin-coated plates to be used with either 3' SL-PAA-biot or 6 ' SLN-PAA-biot. Use at least two replicate rows per virus and two rows for the negative control, 0.05 ml of TBS (Fig.3, left panel) (see Note 12).

3. Incubate, wash, and block virus-coated plates (see steps 2-4 in Subheading 3.1.7).

Virus dilution

Fig. 2. Determination of working dilutions of viruses for adsorption (Subheading 3.1.7). Serial twofold dilutions of viruses were adsorbed in the wells of fetuin-coated plate and incubated with Fet-HRP followed by washing and detection of peroxidise activity in the wells. Viruses A/Hong Kong/156/97 (H5N1) (HK/97, plus symbols) and A/Duck/Ukraine/1/63 (H3N8) (filled diamonds) were adsorbed from allantoic fluid; A/X31 (H3N2) (open circles) was partially purified before adsorption (see Subheading 3.1.1). Arrows depict dilutions of the virus stocks that were chosen for the assay.

Virus dilution

Fig. 2. Determination of working dilutions of viruses for adsorption (Subheading 3.1.7). Serial twofold dilutions of viruses were adsorbed in the wells of fetuin-coated plate and incubated with Fet-HRP followed by washing and detection of peroxidise activity in the wells. Viruses A/Hong Kong/156/97 (H5N1) (HK/97, plus symbols) and A/Duck/Ukraine/1/63 (H3N8) (filled diamonds) were adsorbed from allantoic fluid; A/X31 (H3N2) (open circles) was partially purified before adsorption (see Subheading 3.1.1). Arrows depict dilutions of the virus stocks that were chosen for the assay.

4. Prepare eight serial twofold dilutions of biotinylated SGPs (see Note 13). To study avian viruses, start titration from 0.1 mM of 3'SLN-PAA-biot and 1 mM of 6'SLN-PAA-biot. For human viruses, start from 1 mM of 3'SLN-PAA-biot and 0.1 mM 6'SLN-PAA-biot, respectively. If both human and avian viruses are tested on the same plate, start from 1 mM of each SGP. At least 0.65 ml of each dilution per 96-well plate is required.

5. Remove blocking solution; wash twice with 0.2 ml of ice-cold washing buffer.

6. Add 0.05 ml dilutions of3'SLN-PAA-biot (Plate 1) and 6'SLN-PAA-biot (Plate 2) (see Fig. 3, right panel).

8. Wash five times with 0.2 ml of ice-cold WS.

9. Fill each well with 0.05 ml of peroxidase-labeled streptavidin diluted 1/1,000 in RS (see Note 14).

10. Incubate, wash, and determine peroxidase activity in the wells; save the absorbency data (see steps 8-12 in Subheading 3.1.6).

3.2.1.1. Analysis of Results 1. Using either Microsoft Excel or another spreadsheet software, calculate mean values of A450 for each dilution of SGP in the negative control without the virus. Subtract these values from the values of A450 in virus-coated wells that were incubated with corresponding dilutions of SGP. Use background-corrected values of A450 in all subsequent calculations.

Fig. 3. Layout of the direct binding assay (Subheading 3.2.1). Left panel: Virus adsorption in the fetuin-coated plate with five virus specimens numbered 1-5 (two replicates of each) and two replicate rows of control wells incubated with TBS instead of the virus. Prepare two replicate plates to be used with two SGPs (either 3'SL-PAA-biot or 6'SLN-PAA-biot). Right pane: Titration of SGP in the virus-coated plate. Numbers on top depict serial twofold dilutions; the concentration of the first dilution is chosen as described in the text.

Fig. 3. Layout of the direct binding assay (Subheading 3.2.1). Left panel: Virus adsorption in the fetuin-coated plate with five virus specimens numbered 1-5 (two replicates of each) and two replicate rows of control wells incubated with TBS instead of the virus. Prepare two replicate plates to be used with two SGPs (either 3'SL-PAA-biot or 6'SLN-PAA-biot). Right pane: Titration of SGP in the virus-coated plate. Numbers on top depict serial twofold dilutions; the concentration of the first dilution is chosen as described in the text.

2. Calculate concentration of SGP (C) in mM sialic acid for each dilution.

3. Calculate values of A450/C for each virus-coated well of the plate.

4. Prepare Scatchard plots (A450/C vs. A450) for each virus and each replicate titration of SGP (Fig. 4). Draw trendlines (if required, ignore experimental points with A450 less than 0.1).

5. Determine intercept of the trendlines with the Y and X axes (Y and A , respectively). Because 3'SL-PAA-biot and 6'SLN-

PAA-biot have identical molecular masses and molar contents of sialic acid (20%) and biotin (2.5%), we assume that Scatchard plots for these SGPs should have identical values of A .

Therefore, the trendline for a weakly binding polymer is drawn to have the same A as the trendlines of the high-avidity max counterpart (see Fig. 4).

6. Calculate apparent association constants, K = Y/A .

Average values of Kass from the replicate performed on the same day. Higher values of Kass reflect a stronger binding (see Notes 15 and 16).

3.2.2. Binding to Two monospecific preparations of fetuin are made by resialylation

3-Fet-HRP and 6-Fet-HRP of the same original HRP-labeled asialofetuin (see Subheading 3.1.5).

As a result, 3-Fet-HRP and 6-Fet-HRP differ only by the type of

•QffrHft

•QffrHft

A450

Fig. 4. Scatchard plots for the direct binding of biotinylated SGPs 3'SL-PAA-biot (filled circleS) and 6'SLN-PAA-biot (open circleS) to influenza viruses A/Duck/Alberta/119/98 (H1N1) (a), A/Hong Kong/1/68 (H3N2) (b), and A/Memphis/14/96-M (H1N1) (c). Imax, Yo, and Yo depict intercepts of the trendlines with the axes. Note that the trendlines for the weak binding of 6'SLN-PAA-biot to Dk/Alberta/98 and of 3 ' L-PAA-biot to Memphis/96 are drawn to have the same Imax with the high-avidity SGP counterpart. Apparent association constants (in mM-1) can be determined from the plots as the ratio Yo / Imax

A450

Fig. 4. Scatchard plots for the direct binding of biotinylated SGPs 3'SL-PAA-biot (filled circleS) and 6'SLN-PAA-biot (open circleS) to influenza viruses A/Duck/Alberta/119/98 (H1N1) (a), A/Hong Kong/1/68 (H3N2) (b), and A/Memphis/14/96-M (H1N1) (c). Imax, Yo, and Yo depict intercepts of the trendlines with the axes. Note that the trendlines for the weak binding of 6'SLN-PAA-biot to Dk/Alberta/98 and of 3 ' L-PAA-biot to Memphis/96 are drawn to have the same Imax with the high-avidity SGP counterpart. Apparent association constants (in mM-1) can be determined from the plots as the ratio Yo / Imax a the glycosidic linkage between the terminal Neu5Ac residue and the penultimate Gal residue (a2-3 or a2-6). Direct binding assay with this pair of receptor analogues was previously employed to characterize changes in the receptor specificity during egg adaptation of seasonal human H1N1 influenza virus (37).

1. Prepare two replicate plates coated with the viruses (see steps 1-3 in Subheading 3.2.1).

2. Prepare eight serial twofold dilutions of 3-Fet-HRP and 6-Fet-HRP in RS that cover the optimal range of concentrations determined in Subheading 3.1.6. At least 0.65 ml of each dilution per 96-well plate is required.

3. Remove blocking solution; wash twice with 0.2 ml of ice-cold washing buffer.

4. Add 0.05 ml of 3-Fet-HRP dilutions (see Note 9).

5. Repeat steps 3 and 4 to fill the replicate plate with 6-Fet-HRP.

6. Incubate, wash, and determine peroxidase activity in the wells; save the absorbency data (see steps 8-12 in Subheading 3.1.6).

7. Analyze the data and calculate association constants as described for SGPs (see Subheading 3.2.1.1) taking concentration of 3-Fet-HRP and 6-Fet-HRP in stock solutions for 1,000 arbitrary units (U). Higher values of Kass reflect a stronger binding.

3.3. Fetuin-Binding This assay is based on the competition between non-labeled receptor

Inhibition Assay analogue and standard Fet-HRP preparation for the binding site on a solid-phase immobilized virus (14, 15). The assay is performed in two steps. First, Fet-HRP is titrated in a direct binding assay to determine the optimal working concentration. Then, the binding inhibition test is performed using chosen dilution of Fet-HRP.

3.3.1. Determination of Working Concentration of Fet-HRP

1. Prepare 96-well plates coated with the viruses (see Fig. 5 and steps 1-3 in Subheading 3.2.1). Make one replicate plate for titration of Fet-HRP and one replicate plate per each receptor analogue to be tested in FBI.

2. Prepare eight serial twofold dilutions of Fet-HRP in RS that cover the optimal range of concentrations determined in Subheading 3.1.6. At least 0.65 ml of each dilution per 96-well plate is required.

3. Remove blocking solution; wash twice with 0.2 ml of ice-cold washing buffer.

5. Incubate, wash, and determine peroxidase activity in the wells; save the absorbency data (see steps 8-12 in Subheading 3.1.6).

6. Prepare Scatchard plots and determine A as described in

Subheading 3.2.1.1.

7. For each virus and each Fet-HRP dilution, calculate parameter a = (Amax - A450)/Amax, where a is the portion of remaining free binding sites on the virus and, A450 is the absorbency at a given concentration of Fet-HRP and A is the absorbency at infi-

max nite concentration of Fet-HRP (see Fig. 6).

Fig. 5. Layout of the binding inhibition assay (Subheading 3.3.2). Left panel: Virus adsorption in the fetuin-coated plate with five virus specimens numbered 1-5 (two replicates of each) and two replicate rows of control wells incubated with TBS. Prepare separate replicate plate for titration of Fet-HRP and for each receptor analogue to be tested in FBI. Right panel Incubation of dilutions of non-labeled receptor analogue in working solution of Fet-HRP in the wells of virus-coated plate. Numbers on top depict serial twofold dilutions of the analogue in rows 2-11; the concentration of the first dilution is chosen as described in the text. Rows 1, 12 and columns A, B are incubated with working solution of Fet-HRP (controls of nonspecific binding and specific binding without inhibitor, respectively).

Fig. 5. Layout of the binding inhibition assay (Subheading 3.3.2). Left panel: Virus adsorption in the fetuin-coated plate with five virus specimens numbered 1-5 (two replicates of each) and two replicate rows of control wells incubated with TBS. Prepare separate replicate plate for titration of Fet-HRP and for each receptor analogue to be tested in FBI. Right panel Incubation of dilutions of non-labeled receptor analogue in working solution of Fet-HRP in the wells of virus-coated plate. Numbers on top depict serial twofold dilutions of the analogue in rows 2-11; the concentration of the first dilution is chosen as described in the text. Rows 1, 12 and columns A, B are incubated with working solution of Fet-HRP (controls of nonspecific binding and specific binding without inhibitor, respectively).

Fig. 6. Example of the binding inhibition assay: determination of association constants (Kass) with 3'-sialyllactose for viruses A/Swine/Netherlands/3/80 (H1N1), A/Albany/7/57 (H2N2), A/RI/5+/57 (H2N2), A/Duck/Hokkaido/7/82 (H3N?), and A/ Udorn/307/72 (H3N2) (data from ref. 26). The chartsshowdatafor oneof two replicate rows (see Fig. 5). (a) Determination of working dilution of Fet-HRP; arrow depicts chosen working dilution (1/500). (b) Inhibition of Fet-HRP binding by 3'-sialyllactose. Experimental points with binding between 20 and 80% (dotted lines) were used for calculation of apparent association constants as described in Subheading 3.2.2.1.

Fig. 6. Example of the binding inhibition assay: determination of association constants (Kass) with 3'-sialyllactose for viruses A/Swine/Netherlands/3/80 (H1N1), A/Albany/7/57 (H2N2), A/RI/5+/57 (H2N2), A/Duck/Hokkaido/7/82 (H3N?), and A/ Udorn/307/72 (H3N2) (data from ref. 26). The chartsshowdatafor oneof two replicate rows (see Fig. 5). (a) Determination of working dilution of Fet-HRP; arrow depicts chosen working dilution (1/500). (b) Inhibition of Fet-HRP binding by 3'-sialyllactose. Experimental points with binding between 20 and 80% (dotted lines) were used for calculation of apparent association constants as described in Subheading 3.2.2.1.

8. Find dilution of Fet-HRP that corresponds to a around 0.5 for a majority of viruses to be tested simultaneously (see Fig. 6a). Record actual value of a for each virus at the chosen dilution; these values will be used for calculation of apparent association constants in the binding inhibition assay.

1. Prepare working solution of Fet-HRP in RS (6-7 ml per 96-well plate) using dilution determined in Subheading 3.3.1.

2. Prepare six serial twofold dilutions of non-labeled sialic acid-containing compounds in the working solution of Fet-HRP. For the monovalent analogues, start from 4 mM concentration, for sialylglycopolymers, start from 0.02 mM (see Note 17). At least 0.55 ml of each dilution per 96-well plate is required. Cool on ice.

3. Use virus-coated plates prepared at the step 1 of Subheading 3.3.1. Remove blocking solution; wash twice with 0.2 ml of ice-cold washing buffer.

4. Fill the plate with 0.05 ml per well of either working solution of Fet-HRP or its mixtures with non-labeled receptor analogue (see Fig. 5, right panel).

5. Incubate for 1 h at 4°C, wash, and determine peroxidase activity in the wells; save the absorbency data (see steps 8-12 in Subheading 3.1.6).

3.3.2. Binding Inhibition Assay: Determination of Association Constants

3.3.2.1. Analysis of Results 1. Using Microsoft Excel or another spreadsheet program, calculate mean value of A450 in the negative control (no virus). Subtract this value from the absorbency values in virus-coated wells. Use background-corrected values of A450 in all subsequent calculations.

2. Calculate mean value of absorbency in virus-coated wells without inhibitor (A0). Taking A0 for 100%, calculate percentages of Fet-HRP binding (B) in the presence of the inhibitor: B. = 100 X A/A (see Fig. 6b).

3. For each experimental point with B. in the range from 20 to 80%, calculate value of association constant for the virus-inhibitor complex:

Kass = (100-B.)/a*B.*G, where B. is the percent of Fet-HRP binding at inhibitor concentration C , a is a portion of free binding sites determined for this virus and working dilution of Fet-HRP (see Subheading 3.3.1).

4. Calculate mean value of K and standard deviation using ass ^

values of K determined at different concentrations of the ass inhibitor. Higher values of Kass reflect a stronger binding.

3.3.3. Binding Inhibition Assay: Determination of Binding Patterns

This is a variant of FBI, which was previously used to compare virus binding to a panel of 5-10 SGPs containing the same terminal Neu5Aca2-3Gal moiety but differing by the structure of penultimate sugar residue(s). The patterns of binding to such receptor analogues were found to differ between duck, gull, and poultry viruses indicating that these viruses have different receptor specificity (29-31, 34) (for an example, see Fig. 7).

The only difference from the assay variant described in Subheading 3.3.2 is that each virus is tested for binding inhibition

Concentration of SGP, ^M Neu5Ac

Concentration of SGP, ^M Neu5Ac

Concentration of SGP, ^M Neu5Ac

Concentration of SGP, ^M Neu5Ac

Concentration of SGP, ^M Neu5Ac -•-Slex -e-Su-Slex

Fig. 7. Example of the binding inhibition assay: determination of binding patterns for viruses A/Mallard New York/670/78 (H4N6) (a), A/Gull/Moscow/3100/06 (H6N2) (b), and A/Chicken/Vietnam/NCVD-11/03 (H5N1) (c) (data from ref. 31). SGPs: SLec-PAA (plus symbols), 3'SLN-PAA (filled triangles), Su-3'SLN-PAA (open circles), SLex-PAA (filled squares), and Su-SLex-PAA (open squares).

by all SGPs on the same 96-well plate. This assay format serves the goal of focusing on the distinctions in binding of different analogues to a single virus, rather than on binding of the same analogue to different viruses (Subheading 3.3.2).

1. Determine working concentration of Fet-HRP exactly as described in Subheading 3.3.1.

2. Prepare virus-coated plates for FBI. For each virus, fill rows 2-11 of the fetuin-coated plate with 0.05 ml of chosen virus dilutions in TBS. Fill row 1 with 0.05 ml TBS (control of nonspecific binding). One plate is sufficient to test two replicate titrations of five different SGPs. Fill additional plates for more replicates or more SGPs. Incubate, wash, and block virus-coated plates (see steps 2-4 in Subheading 3.1.7).

3. Prepare working solution of Fet-HRP in RS (6-7 ml per 96-well plate).

4. Prepare eight serial twofold dilutions of each SGP in the working solution of Fet-HRP starting from 0.02 mM (see Note 17). At least 0.15 ml of each dilution is required for two replicate titrations of one SGP with each virus. Cool on ice.

5. Use virus-coated plates prepared at the step 2. Remove blocking solution; wash twice with 0.2 ml of ice-cold washing buffer.

6. Fill rows 1 and 12 of the plate with 0.05 ml per well working solution of Fet-HRP without SGPs (controls of nonspecific binding and 100% binding (A0), respectively). Fill rows 2-11 with dilutions of SGPs in working solution of Fet-HRP.

7. Incubate for 1 h at 4°C, wash, and determine peroxidase activity in the wells; save the absorbency data (see steps 8-12 in Subheading 3.1.6). Analyze the data and determine Kass as described in Subheading 3.3.2.1 (see Note 18).

Figure 7 shows example of FBI with three different avian viruses and five SGPs. The assay reveals the following species-specific features of duck, gull, and highly pathogenic chicken viruses (31). The duck virus displays the highest binding to SLec and does not bind to fucosylated sialyloligosaccharides SLex and Su-SLex. The gull virus efficiently binds to fucosylated receptors SLex and Su-SLex. The H5N1 chicken virus binds strongly to sulfated sialyloligosaccharides Su-3'SLN and Su-SLex.

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