This project was funded by intramural PanFlu funds from the Center for Biologics Evaluation and Research (CBER). Plasmids used to prepare reassortant viruses were kindly provided by Robert Webster, St Jude Children's Research Hosptial, Memphis, TN. We are indebted to Ewan Plant and Haruhiko Murata for critical review of the manuscript.

All live reassortant viruses with avian components must be handled using biosafety level 2 (BSL2)-enhanced practices in a laboratory approved for use by the United States Department of Agriculture (USDA) and the institutional biosafety committee.
1. Preparation of Reagents and Starting Material
Note: Refer to the Materials Table to obtain the source of all reagents.
<ol>
	<li>Fetuin-coated Plates
	<ol>
		<li>Prepare coating buffer by mixing 10 ml 10x coating buffer with 90 ml deionized H2O.</li>
		<li>Prepare a stock solution of fetuin at 25 mg/ml in 1x coating buffer and store in 500 &#181;l aliquots at -20 &#176;C.</li>
		<li>Prepare a working solution of fetuin (25 &#181;g/ml) immediately before coating plates by diluting the stock solution 1,000-fold in 1x coating buffer.</li>
		<li>Use a multichannel pipette to dispense 100 &#181;l of the working solution of fetuin into all wells of a 96-well plate that has high protein-binding capacity.</li>
		<li>Cover each plate with a plate sealer then stack the plates in groups of 10 and wrap them in foil.</li>
		<li>Place the plates in a refrigerator (2-8 &#176;C) for at least 18 hr before performing an assay. 
		Note: Plates may be coated in advance and stored with the coating solution at 2-8 &#176;C for up to 2 months.</li>
	</ol>
	</li>
	<li>Diluents
	<ol>
		<li>To prepare the diluent to make virus and sample dilutions (2-(N-morpholino)ethanesulfonic acid (MES), pH 6.5, 20 mM CaCl2, 1% bovine serum albumin (BSA) and 0.5% Tween 20), mix 94.2 ml 1X MES, pH 6.5 with 2 ml CaCl2 (10 mg/ml), 3.3 ml 30% BSA and 0.5 ml Tween 20.</li>
		<li>To prepare the diluent for PNA-HRPO (MES, pH 6.5 with 20 mM CaCl2 and 1% BSA), mix 94.7 ml 1x MES, pH 6.5 with 2 ml CaCl2 (10 mg/ml), and 3.3 ml 30% BSA.</li>
	</ol>
	</li>
	<li>Neuraminidase (NA) 
	Note: H6Nx virus reassortants (these have HA of H6 subtype and the NA of interest), are generated following published methods 5,10. Request vials of inactivated reassortant virus from a collaborator if they are not available in-house. When using inactivated virus, confirm that the preparation is suitable for use in the ELLA through demonstration that the inactivation process did not have a significant impact on NA activity.
	<ol>
		<li>Culture a stock of H6Nx virus in embryonated chicken eggs 11 and store 0.5 ml aliquots of neat allantoic fluid at -80 &#176;C.</li>
		<li>Use a new aliquot of virus for each assay. Do not freeze and thaw the aliquots.</li>
	</ol>
	</li>
	<li>Serum Samples and Controls
	<ol>
		<li>Heat-inactivate all sera (test samples e.g., pre- and post-vaccination sera, as well as controls, e.g., sample with known NI titer) in a water bath at 56 &#176;C for 45-60 min. Store the sera at -20 to -70 &#176;C before or after the heat treatment. If the samples are to be tested repeatedly, make several aliquots before freezing so that the sample is not repeatedly frozen and thawed.</li>
		<li>Use the ELLA to measure the NI titers of human sera that are available in reasonable quantity (&#62;5 ml) to identify at least one with low titer and another with a high titer. Store 100 &#181;l aliquots as &#8804;-20 &#176;C so that the same sample can be used as a control in many assays in order to track assay performance.</li>
	</ol>
	</li>
	<li>Peanut Agglutinin (PNA)-Horseradish Peroxidase (HRPO)
	<ol>
		<li>Prepare the PNA-HRPO diluent (MES, pH 6.5 with CaCl2 and 1% BSA).</li>
		<li>Prepare a PNA-HRPO stock solution by dissolving 1 mg of PNA-HRPO in 1 ml of diluent. Store 20-200 &#181;l aliquots at -20 &#176;C.</li>
		<li>Before using a new PNA-HRPO lot, test 1:500, 1:750, 1:1,000 and 1:2000 dilutions of this reagent to identify the amount that results in maximal OD with the positive control (virus only) and a background (no virus) that is &#60;10% of the positive signal.
		<ol>
			<li>Make quadruplicate virus dilutions as described in section 2.1.</li>
			<li>Transfer the dilutions to a fetuin-coated plate as described in section 2.2 and incubate the plate at 37 &#176;C.</li>
			<li>Wash the plate 16-18 hr later and add 1:500, 1:750, 1:1,000 and 1:2,000 dilutions of PNA-HRPO (100 &#181;l/well) to duplicate rows of the plate.</li>
			<li>Complete the assay as described in steps 2.3.4 to 2.3.9.</li>
			<li>Review the data to select the dilution that resulted in a maximum signal that is &#62;10 times the background.</li>
		</ol>
		</li>
		<li>Immediately before use, prepare the optimal dilution of PNA-HRPO identified in 1.5.3.</li>
	</ol>
	</li>
	<li>Prepare a large volume of Wash buffer (0.01 M phosphate buffered saline (PBS), pH 7.4, 0.05% Tween 20 (PBS-T)). Store at RT.</li>
	<li>Prepare the peroxidase substrate (o-Phenylenediamine dihydrochloride (OPD)): Note: alternative peroxidase substrates such as 3,3&#8242;,5,5&#8242;-Tetramethylbenzidine (TMB), may be used
	<ol>
		<li>Prepare phosphate-citrate buffer by dissolving 1 capsule in 100 ml dH2O on the day of assay.</li>
		<li>Dissolve 1 OPD tablet (10 mg) in 20 ml of phosphate-citrate buffer immediately before use.</li>
	</ol>
	</li>
	<li>Prepare the stop solution (1 N H2SO4): add 27.2 ml stock 98% H2SO4 to 973 ml dH2O. Mix and then store at RT.</li>
</ol>
2. Determination of the Amount of NA to Use in ELLA
<ol>
	<li>Prepare Virus Dilutions in a 96-well Plate
	<ol>
		<li>Dispense 120 &#181;l sample diluent (section 1.2) into columns 1-11 of the dilution plate.</li>
		<li>To column 1, add an additional 96 &#181;l sample diluent.</li>
		<li>Thaw and then vortex the vial of virus before adding 24 &#181;l to duplicate wells in column 1. This gives a 1:10 dilution of virus.</li>
		<li>Make serial 2-fold dilutions of virus in sample diluent, by transferring 120 &#181;l from one well to the next using clean pipette tips for each dilution.</li>
	</ol>
	</li>
	<li>Transfer Virus Dilutions to a Fetuin-coated Plate
	<ol>
		<li>Wash the fetuin-coated plate 3 times with PBS-T (section 1.6) and then blot each plate onto absorbent paper towel to remove any excess wash buffer.</li>
		<li>Add 50 &#181;l of sample diluent (section 1.2.1) to each well in columns 1 to 11 of the fetuin-coated plate.</li>
		<li>Add 100 &#181;l of sample diluent to column 12 (these wells are the negative control).</li>
		<li>Transfer 50 &#181;l of diluted virus from columns 1-11 of the dilution plate to the corresponding wells of the fetuin-coated plate.</li>
		<li>Cover the plate with a plate sealer and then place it in a humidified incubator at 37 &#176;C.</li>
		<li>Make note of the time at which remaining steps will be performed (16-18 hr later).</li>
	</ol>
	</li>
	<li>Complete the NA Determination
	<ol>
		<li>When the incubation is complete (16-18 hr), transfer the plate to the bench and remove the plate sealer.</li>
		<li>Wash the plate 6 times with PBS-T (section 1.7) and then invert and pat on absorbent paper towels to ensure all liquid has been removed from the wells.</li>
		<li>Add 100 &#181;l/well PNA-HRPO solution (at the dilution determined in 1.5.3) to all wells and incubate the plate for 2 hr at RT.</li>
		<li>Less than 15 min before the incubation is to end, prepare the OPD solution as described in section 1.7.</li>
		<li>Wash the test plates 3 times to remove the PNA-HRPO and blot dry before adding 100 &#181;l of the OPD substrate to each well.</li>
		<li>Incubate the plate for exactly 10 min at RT.</li>
		<li>Stop the reaction by adding 100 &#181;l/well of 1N sulfuric acid.</li>
		<li>Use a plate reader to measure the Optical Density (OD) at 490 nm for 0.1 sec.</li>
		<li>Save and export all data files.</li>
	</ol>
	</li>
	<li>Select the Virus Dilution that will be Used for Serology
	<ol>
		<li>Draw a graph that plots OD490nm values at each virus dilution. Inspect the titration curve to identify the maximum signal (this is usually the signal that corresponds to a plateau at the beginning of the titration curve), the minimum signal (wells that contain no virus in column 12 of the plate provide the background control), and the dilutions of virus that result in OD that is proportional to the input dilution (i.e., linear region of the curve).</li>
		<li>Select the virus dilution that gives approximately 90% of the maximum signal and is within the linear range. Confirm that the OD at the selected dilution is at least 10-fold greater than the background signal. Use the selected virus dilution for all assays that employ this particular virus stock. 
		Note: Alternatively, measure the NA activity of the virus and use ~15-20 &#181;U NA activity/ml in the ELLA.</li>
	</ol>
	</li>
</ol>
3. Enzyme-linked Lectin Assay
Note: Figure 2 shows the setup of the dilution and assay plates.
<ol>
	<li>Make Sample Dilutions
	<ol>
		<li>Place the heat-inactivated samples on ice. 
		Note: 8 sera can be diluted in each plate.</li>
		<li>For a set up using dilutions starting at 1:10 across the plate, add 120 &#181;l sample diluent to all wells in columns 3-11.</li>
		<li>To column 2, add 216 &#181;l of sample diluent and 24 &#181;l of each sample. Mix the sample in the well by pipetting up and down 3 times and then transfer 120 &#181;l to the next column.</li>
		<li>After changing pipette tips, mix the contents of the well by pipetting up and down and then transfer 120 &#181;l to the next column.</li>
		<li>Repeat step 3.1.4 until the sample has been transferred to column 11 and the remaining 120 &#181;l discarded.</li>
	</ol>
	</li>
	<li>Add Samples and Virus to the Fetuin-coated Plate
	<ol>
		<li>Thaw a vial of virus, vortex and resuspend the virus in the diluent (section 1.2) at the dilution that was selected in step 2.4.2. Prepare at least 5 ml of virus for each assay plate. Keep the diluted virus on ice until the plates are washed and serum samples have been added to the plate.</li>
		<li>Decide on the number of fetuin-coated plates that are needed for the assay (generally apply 4 sera per plate). Wash the fetuin-coated plate 3 times with PBS-T and then invert each plate and blot onto absorbent paper towel to remove excess wash buffer.</li>
		<li>Use a multichannel pipette to transfer 50 &#181;l of each serum control or sample dilution from the dilution plate into duplicate wells in columns 2-11.</li>
		<li>Add 50 &#181;l of diluted virus to all wells except for the negative control (column 12).</li>
		<li>Add 50 &#181;l of sample diluent to wells in column 1 and add 100 &#181;l of sample diluent to column 12.</li>
		<li>Cover the wells with a plate sealer and then mix by gently tapping sides of plate or placing on a plate shaker at moderate speed for 10 sec.</li>
		<li>Place the plate in a humidified incubator at 37 &#176;C for 16-18 hr.</li>
	</ol>
	</li>
	<li>Add PNA-HRPO and complete the assay as described in section 2.3</li>
</ol>
4. Data Analysis
<ol>
	<li>Determine the Validity of the Assay Results
	<ol>
		<li>Confirm that the background values (no virus) are less than 10% of the positive control (virus and no serum).</li>
		<li>Confirm that the titers of control sera run in different assays using the same conditions are within 2-fold of the median titer.</li>
		<li>Confirm that OD measurements of control wells are consistent (&#8804;20% different) and that OD measurements of duplicate sample wells are consistent (&#8804;10% different).</li>
		<li>Determine the root cause of invalid results and repeat the assay if the criteria listed in 4.1.1, 4.1.2 or 4.1.3, are not met. Consider the factors presented in Table 1 when trying to troubleshoot.</li>
	</ol>
	</li>
	<li>Assign a 50% End-point Titer
	<ol>
		<li>For each assay plate, subtract the average background (no antigen added to wells) from all readings.</li>
		<li>Calculate the percent inhibition at each serum dilution using the formula: 100 x (ODvirus only control - ODtest sample)/ ODvirus only control.</li>
		<li>Identify the highest dilution that resulted in at least 50% inhibition of the maximum signal.</li>
		<li>Report the reciprocal of this dilution as the 50% end-point titer. 
		Note: If 50% inhibition was not achieved at any dilution, the titer is less than the first dilution tested, e.g., &#60;10 when 1:10 is the first dilution tested.</li>
	</ol>
	</li>
	<li>Calculate the 50% Inhibitory Concentration (IC50)
	<ol>
		<li>Use four parameter logistic regressions to determine the IC50 titer as follows: subtract the average background from all readings and then transfer the results to a program that performs regression analysis.</li>
		<li>Use non-linear regression, with the maximum set to the virus only control and the minimum set to zero, to determine the dilution that corresponds with exactly 50% inhibition.</li>
		<li>Report the reciprocal of this dilution as the IC50 titer.</li>
	</ol>
	</li>
</ol>

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D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Protective+effects+of+specific+immunity+to+viral+neuraminidase+on+influenza+virus+infection+of+mice.">Protective effects of specific immunity to viral neuraminidase on influenza virus infection of mice.</a> J Virol. 2 (8), 778-786 (1968).</li><li>Murphy, B. R., Kasel, J. A., Chanock, R. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Association+of+serum+anti-neuraminidase+antibody+with+resistance+to+influenza+in+man.">Association of serum anti-neuraminidase antibody with resistance to influenza in man.</a> N Engl J Med. 286 (25), 1329-1332 (1972).</li><li>Sandbulte, M. R., Gao, J., Straight, T. M., Eichelberger, M. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+miniaturized+assay+for+influenza+neuraminidase-inhibiting+antibodies+utilizing+reverse+genetics-derived+antigens.">A miniaturized assay for influenza neuraminidase-inhibiting antibodies utilizing reverse genetics-derived antigens.</a> Influenza Other Respi Viruses. 3 (5), 233-240 (2009).</li><li>Lambre, C. R., Terzidis, H., Greffard, A., Webster, R. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Measurement+of+anti-influenza+neuraminidase+antibody+using+a+peroxidase-linked+lectin+and+microtitre+plates+coated+with+natural+substrates.">Measurement of anti-influenza neuraminidase antibody using a peroxidase-linked lectin and microtitre plates coated with natural substrates.</a> J Immunol Methods. 135 (1-2), 49-57 (1990).</li><li>Couzens, L., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=An+optimized+enzyme-linked+lectin+assay+to+measure+influenza+A+virus+neuraminidase+inhibition+antibody+titers+in+human+sera.">An optimized enzyme-linked lectin assay to measure influenza A virus neuraminidase inhibition antibody titers in human sera.</a> J Virol Methods. 210C, 7-14 (2014).</li><li>Eichelberger, M. C., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Comparability+of+neuraminidase+inhibition+antibody+titers+measured+by+enzyme-linked+lectin+assay+(ELLA)+for+the+analysis+of+influenza+vaccine+immunogenicity.">Comparability of neuraminidase inhibition antibody titers measured by enzyme-linked lectin assay (ELLA) for the analysis of influenza vaccine immunogenicity.</a> Vaccine. 34 (4), 458-465 (2016).</li><li>Westgeest, K. B., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Optimization+of+an+enzyme-linked+lectin+assay+suitable+for+rapid+antigenic+characterization+of+the+neuraminidase+of+human+influenza+A(H3N2)+viruses.">Optimization of an enzyme-linked lectin assay suitable for rapid antigenic characterization of the neuraminidase of human influenza A(H3N2) viruses.</a> J Virol Methods. 217, 55-63 (2015).</li><li>Hoffmann, E., Neumann, G., Kawaoka, Y., Hobom, G., Webster, R. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+DNA+transfection+system+for+generation+of+influenza+A+virus+from+eight+plasmids.">A DNA transfection system for generation of influenza A virus from eight plasmids.</a> Proc Natl Acad Sci U S A. 97 (11), 6108-6113 (2000).</li><li>WHO. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Manual of Animal Influenza Diagnosis and Surveillance. , (2002).</li><li>Cate, T. R., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+high+dosage+influenza+vaccine+induced+significantly+more+neuraminidase+antibody+than+standard+vaccine+among+elderly+subjects.">A high dosage influenza vaccine induced significantly more neuraminidase antibody than standard vaccine among elderly subjects.</a> Vaccine. 28 (9), 2076-2079 (2010).</li><li>Couch, R. B., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Antibody+correlates+and+predictors+of+immunity+to+naturally+occurring+influenza+in+humans+and+the+importance+of+antibody+to+the+neuraminidase.">Antibody correlates and predictors of immunity to naturally occurring influenza in humans and the importance of antibody to the neuraminidase.</a> J Infect Dis. 207 (6), 974-981 (2013).</li><li>Fritz, R., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Neuraminidase-Inhibiting+Antibody+Response+to+H5N1+Virus+Vaccination+in+Chronically+Ill+and+Immunocompromised+Patients.">Neuraminidase-Inhibiting Antibody Response to H5N1 Virus Vaccination in Chronically Ill and Immunocompromised Patients.</a> Open Forum Infect Dis. 1 (2), (2014).</li><li>Fries, L. F., Smith, G. E., Glenn, G. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+recombinant+viruslike+particle+influenza+A+(H7N9)+vaccine.">A recombinant viruslike particle influenza A (H7N9) vaccine.</a> N Engl J Med. 369 (26), 2564-2566 (2013).</li><li>Monto, A. 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The authors have no conflicts of interest.

The enzyme-linked lectin assay is a practical method to measure NI antibody titers in sera. Although ELLA was described by Lambre et al., in 1990, its acceptance as a standard serologic assay has been more recent, with numerous laboratories performing the assay to measure NI antibody titers of clinical samples 12-16. Some modifications can be made to protocol steps without a large impact to the NI titers that are measured. For example, PBS can be used as diluent, however, it is very helpful to compare the virus titration curves in the recommended buffer (MES, pH 6.5) and PBS before a decision is made, as some NA subtypes have significantly reduced enzyme activity at pH &#62;7.0. When the optimal pH is not used, the maximum signal of virus may be reduced, resulting in the use of an excessive amount of antigen (i.e., virus) in the assay; under these conditions, the assay may have reduced sensitivity.
Some non-specific inhibition is observed when untreated sera are tested in the ELLA. This is removed by heat-treatment (56 &#176;C for 45 min), indicating the presence of thermolabile &#946;-inhibitors. Other non-specific inhibitors (&#945; and &#947;-class) of influenza HA have been described, particularly in relation to H3N2 virus hemagglutination and infectivity. Indeed, non-specific inhibition is also observed in ELLA when H3N2 viruses are used as the source of NA; this inhibition is removed by treatment of the serum samples with a small amount of sialidase 9.
As for other serologic assays, negative and positive controls should be included in each assay to provide a means to evaluate assay performance. When the assay has not met acceptance criteria, the reason should be identified. Table 1 provides a list of potential steps in the assay that can be addressed to troubleshoot problems.
The ELLA protocol provided in this report uses reassortant viruses that contain the HA originating from an avian virus, as the source of NA. This presents a limitation to performing the assay because a permit is required to work with low pathogenic avian viruses. H6Nx reassortant viruses can be shared between laboratories after they have been inactivated. Therefore the assay can be conducted through collaborations once the laboratory generating the reassortant virus has demonstrated that the inactivation is complete and the preparation has retained its NA activity. The constraint of using H6Nx reassortant viruses can be overcome by using non-infectious sources of NA. For example, some laboratories have used purified recombinant NA 17, while others have used virus-like particles (VLPs) 15. However, neither recombinant NA nor VLPs are readily available and therefore we continue to use influenza viruses with an antigenically-mismatched avian HA.
The traditional method to measure NI antibody titers is not practical for several reasons; of most concern are the harmful chemicals that are needed to produce a color reaction. In addition, large volumes of sample are needed and the assay is cumbersome to perform. In contrast, the ELLA is easy to perform and is in a format that allows titration of a reasonable number of samples. Data from studies that examined ELLA variability show that the assay yields reproducible results 7,8. The ELLA has consequently made routine measurement of NI antibody titers possible, and it is anticipated that it will be used more frequently by laboratories conducting serologic studies.
There are several critical steps that need to be considered when performing the ELLA. First, non-specific inhibitors of NA activity must be removed. These inhibitors are generally thermolabile and are destroyed by heat treatment at 56 &#176;C for 45 min. Heat treatment is sufficient to remove non-specific inhibitors from samples when H6 reassortant viruses are used as the source of NA, however, when H3N2 viruses are used in the ELLA, serum factors that bind to hemagglutinin also interfere with the ELLA and should be removed by treatment with a small amount of sialidase prior to heat treatment 9. Second, an excessive amount of antigen, i.e., reassortant H6Nx virus, should not be used as this reduces the sensitivity of the assay. Careful titration of virus is therefore an essential step; the amount of antigen used in each assay must provide a signal that is well above the background, and must be within the linear range of the titration curve, i.e., the signal (optical density) must be proportional to the virus dilution.
In addition to routine serology, the ELLA can be used to evaluate antigenic differences between NAs of seasonal influenza viruses. This information may be very helpful when selecting virus strains for inclusion as vaccine candidates and will facilitate a greater understanding of immune pressures that result in antigenic drift of NA. In addition, antigenic analysis of NAs from swine or avian influenza viruses may provide critical information in determining the pandemic potential of emerging strains.

Neuraminidase (NA) is a glycoprotein expressed on the surface of influenza virions. Its enzyme activity is essential for release of newly formed virus particles from infected cells 1,2. Antibodies that inhibit NA activity reduce virus titers and disease symptoms in animal models 3 and correlate with resistance against disease in humans 4. An increase in NA inhibition (NI) titers following vaccination may therefore serve an indicator of the vaccine&#39;s efficacy, however many past influenza immunogenicity studies did not include this end-point because the traditional assay to measure NI antibody titers is impractical for routine serology.
The traditional method to measure NI titers is based on quantifying the amount of sialic acid that is cleaved from glycoconjugates by NA 1. This method, often referred to as the thiobarbituric acid (TBA) method, uses hazardous chemicals to convert sialic acid to a chromophore that can be quantified by spectrometry. The assay is not suitable for testing large numbers of samples because individual glass tubes are used, making the assay cumbersome. Miniaturization of the assay to a 96 well plates provides a more practical format 5, however this assay still requires the use of toxic chemicals and therefore is not ideal.
An alternate assay to measure NI antibody titers was developed by Lambr&#233; et al. 6. This assay quantifies enzyme activity by measuring the amount of the penultimate sugar of glycoproteins, galactose, which becomes exposed when sialic acid is released by NA. Since peanut-agglutinin (PNA) binds specifically to galactose, a PNA-horseradish peroxidase (HRPO) conjugate can be used to obtain a colorimetric read-out. The optical density that is measured is therefore proportional to the NA activity in the sample. NI titers are measured by determining the highest dilution of serum that inhibits at least 50% of the NA activity. This enzyme-linked lectin assay (ELLA) is performed in 96-well plates coated with fetuin, a highly glycosylated serum protein, as the substrate for NA.
An important consideration for assays that measure NI titers, is the source of NA. This is because sera from vaccinated or infected individuals contain antibodies to hemagglutinin (HA) as well as antibodies to NA. Antibodies that bind to HA can interfere with NA activity and therefore, to avoid non-specific inhibition by HA-specific antibodies, purified NA or whole virus containing an antigenically-mismatched HA should be used in the assay. These viruses can be generated by classic reassortment or by reverse genetics; the objective is to rescue a virus that contains HA of a subtype that is not related to the target strain, and NA of the virus that is being studied. The assay described in this article employs influenza A viruses which are generated by reverse genetics to contain HA of subtype H6 and the targeted NA from influenza A viruses, subtypes H1N1 and H3N2 5.
Results generated by ELLA and miniaturized TBA assays show these methods are comparable, with similar subtype specificity and sensitivity 7. ELLA does not require the use of hazardous chemicals and therefore is the preferred method to measure NI titers. It is easy to perform, with only a few steps (Figure 1): samples are diluted and transferred to a fetuin-coated plate to which virus (the source of NA) is added. The plate is incubated O/N and washed before adding PNA-HRPO. After a 2 hr incubation, the plate is washed and peroxidase substrate is added; the color reaction is stopped and finally the optical density is measured and the reciprocal of the sample dilution that resulted in at least 50% inhibition of NA activity is reported as the 50% end-point titer. An intra-laboratory study to assess reproducibility of the ELLA, showed that plate-to-plate variability is minimal and operator-to-operator repeatability resulted in no more than 2-fold differences in titer 7. A subsequent inter-laboratory study of ELLA variability showed that the assay has good reproducibility when performed in different laboratories and that inclusion of a standard can further reduce variability in results 8. The ELLA is suitable for measuring NI antibody titers in serum panels from preclinical and clinical influenza vaccine studies 7 and can also be used to evaluate antigenic differences between the NAs of influenza viruses 9.

<table border="0" cellpadding="0" cellspacing="0" width="648">
	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:268px;">coating buffer&#160;</td>
			<td style="width:267px;">KPL</td>
			<td style="width:113px;">50-84-01</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">&#160;fetuin</td>
			<td>Sigma</td>
			<td>F3385</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">5X MES, pH 6.5&#160;</td>
			<td>KD-Medical</td>
			<td>PBS-0134</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">CaCl2</td>
			<td>Sigma</td>
			<td>C7902</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">30% BSA</td>
			<td>Sigma</td>
			<td>A8327</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Tween 20</td>
			<td>Sigma</td>
			<td>P1379</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Lectin PNA-HRPO</td>
			<td>Sigma</td>
			<td>&#160;L7759</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">PBS-T</td>
			<td>Sigma</td>
			<td>P3563-10PAK</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">o-Phenylenediamine dihydrochloride</td>
			<td>Sigma</td>
			<td>P8287</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">phosphate-citrate buffer</td>
			<td>Sigma</td>
			<td>P4922</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Sulfuric acid</td>
			<td>Sigma</td>
			<td>258105</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">96-well&#160; Maxisorp plates</td>
			<td>NUNC</td>
			<td>439454</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">96-well round bottom well plates</td>
			<td>NUNC</td>
			<td>267245</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Plate sealers</td>
			<td>Thermo Scientific&#160;</td>
			<td>14-245-192B</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">chicken eggs</td>
			<td>Charles River Laboratories</td>
			<td>9 day old embryonated specific pathogen free (SPF) chicken eggs</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">multichannel pipette</td>
			<td>Variety of suppliers e.g., Rainin, Eppendorf</td>
			<td>8 or 12 channel manual pipettem 50-250 &#181;l volume</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Pipette tips</td>
			<td>Depends on pipettor brand</td>
			<td>Depends on pipettor brand</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Plate reader, with 490 nm filter</td>
			<td>Perkin Elmer</td>
			<td>Victor V</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">water bath set to 37 &#176;C</td>
			<td>Variety of suppliers&#160;</td>
			<td>Variety of models</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">water bath set to 56 &#176;C</td>
			<td>Variety of suppliers&#160;</td>
			<td>Variety of models</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">refrigerator set to 4 &#176;C</td>
			<td>Variety of suppliers&#160;</td>
			<td>Variety of models</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">incubator set to 37 &#176;C</td>
			<td>Variety of suppliers&#160;</td>
			<td>Variety of models</td>
		</tr>
	</tbody>
</table>

measuring influenza neuraminidase inhibition antibody titers by enzyme-linked lectin assay

Antibodies to neuraminidase (NA), the second most abundant surface protein on influenza virus, contribute toward protection against influenza. Traditional methods to measure NA inhibiting (NI) antibody titers are not practical for routine serology. This protocol describes the enzyme-linked lectin assay (ELLA), a practical alternative method to measure NI titers that is performed in 96 well plates coated with a large glycoprotein substrate, fetuin. NA cleaves terminal sialic acids from fetuin, exposing the penultimate sugar, galactose. Peanut agglutinin (PNA) is a lectin with specificity for galactose and therefore the extent of desialylation can be quantified using a PNA-horseradish peroxidase conjugate, followed by addition of a chromogenic peroxidase substrate. The optical density that is measured is proportional to NA activity. To measure NI antibody titers, serial dilutions of sera are incubated at 37 &#176;C O/N on fetuin-coated plates with a fixed amount of NA. The reciprocal of the highest serum dilution that results in &#8805;50% inhibition of NA activity is designated as the NI antibody titer. The ELLA provides a practical format for routine evaluation of human antibody responses following influenza infection or vaccination.

The assay presented in this manuscript is shown diagrammatically in Figure 1. Figure 2 shows the 96 well plate layout, and indicates that serial dilutions of the serum samples are prepared in a dilution plate before transferring them to the fetuin-coated plate. A critical parameter of the assay is the amount of neuraminidase that is used in the assay; this is determined through titration of the reassortant virus. Examples of H6N1 and H6N2 virus titrations are shown in Figure 3. At 1:10, 1:20 and 1:40 dilutions of H6N1, the optical density was similar, indicating that the conditions were such that a maximum reading was obtained. At a 1:80 dilution, the read-out was approximately 90% of the maximum and therefore this dilution of virus was used in subsequent assays. Examples of serum titrations are shown in Figure 4. The figure shows a human serum sample that was titrated against H6N1 and H6N2 viruses. Each data point shows the percent inhibition of NA activity that was achieved by serial dilutions of serum; the first serum dilution in the H6N1 assay was 1:80; the first dilution of serum in the H6N2 assay was 5. Since the highest dilution that results in &#8805;50% inhibition is the NI antibody titer, the titer of the serum shown in this example is 640 against the NA of NC/99 (N1) and 160 against the NA of WI/05 (N2).
<img alt="Figure 1" src="/files/ftp_upload/54573/54573fig1.jpg" /> 
Figure 1. A schematic of the ELLA protocol. (A) Determination of the dilution of antigen (virus) to use in the assay (step 2 of the protocol): Virus dilutions are added to a fetuin coated plate and the NA activity measured by quantifying terminal galactose residues through binding of PNA-HRPO as described in step 2.3 of the protocol; (B) Determination of serum NI antibody titers (step 3 of the protocol). Samples are diluted and then transferred to a fetuin-coated plate where virus is added. The plate is incubated O/N before adding PNA-HRPO and completing the steps needed to generate a color reaction. Finally, the color reaction is stopped and the optical density is measured. The reciprocal of the sample dilution that results in at least 50% inhibition of NA activity is reported as the 50% end-point titer. <a href="https://www.jove.com/files/ftp_upload/54573/54573fig1large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 2" src="/files/ftp_upload/54573/54573fig2.jpg" /> 
Figure 2. Diagram to show the ELLA plate set up. Serial dilutions of serum samples are made in a dilution plate and then transferred to a fetuin-coated plate. <a href="https://www.jove.com/files/ftp_upload/54573/54573fig2large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 3" src="/files/ftp_upload/54573/54573fig3.jpg" /> 
Figure 3. Examples of H6N1 and H6N2 virus titrations. Serial dilutions of H6N1BR/07 (NA of A/Brisbane/59/2007 (H1N1) shown in blue symbols) and H6N2UR/07 (NA of A/Uruguay/716/2007 (H3N2) shown in red symbols) were incubated for 18 hr in fetuin-coated plates and the reactivity with PNA-HRPO determined as described. Average OD490nm of 2 wells is plotted against the reciprocal of the virus dilution. The dilution of H6N1 selected for use in the ELLA was 1:80 and the dilution of H6N2 selected was 1:40 because these dilutions resulted in approximately 90% of the maximum optical density and were within the linear range. <a href="https://www.jove.com/files/ftp_upload/54573/54573fig3large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 4" src="/files/ftp_upload/54573/54573fig4.jpg" /> 
Figure 4. Titration of serum against NA of N1 (red symbols) and N2 (blue symbols) subtypes. NA inhibition titers were measured against reassortant viruses H6N1NC/99 (contains the NA of A/New Caledonia/20/1999 (H1N1)) and H6N2WI/05 (contains the NA of A/Wisconsin/67/2005 (H3N2)). Percent enzyme inhibition for serial dilutions of serum is shown with the dashed horizontal line indicating 50% inhibition. The NA inhibition titers against the NAs of A/New Caledonia/20/1999 (H1N1) (NC/99) and A/Wisconsin/67/2005 (H3N2) (WI/05) were 29.3 (640) and 27.3(160), respectively. <a href="https://www.jove.com/files/ftp_upload/54573/54573fig4large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>Problem</td>
			<td>Possible cause(s)</td>
			<td>Solution</td>
		</tr>
		<tr>
			<td>Weak signal or no plateau reached in virus titration</td>
			<td>i) low NA enzyme activity 
			ii) virus stock not stored under optimal conditions</td>
			<td>i) confirm that the diluent has pH that is optimal for NA activity; if pH is optimal,&#160; prepare a new virus stock or concentrate virus 
			ii) Regrow and aliquot stock; snap-freeze vials on dry ice before storing at -80 &#176;C</td>
		</tr>
		<tr>
			<td>Weak or no color in positive cell control wells</td>
			<td>i)&#160;&#160; Virus dilution incorrectly assigned 
			ii) Vial-to-vial variability in frozen virus aliquots 
			iii)&#160; PNA- HRPO denatured or diluted too much 
			iv) OPD incorrectly prepared</td>
			<td>i)&#160; Repeat virus titration 
			ii) Titrate several vials from the same batch to ensure there is no variability. If significant variability, prepare fresh aliquots 
			iii) Use optimum virus dilution to retitrate PNA- HRPO 
			iv) Repeat with fresh preparation of OPD</td>
		</tr>
		<tr>
			<td>Weak or no inhibition by positive control sera</td>
			<td>i)&#160;&#160; Too much virus used in assay 
			ii)&#160; Serum deteriorated</td>
			<td>i)&#160;&#160; Repeat virus titration 
			ii)&#160; Obtain new antisera &#160;&#160;&#160;&#160; Check storage conditions</td>
		</tr>
		<tr>
			<td>Inhibition by negative control sera</td>
			<td>i)&#160; Inadequate&#160; heat-treatment of serum 
			ii) Too little virus used in assay</td>
			<td>i)&#160; Repeat heat-inactivation of serum 
			ii) Repeat virus titration</td>
		</tr>
		<tr>
			<td>High background</td>
			<td>i) Possible contamination of plates 
			ii) PNA- HRPO concentration too high/too low</td>
			<td>i) Repeat using freshly coated plates 
			ii) Titrate PNA-HRPO to identify correct dilution to use</td>
		</tr>
		<tr>
			<td>Virus titration shows apparent inhibition of NA activity at low dilutions</td>
			<td>i) Allantoic fluid may contain substrate for NA</td>
			<td>ii) Use virus that has been pelleted through a sucrose cushion</td>
		</tr>
	</tbody>
</table>
Table 1. Root cause analysis of assays that do not meet performance criteria. This table provides possible causes and solutions for problems that may occur when performing the ELLA.

Watch this Scientific Journal Video about Measuring Influenza Neuraminidase Inhibition Antibody Titers by Enzyme-linked Lectin Assay at JoVE.com

Measuring Influenza Neuraminidase Inhibition Antibody Titers by Enzyme-linked Lectin Assay

We describe the enzyme-linked lectin assay (ELLA) for measuring influenza neuraminidase (NA)-inhibition antibody titers in sera. The assay uses peanut agglutinin to quantify galactose residues that become accessible when NA removes sialic acid from fetuin-coated, 96-well plates.

Antibodies to neuraminidase (NA), the second most abundant surface protein on influenza virus, contribute toward protection against influenza. Traditional ...

measuring-influenza-neuraminidase-inhibition-antibody-titers-enzyme

Research

JoVE Journal

Immunology and Infection

18.4K Views.  Food and Drug Administration. We describe the enzyme-linked lectin assay (ELLA) for measuring influenza neuraminidase (NA)-inhibition antibody titers in sera. The assay uses peanut agglutinin to quantify galactose residues that become accessible when NA removes sialic acid from fetuin-coated, 96-well plates.

Video: Measuring Influenza Neuraminidase Inhibition Antibody Titers by Enzyme-linked Lectin Assay

Enzyme-linked Immunospot Assay (ELISPOT):  Quantification of Th-1 Cellular Immune Responses Against Microbial Antigens

Adaptive immunity is an important component to clearance of intracellular pathogens. The ability to detect and quantify these responses in humans is an important diagnostic tool. The enzyme-linked immunospot assay (ELISPOT) is gaining popularity for its ability to identify cellular immune responses against microbial antigens, including immunosuppressed populations such as those with HIV infection, transplantation, and steroid use. This assay has the capacity to quantify the immune responses against specific microbial antigens, as well as distinguish if these responses are Th1 or Th2 in character. ELISPOT is not limited to the site of inflammation. It is versatile in its ability to assess for immune responses within peripheral blood, as well as sites of active involvement such as bronchoalveolar lavage, cerebral spinal fluid, and ascites. Detection of immune responses against a single or multiple antigens is possible, as well as specific epitopes within microbial proteins. This assay facilitates detection of immune responses over time, as well as distinctions in antigens recognized by host T cells. Dual color ELISPOT assays are available for detection of simultaneous expression of two cytokines. Recent applications for this technique include diagnosis of extrapulmonary tuberculosis, as well as investigation of the contribution of infectious antigens to autoimmune diseases.

Enzyme-linked Immunospot Assay ELISPOT:  Quantification of Th-1 Cellular Immune Responses Against Microbial Antigens

Identification of microbial targets of adaptive immunity in idiopathic diseases can be accomplished by the use of the enzyme-linked immunospot assay.

Adaptive immunity is an important component to clearance of intracellular pathogens.  The ability to detect and quantify these responses in humans is an ...

Use of Interferon-&gamma; Enzyme-linked Immunospot Assay to Characterize Novel T-cell Epitopes of Human Papillomavirus

A protocol has been developed to overcome the difficulties of isolating and characterizing rare T cells specific for pathogens, such as human papillomavirus (HPV), that cause localized infections. The steps involved are identifying region(s) of HPV proteins that contain T-cell epitope(s) from a subject, selecting for the peptide-specific T cells based on interferon-&gamma; (IFN-&gamma;) secretion, and growing and characterizing the T-cell clones (Fig. 1). Subject 1 was a patient who was recently diagnosed with a high-grade squamous intraepithelial lesion by biopsy and underwent loop electrical excision procedure for treatment on the day the T cells were collected1. A region within the human papillomavirus type 16 (HPV 16) E6 and E7 proteins which contained a T-cell epitope was identified using an IFN- g enzyme-linked immunospot (ELISPOT) assay performed with overlapping synthetic peptides (Fig. 2). The data from this assay were used not only to identify a region containing a T-cell epitope, but also to estimate the number of epitope specific T cells and to isolate them on the basis of IFN- &gamma; secretion using commercially available magnetic beads (CD8 T-cell isolation kit, Miltenyi Biotec, Auburn CA). The selected IFN-&gamma; secreting T cells were diluted and grown singly in the presence of an irradiated feeder cell mixture in order to support the growth of a single T-cell per well. These T-cell clones were screened using an IFN- &gamma; ELISPOT assay in the presence of peptides covering the identified region and autologous Epstein-Barr virus transformed B-lymphoblastoid cells (LCLs, obtained how described by Walls and Crawford)2 in order to minimize the number of T-cell clone cells needed. Instead of using 1 x 105 cells per well typically used in ELISPOT assays1,3, 1,000 T-cell clone cells in the presence of 1 x 105 autologous LCLs were used, dramatically reducing the number of T-cell clone cells needed. The autologous LCLs served not only to present peptide antigens to the T-cell clone cells, but also to keep a high cell density in the wells allowing the epitope-specific T-cell clone cells to secrete IFN-&gamma;. This assures successful performance of IFN-&gamma; ELISPOT assay. Similarly, IFN- &gamma; ELISPOT assays were utilized to characterize the minimal and optimal amino acid sequence of the CD8 T-cell epitope (HPV 16 E6 52-61 FAFRDLCIVY) and its HLA class I restriction element (B58). The IFN- &gamma; ELISPOT assay was also performed using autologous LCLs infected with vaccinia virus expressing HPV 16 E6 or E7 protein. The result demonstrated that the E6 T-cell epitope was endogenously processed. The cross-recognition of homologous T-cell epitope of other high-risk HPV types was shown. This method can also be used to describe CD4 T-cell epitopes4.

Use of Interferon-&#947; Enzyme-linked Immunospot Assay to Characterize Novel T-cell Epitopes of Human Papillomavirus

Characterizing T-cell epitopes of pathogens that cause localized infections such as human papillomavirus is a challenge because of limited number of T cells in circulation. A method is described in which rare T cells were isolated and were characterized starting with a very small number of cells.

A protocol has been developed to overcome the difficulties of isolating and characterizing rare T cells specific for pathogens, such as human ...

Expression of Functional Recombinant Hemagglutinin and Neuraminidase Proteins from the Novel H7N9 Influenza Virus Using the Baculovirus Expression System

The baculovirus expression system is a powerful tool for expression of recombinant proteins. Here we use it to produce correctly folded and glycosylated versions of the influenza A virus surface glycoproteins - the hemagglutinin (HA) and the neuraminidase (NA). As an example, we chose the HA and NA proteins expressed by the novel H7N9 virus that recently emerged in China. However the protocol can be easily adapted for HA and NA proteins expressed by any other influenza A and B virus strains. Recombinant HA (rHA) and NA (rNA) proteins are important reagents for immunological assays such as ELISPOT and ELISA, and are also in wide use for vaccine standardization, antibody discovery, isolation and characterization. Furthermore, recombinant NA molecules can be used to screen for small molecule inhibitors and are useful for characterization of the enzymatic function of the NA, as well as its sensitivity to antivirals. Recombinant HA proteins are also being tested as experimental vaccines in animal models, and a vaccine based on recombinant HA was recently licensed by the FDA for use in humans. The method we describe here to produce these molecules is straight forward and can facilitate research in influenza laboratories, since it allows for production of large amounts of proteins fast and at a low cost. Although here we focus on influenza virus surface glycoproteins, this method can also be used to produce other viral and cellular surface proteins.

Here we describe a way to express correctly folded and functional influenza virus surface antigens derived from the novel Chinese H7N9 virus in insect cells. The technique can be adapted to express ectodomains of any viral or cellular surface proteins.

The baculovirus expression system is a powerful tool for expression of  recombinant proteins. Here we use it to produce correctly folded and  glycosylated ...

Application of Long-term cultured Interferon-&#947; Enzyme-linked Immunospot Assay for Assessing Effector and Memory T Cell Responses in Cattle

Effector and memory T cells are generated through developmental programing of na&#239;ve cells following antigen recognition. If the infection is controlled up to 95 % of the T cells generated during the expansion phase are eliminated (i.e., contraction phase) and memory T cells remain, sometimes for a lifetime. In humans, two functionally distinct subsets of memory T cells have been described based on the expression of lymph node homing receptors. Central memory T cells express C-C chemokine receptor 7 and CD45RO and are mainly located in T-cell areas of secondary lymphoid organs. Effector memory T cells express CD45RO, lack CCR7 and display receptors associated with lymphocyte homing to peripheral or inflamed tissues. Effector T cells do not express either CCR7 or CD45RO but upon encounter with antigen produce effector cytokines, such as interferon-&#947;. Interferon-&#947; release assays are used for the diagnosis of bovine and human tuberculosis and detect primarily effector and effector memory T cell responses. Central memory T cell responses by CD4+ T cells to vaccination, on the other hand, may be used to predict vaccine efficacy, as demonstrated with simian immunodeficiency virus infection of non-human primates, tuberculosis in mice, and malaria in humans. Several studies with mice and humans as well as unpublished data on cattle, have demonstrated that interferon-&#947; ELISPOT assays measure central memory T cell responses. With this assay, peripheral blood mononuclear cells are cultured in decreasing concentration of antigen for 10 to 14 days (long-term culture), allowing effector responses to peak and wane; facilitating central memory T cells to differentiate and expand within the culture.

Long-term cultured interferon-&#947; enzyme-linked immunospot assay is used as a measure of central memory responses and correlates with protective anti-mycobacterial vaccine responses. With this assay, peripheral blood mononuclear cells are stimulated with mycobacterial antigens and interleukin-2 for 14 days, enabling differentiation and expansion of central memory T cells.

Effector and memory T cells are generated through developmental programing of na&#239;ve cells following antigen recognition. If the infection is ...

Measuring Attachment and Internalization of Influenza A Virus in A549 Cells by Flow Cytometry

Attachment to target cells followed by internalization are the very first steps of the life cycle of influenza A virus (IAV). We provide here a detailed protocol for measuring relative changes in the amount of viral particles that attach to A549 cells, a human lung epithelial cell line, as well as in the amount of particles that are internalized into the cell. We use biotinylated virus which can be easily detected following staining with Cy3-labeled streptavidin (STV-Cy3). We describe the growth, purification and biotinylation of A/WSN/33, a widely used IAV laboratory strain. Cold-bound biotinylated IAV particles on A549 cells are stained with STV-Cy3 and measured using flow cytometry. To investigate uptake of viral particles, cold-bound virus is allowed to internalize at 37 &#176;C. In order to differentiate between external and internalized viral particles, a blocking step is applied: Free binding spots on the biotin of attached virus on the cell surface are bound by unlabeled streptavidin (STV). Subsequent cell permeabilization and staining with STV-Cy3 then enables detection of internalized viral particles. We present a calculation to determine the relative amount of internalized virus. This assay is suitable to measure effects of drug-treatments or other manipulations on attachment or internalization of IAV.

We present a protocol describing a semi-quantitative method for measuring both, the attachment of influenza A virus to A549 cells, as well as the internalization of virus particles into the target cells by flow cytometry.

Attachment to target cells followed by internalization are the very first steps of the life cycle of influenza A virus (IAV). We provide here a detailed ...

Measuring Phagosome pH by Ratiometric Fluorescence Microscopy

Phagocytosis is a fundamental process through which innate immune cells engulf bacteria, apoptotic cells or other foreign particles in order to kill or neutralize the ingested material, or to present it as antigens and initiate adaptive immune responses. The pH of phagosomes is a critical parameter regulating fission or fusion with endomembranes and activation of proteolytic enzymes, events that allow the phagocytic vacuole to mature into a degradative organelle. In addition, translocation of H+ is required for the production of high levels of reactive oxygen species (ROS), which are essential for efficient killing and signaling to other host tissues. Many intracellular pathogens subvert phagocytic killing by limiting phagosomal acidification, highlighting the importance of pH in phagosome biology. Here we describe a ratiometric method for measuring phagosomal pH in neutrophils using fluorescein isothiocyanate (FITC)-labeled zymosan as phagocytic targets, and live-cell imaging. The assay is based on the fluorescence properties of FITC, which is quenched by acidic pH when excited at 490 nm but not when excited at 440 nm, allowing quantification of a pH-dependent ratio, rather than absolute fluorescence, of a single dye. A detailed protocol for performing in situ dye calibration and conversion of ratio to real pH values is also provided. Single-dye ratiometric methods are generally considered superior to single wavelength or dual-dye pseudo-ratiometric protocols, as they are less sensitive to perturbations such as bleaching, focus changes, laser variations, and uneven labeling, which distort the measured signal. This method can be easily modified to measure pH in other phagocytic cell types, and zymosan can be replaced by any other amine-containing particle, from inert beads to living microorganisms. Finally, this method can be adapted to make use of other fluorescent probes sensitive to different pH ranges or other phagosomal activities, making it a generalized protocol for the functional imaging of phagosomes.

Phagosomal pH influences phagosome maturation, oxidant production, phagosomal killing as well as antigen presentation. Here we describe a ratiometric method for measuring time-course and endpoint pH changes in individual phagosomes in living phagocytes using fluorescence microscopy.

Phagocytosis is a fundamental process through which innate immune cells engulf bacteria, apoptotic cells or other foreign particles in order to kill or ...

In Vitro Disassembly of Influenza A Virus Capsids by Gradient Centrifugation

Acid-triggered molecular processes closely control cell entry of many viruses that enter through the endocytic system. In the case of influenza A virus (IAV), virus fusion with the endosomal membrane as well as the subsequent disassembly of the viral capsid, called uncoating, is governed by the ionic conditions inside endocytic vesicles. The early steps in the virus life cycle are hard to study because endosomes cannot be directly accessed experimentally, creating the need for an in vitro approach. Here, we describe a method based on velocity gradient centrifugation of purified virions through a two-layer glycerol gradient, which enables analysis of the IAV core and its stability. The gradient contains a non-ionic detergent (NP-40) in its lower layer to remove the viral membrane by solubilization as the virus sediments toward the bottom. At neutral pH, viral cores are pelleted as stable structures. The major core components, matrix protein (M1) and the viral ribonucleoproteins (vRNPs), can be clearly identified in the pellet fraction by SDS-PAGE. Decreasing the pH to 6.0 or lower in the bottom layer selectively removes M1 from the pellet followed by release of vRNPs at more acidic conditions. Viral protein bands on Coomassie-stained gels can be subjected to densitometric quantification to monitor intermediate states of IAV disassembly. Besides pH, other factors that influence viral core stability can be assessed, such as salt concentration and putative viral uncoating factors, simply by modifying the detergent-containing glycerol layer accordingly. Taken together, the presented technique allows highly reproducible and quantitative analysis of viral uncoating in vitro. It can be applied to other enveloped viruses that undergo complex uncoating processes.

In Vitro Disassembly of Influenza A Virus Capsids by Gradient Centrifugation

Disassembly of influenza A virus cores during virus entry into host cells is a multistep process. We describe an in vitro method to analyze the early stages of viral uncoating. In this approach, velocity gradient centrifugation is used to biochemically dissect the steps that initiate uncoating under defined conditions.

Acid-triggered molecular processes closely control cell entry of many viruses that enter through the endocytic system. In the case of influenza A virus ...

Fluorescence-based Neuraminidase Inhibition Assay to Assess the Susceptibility of Influenza Viruses to The Neuraminidase Inhibitor Class of Antivirals

The neuraminidase (NA) inhibitors are the only class of antivirals approved for the treatment and prophylaxis of influenza that are effective against currently circulating strains. In addition to their use in treating seasonal influenza, the NA inhibitors have been stockpiled by a number of countries for use in the event of a pandemic. It is therefore important to monitor the susceptibility of circulating influenza viruses to this class of antivirals. There are different types of assays that can be used to assess the susceptibility of influenza viruses to the NA inhibitors, but the enzyme inhibition assays using either a fluorescent substrate or a chemiluminescent substrate are the most widely used and recommended. This protocol describes the use of a fluorescence-based assay to assess influenza virus susceptibility to NA inhibitors. The assay is based on the NA enzyme cleaving the 2&#8242;-(4-Methylumbelliferyl)-&#945;-D-N-acetylneuraminic acid (MUNANA) substrate to release the fluorescent product 4-methylumbelliferone (4-MU). Therefore, the inhibitory effect of an NA inhibitor on the influenza virus NA is determined based on the concentration of the NA inhibitor that is required to reduce 50% of the NA activity, given as an IC50 value.

We describe the use of a phenotypic fluorescence-based neuraminidase inhibition assay to assess the susceptibility of influenza A and B viruses to the neuraminidase inhibitor class of antivirals.

The neuraminidase (NA) inhibitors are the only class of antivirals approved for the treatment and prophylaxis of influenza that are effective against ...

Measuring Influenza Neutralizing Antibody Responses to A(H3N2) Viruses in Human Sera by Microneutralization Assays Using MDCK-SIAT1 Cells

Neutralizing antibodies against hemagglutinin (HA) of influenza viruses are considered the main immune mechanism that correlates with protection for influenza infections. Microneutralization (MN) assays are often used to measure neutralizing antibody responses in human sera after influenza vaccination or infection. Madine Darby Canine Kidney (MDCK) cells are the commonly used cell substrate for MN assays. However, currently circulating 3C.2a and 3C.3a A(H3N2) influenza viruses have acquired altered receptor binding specificity. The MDCK-SIAT1 cell line with increased &#945;-2,6 sialic galactose moieties on the surface has proven to provide improved infectivity and more faithful replications than conventional MDCK cells for these contemporary A(H3N2) viruses. Here, we describe a MN assay using MDCK-SIAT1 cells that has been optimized to quantify neutralizing antibody titers to these contemporary A(H3N2) viruses. In this protocol, heat inactivated sera containing neutralizing antibodies are first serially diluted, then incubated with 100 TCID50/well of influenza A(H3N2) viruses to allow antibodies in the sera to bind to the viruses. MDCK-SIAT1 cells are then added to the virus-antibody mixture, and incubated for 18 - 20 h at 37 &#176;C, 5% CO2 to allow A(H3N2) viruses to infect MDCK-SIAT1 cells. After overnight incubation, plates are fixed and the amount of virus in each well is quantified by an enzyme-linked immunosorbent assay (ELISA) using anti-influenza A nucleoprotein (NP) monoclonal antibodies. Neutralizing antibody titer is defined as the reciprocal of the highest serum dilution that provides &#8805;50% inhibition of virus infectivity.

Measuring Influenza Neutralizing Antibody Responses to AH3N2 Viruses in Human Sera by Microneutralization Assays Using MDCK-SIAT1 Cells

Influenza neutralizing antibodies correlate with protection of influenza infections. Microneutralization assays measure neutralizing antibodies in human sera and are often used for influenza human serology. We describe a microneutralization assay using MDCK-SIAT1 cells to measure neutralizing antibody titers to contemporary 3C.2a and 3C.3a A(H3N2) viruses following influenza vaccination or infection.

Neutralizing antibodies against hemagglutinin (HA) of influenza viruses are considered the main immune mechanism that correlates with protection for ...

Development and Validation of an Ultrasensitive Single Molecule Array Digital Enzyme-linked Immunosorbent Assay for Human Interferon-&#945;

The main aim of this protocol is to describe the development and validation of an interferon (IFN)-&#945; single molecule array digital Enzyme-Linked ImmunoSorbent Assay (ELISA) assay. This system enables the quantification of human IFN-&#945; protein with unprecedented sensitivity, and with no cross-reactivity for other species of IFN.
The first key step of the protocol is the choice of the antibody pair, followed by the conjugation of the capture antibody to paramagnetic beads, and biotinylation of the detection antibody. Following this step, different parameters such as assay configuration, detector antibody concentration, and buffer composition can be modified until optimum sensitivity is achieved. Finally, specificity and reproducibility of the method are assessed to ensure confidence in the results. Here, we developed an IFN-&#945; single molecule array assay with a limit of detection of 0.69 fg/mL using high-affinity autoantibodies isolated from patients with biallelic mutations in the autoimmune regulator (AIRE) protein causing autoimmune polyendocrinopathy syndrome type 1/autoimmune polyendocrinopathy-candidiasis-ectodermal dystrophy (APS1/APECED). Importantly, these antibodies enabled detection of all 13 IFN-&#945; subtypes.
This new methodology allows the detection and quantification of IFN-&#945; protein in human biological samples at attomolar concentrations for the first time. Such a tool will be highly useful in monitoring the levels of this cytokine in human health and disease states, most particularly infection, autoimmunity, and autoinflammation.

Here we present a protocol to describe the development and validation of a single molecule array digital ELISA assay, which enables the ultra-sensitive detection of all IFN-&#945; subtypes in human samples.

The main aim of this protocol is to describe the development and validation of an interferon (IFN)-&#945; single molecule array digital Enzyme-Linked ...

Measuring Influenza Neuraminidase Inhibition Antibody Titers by Enzyme-linked Lectin Assay

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Measuring Phagosome pH by Ratiometric Fluorescence Microscopy

In Vitro Disassembly of Influenza A Virus Capsids by Gradient Centrifugation

Fluorescence-based Neuraminidase Inhibition Assay to Assess the Susceptibility of Influenza Viruses to The Neuraminidase Inhibitor Class of Antivirals

Measuring Influenza Neutralizing Antibody Responses to A(H3N2) Viruses in Human Sera by Microneutralization Assays Using MDCK-SIAT1 Cells

Development and Validation of an Ultrasensitive Single Molecule Array Digital Enzyme-linked Immunosorbent Assay for Human Interferon-α