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	<li>. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Prevention+and+control+of+seasonal+influenza+with+vaccines.+Recommendations+of+the+Advisory+Committee+on+Immunization+Practices--United+States,+2013-2014.">Prevention and control of seasonal influenza with vaccines. Recommendations of the Advisory Committee on Immunization Practices--United States, 2013-2014.</a> MMWR Recomm Rep. 62, 1-43 (2013).</li><li>Dominguez-Cherit, G., 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=Critically+Ill+patients+with+2009+influenza+A(H1N1)+in+Mexico.">Critically Ill patients with 2009 influenza A(H1N1) in Mexico.</a> JAMA. 302 (17), 1880-1887 (2009).</li><li>Fox, B. D., 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=Pandemic+influenza+(H1N1):+impact+on+lung+transplant+recipients+and+candidates.">Pandemic influenza (H1N1): impact on lung transplant recipients and candidates.</a> J Heart Lung Transplant. 29 (9), 1034-1038 (2010).</li><li>Piercy, J., Miles, A., Krankheiten, S. B. f. G. S. V., Values, 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> The Economic Impact of Influenza in Switzerland: Interpandemic Situation. , (2003).</li><li>Barclay, V. 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=Positive+network+assortativity+of+influenza+vaccination+at+a+high+school:+implications+for+outbreak+risk+and+herd+immunity.">Positive network assortativity of influenza vaccination at a high school: implications for outbreak risk and herd immunity.</a> PLoS One. 9 (2), 87042 (2014).</li><li>Baluch, A., 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=Randomized+controlled+trial+of+high-dose+intradermal+versus+standard-dose+intramuscular+influenza+vaccine+in+organ+transplant+recipients.">Randomized controlled trial of high-dose intradermal versus standard-dose intramuscular influenza vaccine in organ transplant recipients.</a> Am J Transplant. 13 (4), 1026-1033 (2013).</li><li>Haralambieva, I. H., 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=The+Impact+of+Immunosenescence+on+Humoral+Immune+Response+Variation+after+Influenza+A/H1N1+Vaccination+in+Older+Subjects.">The Impact of Immunosenescence on Humoral Immune Response Variation after Influenza A/H1N1 Vaccination in Older Subjects.</a> PLoS One. 10 (3), 0122282 (2015).</li><li>Egli, A., 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=Vaccine+adjuvants--understanding+molecular+mechanisms+to+improve+vaccines.">Vaccine adjuvants--understanding molecular mechanisms to improve vaccines.</a> Swiss Med Wkly. 144, 13940 (2014).</li><li>O'Shea, D., Widmer, L. A., Stelling, J., Egli, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Changing+face+of+vaccination+in+immunocompromised+hosts.">Changing face of vaccination in immunocompromised hosts.</a> Curr Infect Dis Rep. 16 (9), 420 (2014).</li><li>Meulemans, G., Carlier, M. C., Gonze, M., Petit, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Comparison+of+hemagglutination-inhibition,+agar+gel+precipitin,+and+enzyme-linked+immunosorbent+assay+for+measuring+antibodies+against+influenza+viruses+in+chickens.">Comparison of hemagglutination-inhibition, agar gel precipitin, and enzyme-linked immunosorbent assay for measuring antibodies against influenza viruses in chickens.</a> Avian Dis. 31 (3), 560-563 (1987).</li><li>Martins, T. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Development+of+internal+controls+for+the+Luminex+instrument+as+part+of+a+multiplex+seven-analyte+viral+respiratory+antibody+profile.">Development of internal controls for the Luminex instrument as part of a multiplex seven-analyte viral respiratory antibody profile.</a> Clin Diagn Lab Immunol. 9 (1), 41-45 (2002).</li><li>Webster, R., Cox, N., St&#246;hr, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=WHO+Animal+Influenza+Manual.">WHO Animal Influenza Manual.</a> WHO/CDS/CSR/NCS. 2002.5, 1-99 (2002).</li><li>Mittelholzer, C. M., 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=Human+cell+lines+used+in+a+micro+neutralization+test+for+measuring+influenza-neutralizing+antibodies.">Human cell lines used in a micro neutralization test for measuring influenza-neutralizing antibodies.</a> Scand J Immunol. 63 (4), 257-263 (2006).</li><li>Hamilton, R. S., Gravell, M., Major, E. O. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Comparison+of+antibody+titers+determined+by+hemagglutination+inhibition+and+enzyme+immunoassay+for+JC+virus+and+BK+virus.">Comparison of antibody titers determined by hemagglutination inhibition and enzyme immunoassay for JC virus and BK virus.</a> J Clin Microbiol. 38 (1), 105-109 (2000).</li><li>Kumakura, S., 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=Comparison+of+hemagglutination+inhibition+assay+and+enzyme+immunoassay+for+determination+of+mumps+and+rubella+immune+status+in+health+care+personnel.">Comparison of hemagglutination inhibition assay and enzyme immunoassay for determination of mumps and rubella immune status in health care personnel.</a> J Clin Lab Anal. 27 (5), 418-421 (2013).</li><li>Ogundiji, O. T., Okonko, I. O., Adu, F. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Determination+of+measles+hemagglutination+inhibiting+antibody+levels+among+school+children+in+Ibadan,+Nigeria.">Determination of measles hemagglutination inhibiting antibody levels among school children in Ibadan, Nigeria.</a> J Immunoassay Immunochem. 34 (2), 208-217 (2013).</li><li>Cwach, K. T., Sandbulte, H. R., Klonoski, J. M., Huber, V. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Contribution+of+murine+innate+serum+inhibitors+toward+interference+within+influenza+virus+immune+assay.">Contribution of murine innate serum inhibitors toward interference within influenza virus immune assay.</a> Influenza Other Respir Viruses. 6 (2), 127-135 (2012).</li><li>Lee, P. S., 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=Receptor+mimicry+by+antibody+F045-092+facilitates+universal+binding+to+the+H3+subtype+of+influenza+virus.">Receptor mimicry by antibody F045-092 facilitates universal binding to the H3 subtype of influenza virus.</a> Nat Commun. 5, 3614 (2014).</li><li>Blumel, 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=Age-related+prevalence+of+cross-reactive+antibodies+against+influenza+A(H3N2)+variant+virus,+Germany,+2003+to+2010.">Age-related prevalence of cross-reactive antibodies against influenza A(H3N2) variant virus, Germany, 2003 to 2010.</a> Euro Surveill. 20 (32), 16-24 (2015).</li><li>Reber, A. J., 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=Seasonal+Influenza+Vaccination+of+Children+Induces+Humoral+and+Cell-Mediated+Immunity+Beyond+the+Current+Season:+Cross-reactivity+with+Past+and+Future+Strains.">Seasonal Influenza Vaccination of Children Induces Humoral and Cell-Mediated Immunity Beyond the Current Season: Cross-reactivity with Past and Future Strains.</a> J Infect Dis. , (2016).</li></ol>

A.E. was supported by a research grants from the "SNSF Ambizione Score" program (PZ00P3_154709), "Forschungsfond, F&#246;rderung strategischer Projekte" University of Basel, Stiftungsinfektionskrankheiten Basel, and Bangeter Rhyner Stiftung. L.K. was supported by a grant of the Technical University of Graz, Austria. J.L. acknowledges support by an iPhD fellowship of the SystemsX.ch initiative in systems biology program (9th call).

Quantification of pre- and post-vaccination influenza virus specific antibody titers is an important tool necessary for vaccine studies. Based on the surrogate measures of protection against virus infection, such as seroprotection (&#62;1:40) or seroconversion (4-fold titer increase), vaccination strategies can be optimized9. Using the provided protocols can determine: (i) the hemagglutination potential of a particular virus, and (ii) the antibody titers for a virus of interest.
Modification and Troubleshooting:
This protocol is based on the WHO standard12. We modified the protocol by using PCR tube strips for serum preparations (see step 5). This modification helped to significantly reduce the workload and to increase the throughput of the assay. Further, we reduced the antigen amount by one fourth in the antigen titration step, which is cost effective overtime. A lower amount of serum (10 &#181;L) can be used for the RDE treatment, which helps especially when the sample amount is limited (e.g., mouse sera). The back titration and positive control are included in the antibody measurement plate to serve as a proper internal control and to monitor the aging of erythrocytes.
In addition, we used different erythrocyte concentrations than those in the WHO standard12 to set the optimal size of the erythrocyte clot for a good visual readout. To guarantee this we suggest checking the erythrocyte concentration before the assay. Although, we have not optimized this part in our protocol, methods such as absorbance measurement with OD or cell counting could be used.
If the RDE is not completely inactivated, RBCs can be desialylated and reverse HA-positive wells when hemagglutination is measured at room temperature. Although we never observed this problem, in this case, we suggest performing the HI at 4 &#176;C, since RDE activity is significantly lower at 4 &#176;C. However, performing the HI assay at 4 &#176;C is slower.
Limitations of the Technique:
A few critical aspects of the HI assay include the following points: Interestingly, the hemagglutination is strongly dependent on the particular type of erythrocyte (e.g., turkey or guinea pig RBC). The optimal type of blood should be tested before a particular virus strain is evaluated and the same type of blood should be used throughout the assay. Although the induction of cross-reactivity between viruses may generate an immunological advantage in the case of a slightly new virus19,20, this may cause some problems from a diagnostic point of view due to low specificity. Therefore, cross reactivity between similar viruses should be carefully addressed and discussed in studies. By choosing erythrocytes from a specific species, the amount of cross-reactivity can be somewhat lowered.
Significance with Respect to Existing Methods:
The HI is a well-established gold standard method providing highly reproducible and reliable results. Other techniques such as ELISA may detect non-neutralizing antibodies, whereas the HI only detects antibodies which bind to the HA stem loop and thereby correlate with neutralization.
Critical Steps Within the Protocol:
The most critical steps include the serum treatment with RDE to inactivate unspecific inhibitors and binding to the HA of the virus. Another critical step is to control for hemolysis of the erythrocytes as they age over time.
Future Applications:
The protocol may be used for other viruses with hemagglutination potential. Although we have only shown results on human sera samples here, the assay can also be used to measure antibody titers in mouse sera or in cell culture supernatant with stimulated B-cells (data not shown). In summary, the HI allows a rapid and reproducible assessment of vaccine-induced antibody titers.

Infection with influenza virus is associated with considerable morbidity, mortality, and high healthcare costs1,2,3,4. In particular, elderly, newborns, pregnant women, and patients with chronic disease are at risk for more severe clinical outcomes. Therefore, vaccination against circulating influenza virus strains is the primary measure to decrease the burden of disease in these high-risk populations. The increase of the individual immune response after vaccination, e.g., influenza-specific antibodies above a protective threshold, reduces the individual risk of infection and in general the likelihood of viral transmission within a population5. A detailed understanding of the vaccine-induced humoral immune response in different populations and across various age groups is a key element to answer important clinical questions6,7,8,9, such as: Why do some elderly patients have infections despite previous vaccination? What is a &#34;good&#34; and &#34;sufficient&#34; vaccine-induced protection? How often should a vaccine be applied to an immunosuppressed patient to reach protective titers? What is the most effective dosage? What is the impact of a novel adjuvant on post-vaccination antibody titers? The measurement of the vaccine-specific antibody production may help answer these important questions and improve vaccination outcomes.
The quantification of virus-specific antibody titers can be performed with various immunological methods. This includes solid-phase10 or bead-based ELISA11 assays, the HI assay12, and neutralizing assays13. ELISA-based methods allow the screening of relatively large amounts of serum samples against various antigens. Also, pathogen-specific Immunoglobulin (Ig)M and IgG can be separately explored. Although the characteristics of an antigen, e.g., the linear amino acid sequence or virus-like particle may influence the binding of antibodies, the spectrum of potential epitopes is very broad and does not provide information on whether an antibody response has functional relevance.
In contrast, the neutralization assay determines the potential of antibodies to functionally inhibit the infection of cells and therefore reflects the neutralization potential. However, this method is very labor intensive, requires culturing of specific cell lines and live viruses, and therefore, it is time-consuming, expensive, and requires special equipment.
This article describes a step-by-step the World Health Organization (WHO)-based HI protocol12 to quantify influenza-specific antibody titers. Hemagglutination is a characteristic effect of some viruses leading to the agglutination of erythrocytes. The inhibition of this effect with patient sera allows the measurement of inhibitory antibody concentrations, which reflects a neutralizing effect.
We have modified the workflow of the WHO-protocol to allow a more efficient handling of multiple samples at the same time and thereby reducing the required time. The first protocol describes the determination of the agglutination potential of a particular influenza antigen. In doing so, the correct influenza antigen concentration is determined for the second protocol. This part should be repeated with every new viral antigen, as well as each batch of blood.
The second protocol describes the determination of influenza-specific antibody titers. The presented protocols are optimized for the investigation of influenza virus and human serum samples however, it can also be applied for mouse serum samples or cell-culture supernatants from stimulated immune cells, e.g., virus-specific B-cells. Results can be determined as absolute measured titers. In many vaccine studies, the geometric mean titers and the 95% confidence interval are shown for each particular population. For interpretation, seroprotection or seroconversion are often used to describe the susceptibility of a population to a certain virus. Seroprotection is defined as a titer of &#8805;1:40, and seroconversion as a more than 4-fold titer increase with achievement of seroprotective titers between two time points (most commonly pre-vaccination and 30 days post-vaccination are used).
Both protocols are easy to use and they can be adapted to a broad range of research questions. In particular, they can be used to determine reliably and quickly&#160;the antibody titers against various other viruses with the capacity for hemagglutination, such as measles, polyomaviruses, mumps, or rubella14,15,16.

<table><tbody><tr><td>25 ml Disposable Multichannel Pipette Reservoirs</td><td>Integra</td><td>4312</td><td></td></tr><tr><td>8-well PCR tubes</td><td>Brand GMBH</td><td>781332</td><td>For serum aliquots</td></tr><tr><td>96-well microtiter plate, U-shaped</td><td>TPP</td><td>92097</td><td>For HI assay when using mammalian RBCs</td></tr><tr><td>96-well microtiter plate, V-shaped</td><td>Corning Costar</td><td>3897</td><td>For HI assay when using avian RBCs</td></tr><tr><td>Aqua ad iniect. Steril</td><td>Bichsel AG</td><td>1000004</td><td>For preparing influenza antigen and cholera filtrate solutions</td></tr><tr><td>Chicken RBC (10%)</td><td>Cedarlane</td><td>CLC8800</td><td>10% suspension of chicken red blood cells in Alsever&#039;s solution</td></tr><tr><td>Cholera filtrate</td><td>Sigma-Aldrich</td><td>C8772</td><td>Used as receptor destroying enzyme (RDE)</td></tr><tr><td>Dulbecco&#039;s PBS</td><td>Sigma-Aldrich</td><td>D8537</td><td>For diluting the serum samples, RBCs and antigens</td></tr><tr><td>Eppendorf Multichannel pipette, 12-channel, 10-100 &amp;micro;l</td><td>Sigma-Aldrich</td><td>Z683949</td><td></td></tr><tr><td>Eppendorf Multichannel pipette, 8-channel, 10-100 &amp;micro;l</td><td>Sigma-Aldrich</td><td>Z683930</td><td></td></tr><tr><td>Guinea Pig RBC (10%)</td><td>Cedarlane</td><td>CLC1800</td><td>10% suspension of guinea pig red blood cells in Alsever&#039;s solution</td></tr><tr><td>Influenza Anti-A/California/7/09 HA serum&amp;nbsp;</td><td>NIBSC</td><td>14/134&amp;nbsp;</td><td>Used as positive control at the HI assay</td></tr><tr><td>Influenza Anti-A/Switzerland/9715293/2013-like HA serum&amp;nbsp;</td><td>NIBSC</td><td>14/272</td><td>Used as positive control at the HI assay</td></tr><tr><td>Influenza Anti-A/Texas/50/2012-Like HA Serum&amp;nbsp;</td><td>NIBSC</td><td>13/178</td><td>Used as positive control at the HI assay</td></tr><tr><td>Influenza Anti-B/Brisbane/60/2008-HA serum&amp;nbsp;</td><td>NIBSC</td><td>13/254&amp;nbsp;</td><td>Used as positive control at the HI assay</td></tr><tr><td>Influenza Anti-B/Massachusetts/02/2012 HA serum&amp;nbsp;</td><td>NIBSC</td><td>13/182</td><td>Used as positive control at the HI assay</td></tr><tr><td>Influenza antigen A/California/7/09 (H1N1)(NYMC-X181)&amp;nbsp;</td><td>NIBSC</td><td>12/168</td><td>Inactivated, partially purified A/California/7/09 (H1N1)(NYMC-X181)&amp;nbsp; virus (ca. 46&amp;micro;gHA/ml)</td></tr><tr><td>Influenza antigen A/Switzerland/9715293/2013 (NIB88)</td><td>NIBSC</td><td>14/254</td><td>Inactivated, partially purified A/Switzerland/9715293/2013 (NIB88) virus (ca. 55&amp;micro;gHA/ml)</td></tr><tr><td>Influenza antigen A/Texas/50/2012 (H3N2)(NYMCX-223)</td><td>NIBSC</td><td>13/112</td><td>Inactivated, partially purified A/Texas/50/2012 (H3N2)(NYMCX-223) virus (ca. 74&amp;micro;gHA/ml)</td></tr><tr><td>Influenza antigen B/Brisbane/60/2008</td><td>NIBSC</td><td>13/234</td><td>Inactivated, partially purified B/Brisbane/60/2008 virus (ca. 42&amp;micro;gHA/ml)</td></tr><tr><td>Influenza antigen B/Massachusetts/02/2012</td><td>NIBSC</td><td>13/134</td><td>Inactivated, partially purified B/Massachusetts/02/2012 virus (ca. 35&amp;micro;gHA/ml)</td></tr><tr><td>Serum-Tubes</td><td>S-Monovette, Sardstedt</td><td>01.1601.100</td><td>For serum extraction with clotting activator</td></tr><tr><td>Single Donor Human RBC, Type 0</td><td>Innovative Research</td><td>IPLA-WB3&amp;nbsp;</td><td>Suspension of single donor human red blood cells in Alsever&#039;s solution (ca. 26%)</td></tr><tr><td>Turkey RBC (10%)</td><td>Cedarlane</td><td>CLC1180</td><td>10% suspension of turkey red blood cells in Alsever&#039;s solution</td></tr><tr><td>Phosphate Buffered Saline (PBS)</td><td>Gibco</td><td></td><td></td></tr></tbody></table>

an optimized hemagglutination inhibition (hi) assay to quantify influenza-specific antibody titers

Antibody titers are commonly used as surrogate markers for serological protection against influenza and other pathogens. Detailed knowledge of antibody production pre- and post-vaccination is required to understand vaccine-induced immunity. This article describes a reliable point-by-point protocol to determine influenza-specific antibody titers. The first protocol describes a method to specify the antigen amounts required for hemagglutination, which standardizes the concentrations for subsequent usage in the second protocol (hemagglutination assay, HA assay). The second protocol describes the quantification of influenza-specific antibody titers against different viral strains by using a serial dilution of human serum or cell culture supernatants (hemagglutination inhibition assay, HI assay).
As an applied example, we show the antibody response of a healthy cohort, which received a trivalent inactivated influenza vaccine. Additionally, the cross-reactivity between the different influenza viruses is shown and methods to minimize cross-reactivity by using different types of animal red blood cells (RBCs) are explained. The discussion highlights advantages and disadvantages of the presented assays and how the determination of influenza-specific antibody titers can improve the understanding of vaccine-related immunity.

Pre- and post-vaccination induced antibody response against Influenza A H3N2 
The vaccine-induced antibody response was assessed in 26 healthy volunteers who received an inactivated trivalent subunit influenza vaccine containing Influenza A/H1N1/California/2009, A/H3N2/Texas/2012, and B/Massachusetts/02/2012 prior to the 2014/2015 influenza season. Figure 6 shows a representative example of 2 vaccine recipients. Interestingly, during that particular influenza season, A/H3N2/Texas/2012 was not circulating, and instead the season included the somewhat different viral strain: A/H3N2/Switzerland/2013. The viral hemagglutinin of A/H3N2/Texas/2012 and A/H3N2/Switzerland/2013 show 97% sequence identity and differ in only eleven amino acids (see Table 4), whose positions are highlighted in Figure 7.
<img alt="Figure 7" class="xfigimg" src="/files/ftp_upload/55833/55833fig8.jpg" /> 
Figure 7: Hemagglutinin comparison of A/H3N2 influenza strains.&#160;We compared the hemagglutinin of the viral strains A/Texas/50/2012 and A/Switzerland/9715293/2013. Since there are no hemagglutinin crystal structures of these strains, we used the crystal structure of the highly similar hemagglutinin of the influenza strain A/Victoria/361/201118, which shows 98% sequence identity with the Texas strain and 95% sequence identity with the Switzerland strain. The amino acid positions in which the Texas and Switzerland strains differ are highlighted.&#160;<a href="https://cloudflare-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/55833/55833fig8large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
We observed a cross-reactive immune response for the viral strains A/H3N2/Switzerland/2013 and A/H3N2/Texas/2012. HI titers against Influenza A/H3N2/Switzerland/2013 were significantly lower in terms of geometric mean titers and induced seroprotection (Figure 8A) in comparison to Influenza A/H3N2/Texas/2012 (Figure 8B).
<img alt="Figure 8" class="xfigimg" src="/files/ftp_upload/55833/55833fig9.jpg" /> 
Figure 8: Geometric mean antibody-titers of healthy donors.&#160;The geometric mean antibody-titers (GMTs) of 25 healthy donors pre- and post-vaccination are determined using two different antigens. The mean titers of A/H3N2/Switzerland/2013 (A) and A/H3N2/Texas/2012 (B) are shown. An immune response due to the vaccination can be observed as increasing titers after the vaccination (d7-d60), compared to the GMTs before the vaccination (d0). 180 days after the vaccination, the GMTs decrease again. Of note, only A/H3N2/Texas/2012 (which was in the vaccine) reaches protective titers. Bars indicate geometric mean titers, and whiskers indicate the 95% confidence intervals. The dashed line indicates the seroprotection threshold. The % of seroprotected people (titer &#62;1:40) is shown in the graph.&#160;<a href="https://cloudflare-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/55833/55833fig9large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
After vaccination, the antibody titers against A/H3N2/Texas/2012 increased in most subjects; although the A/H3N2/Switzerland/2013 strain was not present in the vaccine, the titer against A/H3N2/Switzerland/2013 increased in some subjects as well. Figure 9 shows the correlation between both titers over all time points with an R2 of 0.745 for a linear regression model. As one would expect, the induction of the antibody response against A/H3N2/Switzerland/2013 was less potent.
<img alt="Figure 9" class="xfigimg" src="/files/ftp_upload/55833/55833fig10.jpg" /> 
Figure 9: Cross-reaction between A/H3N2 influenza strains.&#160;The A/H3N2/Texas titers of every individual and time point are plotted against the corresponding A/H3N2/Switzerland titers. A linear regression model shows an R2&#160;of 0.745.&#160;<a href="https://cloudflare-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/55833/55833fig10large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
Hemagglutination potential is based on the type of blood used 
The viral hemagglutinin shows different species-dependent potential to hemagglutinate erythrocytes. This species-dependent effect also impacts the hemagglutination inhibition assay. To improve the specificity of measured anti-viral titers, we evaluated the best suited type of erythrocytes for five viral antigens (Influenza B/Brisbane/60/2008 and B/Massachusetts/02/2012, Influenza A/H1N1/California/2009, A/H3N2/Texas/2012, and A/H3N2/Switzerland/2013) to achieve the maximum hemagglutination but also the lowest cross-reactivity. We used positive control sera from the National Institute for Biological Standards and Control (NIBSC) against each antigen to perform these assays.
For Influenza B, we could observe that the B/Massachusetts/02/2012 induced antibody response does not provide protection against B/Brisbane/60/2008. In contrast, antibodies against B/Brisbane/60/2008 showed cross-reactivity against B/Massachusetts/02/2012 at a 4-fold lower titer across different erythrocytes (see Table 3). Of interest, guinea pig blood did not properly hemagglutinate with Influenza B. Turkey blood did best in showing the potential to hemagglutinate and the highest titers with relative low cross-reactivity apart from the previously mentioned A/H3N2/Texas and /Switzerland strains.
<table fo:keep-together.within-page="1">
	<tbody>
		<tr>
			<td></td>
			<td>Turkey</td>
			<td>Guinea Pig</td>
			<td>Chicken</td>
			<td>Human type O</td>
		</tr>
		<tr>
			<td>B/Brisbane</td>
			<td>1024</td>
			<td>-</td>
			<td>1024</td>
			<td>1024</td>
		</tr>
		<tr>
			<td>B/Massachusetts</td>
			<td>1024</td>
			<td>384</td>
			<td>768</td>
			<td>1024</td>
		</tr>
		<tr>
			<td>A/H3N2/Switzerland</td>
			<td>1024</td>
			<td>1024</td>
			<td>-</td>
			<td>1024</td>
		</tr>
		<tr>
			<td>A/H3N2/Texas</td>
			<td>1024</td>
			<td>1024</td>
			<td>512</td>
			<td>1024</td>
		</tr>
		<tr>
			<td>A/H1N1/California</td>
			<td>1024</td>
			<td>1024</td>
			<td>768</td>
			<td>768</td>
		</tr>
	</tbody>
</table>
Table 3: Positive control titers against the respective influenza HA antigen across different species.
<table fo:keep-together.within-page="1">
	<tbody>
		<tr>
			<td>No</td>
			<td>&#160;A/H3N2/Texas/2012 strain</td>
			<td>&#160;A/H3N2/Switzerland/2013 strain</td>
			<td>Position</td>
		</tr>
		<tr>
			<td>1</td>
			<td>Asparagine (N)</td>
			<td>Alanine (A)</td>
			<td>128</td>
		</tr>
		<tr>
			<td>2</td>
			<td>Alanine (A)</td>
			<td>Serine (S)</td>
			<td>138</td>
		</tr>
		<tr>
			<td>3</td>
			<td>Isoleucine (I)</td>
			<td>Arginine (R)</td>
			<td>140</td>
		</tr>
		<tr>
			<td>4</td>
			<td>Arginine (R)</td>
			<td>Glycine (G)</td>
			<td>142</td>
		</tr>
		<tr>
			<td>5</td>
			<td>Asparagine (N)</td>
			<td>Serine (S)</td>
			<td>145</td>
		</tr>
		<tr>
			<td>6</td>
			<td>Phenylalanine (F)</td>
			<td>Serine (S)</td>
			<td>159</td>
		</tr>
		<tr>
			<td>7</td>
			<td>Glycine (G)</td>
			<td>Valine (V)</td>
			<td>186</td>
		</tr>
		<tr>
			<td>8</td>
			<td>Proline (P)</td>
			<td>Serine (S)</td>
			<td>198</td>
		</tr>
		<tr>
			<td>9</td>
			<td>Serine (S)</td>
			<td>Phenylalanine (F)</td>
			<td>219</td>
		</tr>
		<tr>
			<td>10</td>
			<td>Asparagine (N)</td>
			<td>Aspartate (D)</td>
			<td>225</td>
		</tr>
		<tr>
			<td>11*</td>
			<td>Lysine (K)</td>
			<td>Arginine (R)</td>
			<td>326</td>
		</tr>
		<tr>
			<td></td>
			<td colspan="3">*not shown in the crystal structure in Figure 8, because the hemagglutinin was cutted at residue 325.&#160;</td>
		</tr>
	</tbody>
</table>
Table 4: List of different amino acids of hemagglutinin between A/H3N2/Texas/2012 and A/H3N2/Switzerland/2013 strains

Watch this Scientific Journal Video about An Optimized Hemagglutination Inhibition HI Assay to Quantify Influenza-specific Antibody Titers at JoVE.com

An Optimized Hemagglutination Inhibition HI Assay to Quantify Influenza-specific Antibody Titers

The presented protocols describe how to perform a hemagglutination inhibition assay to quantify influenza-specific antibody titers from serum samples of influenza vaccine recipients. The first assay determines optimal viral antigen concentrations by hemagglutination. The second assay quantifies influenza-specific antibody titers by hemagglutination inhibition.

An Optimized Hemagglutination Inhibition (HI) Assay to Quantify Influenza-specific Antibody Titers

Antibody titers are commonly used as surrogate markers for serological protection against influenza and other pathogens. Detailed knowledge of antibody ...

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Nanyang Technological University

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35.7K Views.  University of Basel. De gepresenteerde protocollen beschrijven het uitvoeren van een hemagglutination inhibitie-test om te kwantificeren van influenza-specifieke antilichamen titers van serummonsters van influenza vaccinontvangers. De eerste bepaling bepaalt optimale virusantigeen concentraties door hemagglutination. De tweede test kwantificeert influenza-specifieke antilichamen titers door inhibitie van de hemagglutination.

Video: An Optimized Hemagglutination Inhibition HI Assay to Quantify Influenza-specific Antibody Titers

Quantitative Analyses of all Influenza Type A Viral Hemagglutinins and Neuraminidases using Universal Antibodies in Simple Slot Blot Assays

Hemagglutinin (HA) and neuraminidase (NA) are two surface proteins of influenza viruses which are known to play important roles in the viral life cycle and the induction of protective immune responses1,2. As the main target for neutralizing antibodies, HA is currently used as the influenza vaccine potency marker and is measured by single radial immunodiffusion (SRID)3. However, the dependence of SRID on the availability of the corresponding subtype-specific antisera causes a minimum of 2-3 months delay for the release of every new vaccine. Moreover, despite evidence that NA also induces protective immunity4, the amount of NA in influenza vaccines is not yet standardized due to a lack of appropriate reagents or analytical method5. Thus, simple alternative methods capable of quantifying HA and NA antigens are desirable for rapid release and better quality control of influenza vaccines.
Universally conserved regions in all available influenza A HA and NA sequences were identified by bioinformatics analyses6-7. One sequence (designated as Uni-1) was identified in the only universally conserved epitope of HA, the fusion peptide6, while two conserved sequences were identified in neuraminidases, one close to the enzymatic active site (designated as HCA-2) and the other close to the N-terminus (designated as HCA-3)7. Peptides with these amino acid sequences were synthesized and used to immunize rabbits for the production of antibodies. The antibody against the Uni-1 epitope of HA was able to bind to 13 subtypes of influenza A HA (H1-H13) while the antibodies against the HCA-2 and HCA-3 regions of NA were capable of binding all 9 NA subtypes. All antibodies showed remarkable specificity against the viral sequences as evidenced by the observation that no cross-reactivity to allantoic proteins was detected. These universal antibodies were then used to develop slot blot assays to quantify HA and NA in influenza A vaccines without the need for specific antisera7,8. Vaccine samples were applied onto a PVDF membrane using a slot blot apparatus along with reference standards diluted to various concentrations. For the detection of HA, samples and standard were first diluted in Tris-buffered saline (TBS) containing 4M urea while for the measurement of NA they were diluted in TBS containing 0.01% Zwittergent as these conditions significantly improved the detection sensitivity. Following the detection of the HA and NA antigens by immunoblotting with their respective universal antibodies, signal intensities were quantified by densitometry. Amounts of HA and NA in the vaccines were then calculated using a standard curve established with the signal intensities of the various concentrations of the references used. 
Given that these antibodies bind to universal epitopes in HA or NA, interested investigators could use them as research tools in immunoassays other than the slot blot only.

A simple slot blot method was developed for the quantification of influenza viral hemagglutinin and neuraminidase using universal antibodies targeting their most conserved sequences identified through bioinformatics analyses. This innovative approach may provide a useful alternative to quantitative determination of all viral hemagglutinin and neuraminidase.

Hemagglutinin (HA) and neuraminidase (NA) are two surface proteins of influenza viruses which are known to play important roles in the viral life cycle ...

Immunology and Infection

High-throughput Detection Method for Influenza Virus

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

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

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

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.

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 ...

Generation of Escape Variants of Neutralizing Influenza Virus Monoclonal Antibodies

Influenza viruses exhibit a remarkable ability to adapt and evade the host immune response. One way is through antigenic changes that occur on the surface glycoproteins of the virus. The generation of escape variants is a powerful method in elucidating how viruses escape immune detection and in identifying critical residues required for antibody binding. Here, we describe a protocol on how to generate influenza A virus escape variants by utilizing human or murine monoclonal antibodies (mAbs) directed against the viral hemagglutinin (HA). With the use of our technique, we previously characterized critical residues required for the binding of antibodies targeting either the head or stalk of the novel avian H7N9 HA. The protocol can be easily adapted for other virus systems. Analyses of escape variants are important for modeling antigenic drift, determining single nucleotide polymorphisms (SNPs) conferring resistance and virus fitness, and in the designing of vaccines and/or therapeutics.

We describe a method by which we identify critical residues required for the binding of human or murine monoclonal antibodies that target the viral hemagglutinin of influenza A viruses. The protocol can be adapted to other virus surface glycoproteins and their corresponding neutralizing antibodies.

Influenza viruses exhibit a remarkable ability to adapt and evade the host immune response. One way is through antigenic changes that occur on the surface ...

A Method to Assess Fc-mediated Effector Functions Induced by Influenza Hemagglutinin Specific Antibodies

Antibodies play a crucial role in coupling the innate and adaptive immune responses against viral pathogens through their antigen binding domains and Fc-regions. Here, we describe how to measure the activation of Fc effector functions by monoclonal antibodies targeting the influenza virus hemagglutinin with the use of a genetically engineered Jurkat cell line expressing an activating type 1 Fc-Fc&#947;R. Using this method, the contribution of specific Fc-Fc&#947;R interactions conferred by immunoglobulins can be determined using an in vitro assay.

We describe a method to measure the activation of Fc-mediated effector functions by antibodies that target the influenza virus hemagglutinin. This assay can also be adapted to assess the ability of monoclonal antibodies or polyclonal sera targeting other viral surface glycoproteins to induce Fc-mediated immunity.

Antibodies play a crucial role in coupling the innate and adaptive immune responses against viral pathogens through their antigen binding domains and ...

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 ...

Streamlined Preparation of 4&#8212HAU Antigen for Accurate NDV Antibody Detection

Optimization of Antigen Preparation for Hemagglutination Inhibition Assay of Newcastle Disease Virus Serology

Accurate assessment of Newcastle disease virus (NDV) antibody titers is crucial for effective poultry disease control and surveillance. This article introduces an optimized method for preparing 4 hemagglutination units (4-HAU) antigen solution, a key component of the hemagglutination inhibition assay (HI) used in NDV serological detection. Unlike conventional methods, which involve time-consuming and undefined back-titration and adjustment steps, this approach streamlines this process by accurately measuring the HA titer using an initial series of dilutions (1:3, 1:5, 1:7, and 1:9). We also provide a specific method for adjusting or reformulating based on the back-titration results, reducing the need for repeated back-titrations. In addition, we evaluated the effect of the accuracy of the 4-HAU antigen solution on serum HI titer and found that when the titer of the 4-HAU antigen was lower than 3, it resulted in the appearance of false-positive HI samples. By providing an accurate method and minimizing computational tasks, this approach increases test efficiency and reliability, contributing to improved disease surveillance and control in poultry populations.

This article presents a simplified method for preparing 4 hemagglutination units antigen for Newcastle disease virus serological testing. By accurately determining the hemagglutination titer of the antigen, combined with a more rigorous back-titration method, and a well-defined adjustment process, it enhances test efficiency, reduces false positives, and improves disease surveillance in poultry.

Accurate assessment of Newcastle disease virus (NDV) antibody titers is crucial for effective poultry disease control and surveillance. This article ...

Generation of Influenza Virus Escape Variants against Neutralizing Antibodies

Source: Leon, P. E., et al. <a href="https://app-jove-com.remotexs.ntu.edu.sg/v/56067">Generation of Escape Variants of Neutralizing Influenza Virus Monoclonal Antibodies.</a> J. Vis. Exp. (2017).
This video demonstrates a technique for generating escape variants of neutralizing influenza virus monoclonal antibodies. Antibody-neutralized virus suspension is injected into embryonated chicken eggs to propagate escape variants selectively. This results in a virus population predominantly of escape variants, confirmed via a Hemagglutination assay.

NOTE: HA-specific antibodies that inhibit viral replication can generally be categorized into i) ones that bind on or adjacent of the receptor binding ...

JoVE Encyclopedia of Experiments

Immunology

Antibody-Based Technologies

Characterization and Functional Analysis

The Hemagglutination Inhibition Assay to Detect Serum Antibodies Against a Target Antigen

Source: Kaufmann, L. et al., <a href="https://app-jove-com.remotexs.ntu.edu.sg/v/55833">An Optimized Hemagglutination Inhibition (HI) Assay to Quantify Influenza-specific Antibody Titers.</a> J. Vis. Exp. (2017)
The video demonstrates a hemagglutination inhibition assay for detecting serum antibodies against specific antigens. Confirmation of hemagglutination inhibition in the test well and hemagglutination in the control well is achieved through distinct visual patterns. This assay plays a crucial role in assessing immune response, vaccine effectiveness, and studying viral infections in virology and immunology.

All procedures involving human participants have been performed in compliance with the institutional, national, and international guidelines for human ...

Immunodiagnostics

Specialized Assays

An Assay to Measure Influenza Neuraminidase Inhibition Antibody Titers

Source: Gao, J., et al. <a href="https://app-jove-com.remotexs.ntu.edu.sg/v/54573">Measuring Influenza Neuraminidase Inhibition Antibody Titers by Enzyme-linked Lectin Assay.</a> J. Vis. Exp. (2016).
This video demonstrates an assay measuring influenza neuraminidase (NA) inhibiting antibody titers in a serum sample. A serum sample containing antibodies against NA is added to a fetuin-coated microwell plate. Upon adding the virus, the antibodies prevent viral NA from cleaving fetuin and exposing its galactose moiety. A peroxidase-conjugated lectin is then added, which binds to the exposed galactose. The NA-inhibiting antibody titer is determined using a colorimetric test, where a peroxidase substrate gets cleaved by the immobilized lectin-conjugated peroxidase to produce color.

All procedures involving sample collection have been performed in accordance with the institute&#39;s IRB guidelines.
1. Preparation of Reagents and ...

An Optimized Hemagglutination Inhibition (HI) Assay to Quantify Influenza-specific Antibody Titers

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