Name: an optimized hemagglutination inhibition (hi) assay to quantify influenza-specific antibody titers
Uploaded: 2017-12-01T15:00:00
Description: Watch this Scientific Journal Video about An Optimized Hemagglutination Inhibition HI Assay to Quantify Influenza-specific Antibody Titers at JoVE.com

The study protocols were approved through the local ethical review board (www.EKNZ.ch) and written informed consent was obtained from all participants.
1. Serum Collection
<ol>
	<li>Collect serum samples from humans at time points of interest. For this study, we collected sera at days 0 (time of influenza vaccination), +7, +30, +60, and +180 after vaccination.</li>
	<li>To obtain the serum, centrifuge the sample tubes at 1,200 x g for 10 min at room temperature (20 - 25 &#176;C). 
	NOTE...

<ol>
	<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>

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.
<p class="jove...

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 style="border-collapse:collapse;width:716pt" width="955">
	<colgroup>
		<col style="width:263pt" width="351" />
		<col style="width:100pt" width="133" />
		<col style="width:85pt" width="113" />
		<col style="width:269pt" width="358" />
	</colgroup>
	<tbody>
		<tr height="21" style="height:15.75pt">
			<td class="xl67" height="21" style="width:263pt;height:15.75pt" width="351">25 ml Disposable Multichannel Pipette Reservoirs</td>
			<td class="xl67" style="width:100pt" width="133">Integra</td>
			<td class="xl67" style="width:85pt" width="113">4312</td>
			<td class="xl68" style="width:269pt" width="358"></td>
		</tr>
		<tr height="21" style="height:15.75pt">
			<td class="xl63" height="21" style="width:263pt;height:15.75pt" width="351">8-well PCR tubes</td>
			<td class="xl63" style="width:100pt" width="133">Brand GMBH</td>
			<td class="xl63" style="width:85pt" width="113">781332</td>
			<td class="xl66">For serum aliquots</td>
		</tr>
		<tr height="21" style="height:15.75pt">
			<td class="xl63" height="21" style="width:263pt;height:15.75pt" width="351">96-well microtiter plate, U-shaped</td>
			<td class="xl63" style="width:100pt" width="133">TPP</td>
			<td class="xl63" style="width:85pt" width="113">92097</td>
			<td class="xl66">For HI assay when using mammalian RBCs</td>
		</tr>
		<tr height="21" style="height:15.75pt">
			<td class="xl63" height="21" style="width:263pt;height:15.75pt" width="351">96-well microtiter plate, V-shaped</td>
			<td class="xl63" style="width:100pt" width="133">Corning Costar</td>
			<td class="xl63" style="width:85pt" width="113">3897</td>
			<td class="xl66">For HI assay when using avian RBCs</td>
		</tr>
		<tr height="21" style="height:15.75pt">
			<td class="xl63" height="21" style="width:263pt;height:15.75pt" width="351">Aqua ad iniect. Steril</td>
			<td class="xl63" style="width:100pt" width="133">Bichsel AG</td>
			<td align="right" class="xl63" style="width:85pt" width="113">1000004</td>
			<td class="xl66">For preparing influenza antigen and cholera filtrate solutions</td>
		</tr>
		<tr height="21" style="height:15.75pt">
			<td class="xl63" height="21" style="width:263pt;height:15.75pt" width="351">Chicken RBC (10%)</td>
			<td class="xl63" style="width:100pt" width="133">Cedarlane</td>
			<td class="xl63" style="width:85pt" width="113">CLC8800</td>
			<td class="xl66">10% suspension of chicken red blood cells in Alsever&#39;s solution</td>
		</tr>
		<tr height="21" style="height:15.75pt">
			<td class="xl63" height="21" style="width:263pt;height:15.75pt" width="351">Cholera filtrate</td>
			<td class="xl63" style="width:100pt" width="133">Sigma-Aldrich</td>
			<td class="xl63" style="width:85pt" width="113">C8772</td>
			<td class="xl66">Used as receptor destroying enzyme (RDE)</td>
		</tr>
		<tr height="21" style="height:15.75pt">
			<td class="xl63" height="21" style="width:263pt;height:15.75pt" width="351">Dulbecco&#39;s PBS</td>
			<td class="xl63" style="width:100pt" width="133">Sigma-Aldrich</td>
			<td class="xl63" style="width:85pt" width="113">D8537</td>
			<td class="xl66">For diluting the serum samples, RBCs and antigens</td>
		</tr>
		<tr height="42" style="height:31.5pt">
			<td class="xl63" height="42" style="width:263pt;height:31.5pt" width="351">Eppendorf Multichannel pipette, 12-channel, 10-100 &#181;l</td>
			<td class="xl63" style="width:100pt" width="133">Sigma-Aldrich</td>
			<td class="xl67" style="width:85pt" width="113">Z683949</td>
			<td class="xl66"></td>
		</tr>
		<tr height="42" style="height:31.5pt">
			<td class="xl63" height="42" style="width:263pt;height:31.5pt" width="351">Eppendorf Multichannel pipette, 8-channel, 10-100 &#181;l</td>
			<td class="xl63" style="width:100pt" width="133">Sigma-Aldrich</td>
			<td class="xl67" style="width:85pt" width="113">Z683930</td>
			<td class="xl66"></td>
		</tr>
		<tr height="21" style="height:15.75pt">
			<td class="xl63" height="21" style="width:263pt;height:15.75pt" width="351">Guinea Pig RBC (10%)</td>
			<td class="xl63" style="width:100pt" width="133">Cedarlane</td>
			<td class="xl67" style="width:85pt" width="113">CLC1800</td>
			<td class="xl66">10% suspension of guinea pig red blood cells in Alsever&#39;s solution</td>
		</tr>
		<tr height="21" style="height:15.75pt">
			<td class="xl63" height="21" style="width:263pt;height:15.75pt" width="351">Influenza Anti-A/California/7/09 HA serum&#160;</td>
			<td class="xl63" style="width:100pt" width="133">NIBSC</td>
			<td class="xl67" style="width:85pt" width="113">14/134&#160;</td>
			<td class="xl66">Used as positive control at the HI assay</td>
		</tr>
		<tr height="42" style="height:31.5pt">
			<td class="xl63" height="42" style="width:263pt;height:31.5pt" width="351">Influenza Anti-A/Switzerland/9715293/2013-like HA serum&#160;</td>
			<td class="xl63" style="width:100pt" width="133">NIBSC</td>
			<td class="xl67" style="width:85pt" width="113">14/272</td>
			<td class="xl66">Used as positive control at the HI assay</td>
		</tr>
		<tr height="21" style="height:15.75pt">
			<td class="xl63" height="21" style="width:263pt;height:15.75pt" width="351">Influenza Anti-A/Texas/50/2012-Like HA Serum&#160;</td>
			<td class="xl63" style="width:100pt" width="133">NIBSC</td>
			<td class="xl63" style="width:85pt" width="113">13/178</td>
			<td class="xl66">Used as positive control at the HI assay</td>
		</tr>
		<tr height="21" style="height:15.75pt">
			<td class="xl63" height="21" style="width:263pt;height:15.75pt" width="351">Influenza Anti-B/Brisbane/60/2008-HA serum&#160;</td>
			<td class="xl63" style="width:100pt" width="133">NIBSC</td>
			<td class="xl63" style="width:85pt" width="113">13/254&#160;</td>
			<td class="xl66">Used as positive control at the HI assay</td>
		</tr>
		<tr height="21" style="height:15.75pt">
			<td class="xl63" height="21" style="width:263pt;height:15.75pt" width="351">Influenza Anti-B/Massachusetts/02/2012 HA serum&#160;</td>
			<td class="xl63" style="width:100pt" width="133">NIBSC</td>
			<td class="xl63" style="width:85pt" width="113">13/182</td>
			<td class="xl66">Used as positive control at the HI assay</td>
		</tr>
		<tr height="42" style="height:31.5pt">
			<td class="xl63" height="42" style="width:263pt;height:31.5pt" width="351">Influenza antigen A/California/7/09 (H1N1)(NYMC-X181)&#160;</td>
			<td class="xl63" style="width:100pt" width="133">NIBSC</td>
			<td class="xl63" style="width:85pt" width="113">12/168</td>
			<td class="xl66">Inactivated, partially purified A/California/7/09 (H1N1)(NYMC-X181)&#160; virus (ca. 46&#181;gHA/ml)</td>
		</tr>
		<tr height="42" style="height:31.5pt">
			<td class="xl63" height="42" style="width:263pt;height:31.5pt" width="351">Influenza antigen A/Switzerland/9715293/2013 (NIB88)</td>
			<td class="xl63" style="width:100pt" width="133">NIBSC</td>
			<td class="xl63" style="width:85pt" width="113">14/254</td>
			<td class="xl66">Inactivated, partially purified A/Switzerland/9715293/2013 (NIB88) virus (ca. 55&#181;gHA/ml)</td>
		</tr>
		<tr height="42" style="height:31.5pt">
			<td class="xl63" height="42" style="width:263pt;height:31.5pt" width="351">Influenza antigen A/Texas/50/2012 (H3N2)(NYMCX-223)</td>
			<td class="xl63" style="width:100pt" width="133">NIBSC</td>
			<td class="xl63" style="width:85pt" width="113">13/112</td>
			<td class="xl66">Inactivated, partially purified A/Texas/50/2012 (H3N2)(NYMCX-223) virus (ca. 74&#181;gHA/ml)</td>
		</tr>
		<tr height="21" style="height:15.75pt">
			<td class="xl63" height="21" style="width:263pt;height:15.75pt" width="351">Influenza antigen B/Brisbane/60/2008</td>
			<td class="xl63" style="width:100pt" width="133">NIBSC</td>
			<td class="xl63" style="width:85pt" width="113">13/234</td>
			<td class="xl66">Inactivated, partially purified B/Brisbane/60/2008 virus (ca. 42&#181;gHA/ml)</td>
		</tr>
		<tr height="21" style="height:15.75pt">
			<td class="xl63" height="21" style="width:263pt;height:15.75pt" width="351">Influenza antigen B/Massachusetts/02/2012</td>
			<td class="xl63" style="width:100pt" width="133">NIBSC</td>
			<td class="xl63" style="width:85pt" width="113">13/134</td>
			<td class="xl66">Inactivated, partially purified B/Massachusetts/02/2012 virus (ca. 35&#181;gHA/ml)</td>
		</tr>
		<tr height="42" style="height:31.5pt">
			<td class="xl63" height="42" style="width:263pt;height:31.5pt" width="351">Serum-Tubes</td>
			<td class="xl75" style="width:100pt" width="133">S-Monovette, Sardstedt</td>
			<td class="xl63" style="width:85pt" width="113">01.1601.100</td>
			<td class="xl66">For serum extraction with clotting activator</td>
		</tr>
		<tr height="42" style="height:31.5pt">
			<td class="xl63" height="42" style="width:263pt;height:31.5pt" width="351">Single Donor Human RBC, Type 0</td>
			<td class="xl63" style="width:100pt" width="133">Innovative Research</td>
			<td class="xl63" style="width:85pt" width="113">IPLA-WB3&#160;</td>
			<td class="xl66">Suspension of single donor human red blood cells in Alsever&#39;s solution (ca. 26%)</td>
		</tr>
		<tr height="21" style="height:15.75pt">
			<td class="xl63" height="21" style="width:263pt;height:15.75pt" width="351">Turkey RBC (10%)</td>
			<td class="xl63" style="width:100pt" width="133">Cedarlane</td>
			<td class="xl63" style="width:85pt" width="113">CLC1180</td>
			<td class="xl66">10% suspension of turkey red blood cells in Alsever&#39;s solution</td>
		</tr>
		<tr height="21" style="height:15.75pt">
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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 tha...

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 ...

The overall goal of this hemagglutination inhibition assay is to measure the antibody titers against specific viruses of interest. This method can help answer key questions in the vaccinology field about the vaccine-mediated immunity and protection within different populations and across various age and patient groups. The main advantages of this technique are that it is accurate and that it allows the fast titer determination of the presence of neutralizing antibodies.Though this method can provide insight into human antibody titers, it can also be applied to other systems, such as antibody titers for mouse serum, also culture supernatants. Begin by labeling 96 well microtiter plates with the appropriate experimental information. Then turn the plate in the vertical orientation and use a multi-channel pipette to add 25 microliters of PBS to every well, except the first well of the bottom back titration row.Add 50 microliters of the freshly prepared antigen solution of interest to the first well of the back titration row, followed by the addition of 25 microliters of RDE treated serum samples to the first wells of the top ten rows. Add 25 microliters of the appropriate antiserum to the first well of the 11th row as a positive control. The transfer 25 microliters from the first well of each row to their successive wells to perform serial, two-fold dilutions.Pipette up and down 10 to 15 times for each dilution step, discarding the final 25 microliters from the last wells. Next, add 25 microliters of the antigen solution to each well of rows one through 11 and 25 microliters of PBS only to each well of the back titration row. Tap the plate carefully 10 times on all four side to mix.Then cover the plate for 30 minutes at room temperature. At the end of the incubation, add 50 microliters of red blood cell solution to each well and mix the plate with more tapping. Then cover the plate again and incubate the red blood cells according to the species used as outlined in the table.After adding the red blood cells to the plate, adhere to the appropriate incubation time for the type of blood used in the assay. A too short or too long incubation will lead to an incorrect interpretation of the results. At the end of the incubation, assess the hemagglutination by tilting the plate 90 degrees for 25 seconds and mark the results for each well, while the plate is still tilted, on a printed scheme of the 96 well plate.In this experiment, the vaccine-induced antibody response was assessed in 26 healthy volunteers who received an inactivated, trivalent influenza vaccine containing influenza A/H1N1/California/2009, A/H3N2/Texas/2012 and B/Massachusetts/O2/2012 prior to the 24 2015 influenza season. A cross-reactive immune response for A/H3N2/Switzerland/2013 and A/H3N2/Texas/2012 virus strains was observed, with significantly lower geometric mean hemagglutination inhibition titers and induced seroprotection against influenza A/H3N2/Switzerland/2013 compared to influenza A/H3N2/Texas/2012. After vaccination, the antibody titers against both strains increased, even though the A/H3N2/Switzerland/2013 strain was not present in the vaccine.Viral hemagglutinin demonstrates a species dependent potential for erythrocyte hemagglutination. Of interest, guinea pig blood does not properly hemagglutinate with influenza B and turkey blood has the potential for hemagglutination with high titers and a low cross-reactivity. Once mastered, this technique can be completed in the three hours if it's performed properly.While attempting this procedure it's important to remember to serum with RDE to inactivate any unspecific inhibitors and unspecific binding during the hemagglutination of the virus. Following this procedure, other methods like ELISA can be performed to answer additional questions about the role of individual pathogen specific immunoglobulins. After its development, this technique paved the way for researchers in the field of viral immunology to further our understanding of vaccine-related immunity in healthy and immunosuppressed patients within different age groups.After watching this video, you should have a good understanding of how to perform a hemagglutination inhibition assay to quantify influenza strain specific antibody titers on a large scale. Don't forget that working with viral antigens, animal blood, and human serum samples can be extremely hazardous and that precautions such as working in a BSL2 laboratory should always be taken while performing this procedure.

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