Name: The Hemagglutination Inhibition Assay to Detect Serum Antibodies Against a Target Antigen
Uploaded: 2023-11-30T12:47:40
Description: All procedures involving human participants have been performed in compliance with the institutional, national, and international guidelines for human ...

the hemagglutination inhibition assay to detect serum antibodies against a target antigen

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

Begin by labeling 96-well microtiter plates with the appropriate experimental information. Then, turn the plate in the vertical orientation and use a multichannel 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 10 rows.
Add 25 microliters of the appropriate anti-serum to the first well of the 11th row as a positive control. Then, 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 1 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 sides to mix, and 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.

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All procedures involving human participants have been performed in compliance with the institutional, national, and international guidelines for human welfare and have been reviewed by the local institutional review board.
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: Non-centrifuged blood samples should be stored at 4 &#176;C and for no longer than 24 h.</li>
	<li>Aliquot the serum into different tubes (cryo-vials) and freeze at -80 &#176;C until use.</li>
	<li>Perform the subsequent assays batch-wise, including all the time points of one person to reduce variability within a patient.</li>
</ol>
2. Preparation of Antigens
CAUTION: Five different antigens are used (see&#160;Table of Materials). Prepare antigens in a Biosafety Level 2 (BSL-2) laboratory.
<ol>
	<li>According to the manufacturer&#39;s instructions, reconstitute the total contents of one lyophilized influenza antigen ampoule with 1.0 mL of distilled water and allow the dissolved antigen to stand for a minimum of 5 min at room temperature before proceeding.</li>
	<li>Aliquot the antigen solution to 1.5 mL tubes and freeze at -80 &#176;C until further use.</li>
</ol>
3. Preparation of Cholera Filtrate
NOTE: Cholera filtrate is used as a receptor-destroying enzyme (RDE) according to the World Health Organization (WHO) protocol. This removes innate inhibitors from the serum which would interfere with the assay.
<ol>
	<li>Reconstitute the lyophilized RDE according to the manufacturer&#39;s instructions.</li>
	<li>Store the RDE solution in a 15 mL tube at 4 &#176;C until further use.</li>
</ol>
4. HA Assay
NOTE: To ensure that the hemagglutination inhibition (HI) assays are comparable between several plates, the same amount of virus particles must be used for each plate. The hemagglutination (HA) assay (also called HA titration) is performed to quantify the virus particles necessary for hemagglutination and is recorded in HA units. A &#34;unit&#34; of hemagglutination is an operational unit dependent on the method used for HA titration and is not a measurement of an absolute amount of virus. Thus, an HA unit is defined as the amount of virus needed to agglutinate an equal volume of a standardized RBC suspension. According to the WHO, the standard amount used for the HI assay is 4 HA units per 25 &#181;L. For an illustration of the principle of the HA assay see&#160;Figure 1.
NOTE: The red blood cells (RBCs) used are dependent on the type of influenza virus in the assay (Table 1). Further, for various types of 96-well microtiter plates, the incubation time as well as the appearance of the non-agglutinated cells differ (Table 2).
<ol>
	<li>Preparation of the RBC Suspension
	<ol>
		<li>Dilute the RBC stock suspension (10%, v/v; except human type O) (see&#160;Table of Materials) with phosphate-buffered saline (PBS) to make the proper concentrations for avian and mammalian RBCs of 0.75% and 1%, respectively.</li>
	</ol>
	</li>
	<li>Preparation of the 96-well Microtiter Plate 
	NOTE:&#160;See&#160;Figure 2&#160;for an overview of the plate design.&#160;&#160;&#160; 
	<ol>
		<li>Add 25 &#181;L of PBS to wells 1 to 12 of each used row of a 96-well microtiter plate by using a multichannel pipette (Figure 2). Use the V-shaped microtiter plate when working with avian RBCs, like chicken and turkey. Use the U-shaped microtiter plate when working with mammalian RBCs, like guinea pigs and human type O (Table 2).</li>
		<li>Add 25 &#181;L of influenza antigen to the first well of the antigen rows, which are arranged in duplicates. No antigen is added to the control rows. The control rows should not show a hemagglutination effect and serve as negative controls (Figure 2).</li>
		<li>Perform a serial 2-fold dilution by transferring 25 &#181;L from the first well of the antigen rows to successive wells by using a multichannel pipette. Mix each dilution step by pipetting up and down gently 10 times.</li>
		<li>Discard the final 25 &#181;L of the last wells.</li>
		<li>Add 25 &#181;L of PBS to wells 1 to 12 of each used row by using a multichannel pipette, to reach a total volume of 50 &#181;L per well.</li>
		<li>Add 50 &#181;L of the RBC suspension to each used well by using a multichannel pipette.</li>
		<li>Tap the plate carefully 10 times on all four sides to mix.</li>
		<li>Cover the plate with a lid and incubate at room temperature for the appropriate amount of time depending on the RBC species used (see&#160;Table 2). Do not move the plate while incubating.</li>
	</ol>
	</li>
	<li>Reading the plate 
	NOTE:&#160;The readout is slightly different when using avian RBCs compared to mammalian RBCs, because of the different shaped microtiter wells (Figure 3).
	<ol>
		<li>Readout of avian RBCs
		<ol>
			<li>Tilt the plate 90&#176; for 25 s. 
			NOTE: The HA titration endpoint is the last well where complete hemagglutination occurs. This well contains 1 HA unit of virus. Because of the 2-fold dilutions of the antigen, two wells ahead of the HA titration endpoint is the well that contains 4 HA units of virus (Figure 4).</li>
			<li>Mark the results immediately, while the plate is still in a tilted position, on a printed scheme of the 96-well plate. The agglutination patterns of the avian RBCs are shown in&#160;Figure 3.</li>
		</ol>
		</li>
		<li>Readout of mammalian RBCs
		<ol>
			<li>Mark the results on a printed scheme of the 96-well plate, without tilting the plate (horizontal position on the bench). 
			NOTE: When hemagglutination occurs, the agglutinated cells do not settle to the bottom, whereas non-agglutinated cells appear as a halo at the bottom of the well. The halo of the partially agglutinated cells is less intense and has a larger diameter (Figure 3).</li>
		</ol>
		</li>
		<li>Determination of 4 HA units. 
		NOTE: Tilting the plate is crucial for the differentiation of avian patterns because all three different types of agglutination patterns (completely agglutinated, partially agglutinated, and non-agglutinated) appear as a button when not tilted.</li>
	</ol>
	</li>
</ol>
5. HI Assay
NOTE: The workflow of the protocol has been optimized to allow more efficient handling of multiple samples at the same time, by using polymerase chain reaction (PCR) tube stripes and a thermocycler (see below).
<ol>
	<li>Preparation of the Serum Samples&#160;&#160;&#160;&#160;&#160; 
	NOTE: Prepare serum samples in a biosafety level-2 (BSL-2) laboratory.
	<ol>
		<li>Thaw the frozen serum samples of each time point of every person (see step 1.2) at room temperature.</li>
		<li>Add an aliquot of 10 &#181;L of each thawed serum sample to a tube of PCR tube strip (10 tubes in one strip). 
		NOTE: The big advantage of using PCR tube strips is that a multichannel pipette can be used for the following steps in the HI assay; this saves a lot of time when testing a large number of serum samples and when performing repeated measures of the same samples for antibody titers against different virus strains.</li>
		<li>Store the aliquoted serum samples in the PCR tube strips at -80 &#176;C until use.</li>
		<li>One day before the HI assay is performed, thaw the serum sample aliquots of interest at room temperature.</li>
		<li>Add 10 &#181;L of the appropriate anti-serum to an empty PCR tube. 
		NOTE: To serve as a positive control, the anti-serum against a specific virus must match the used virus. The positive control allows for standardization of plate performance over multiple plates.</li>
		<li>Add 30 &#181;L of cholera filtrate solution to each serum aliquot and to the anti-serum (3 volumes of cholera filtrate to 1 volume of serum) by using a multichannel pipette.</li>
		<li>Keep the PCR tubes in a PCR 96-well rack or an empty tip-box and vortex for 5 s.</li>
		<li>Incubate the samples overnight at 37 &#176;C using a thermocycler.</li>
		<li>Incubate the samples at 56 &#176;C for 30 min to inactivate the cholera filtrate using a thermocycler.&#160;&#160;&#160;&#160;&#160;&#160;&#160; 
		NOTE: Depending on the thermocycler, this step can be programmed to further automate the process.</li>
		<li>Keep the PCR tubes in a PCR 96-well rack or an empty tip-box and vortex for 5 s.</li>
		<li>Store the samples at 4 &#176;C in the fridge until use for the HI assay.</li>
	</ol>
	</li>
	<li>HI assay 
	NOTE: For an illustration of the principle of the HI assay see&#160;Figure 1. Depending on the virus, different species of RBCs are used for the assay (Table 1). The different species of RBCs are used in various types of 96-well plates, and the incubation time as well as the appearance of the non-agglutinated cells differs (Table 2). For the HI assay, 4 HA units of virus or antigen are added to the 2-fold dilution series of the samples.
	<ol>
		<li>Preparation of the antigen solution
		<ol>
			<li>Calculate the volume of antigen solution needed according to the number of 96-well plates used (25 &#181;L antigen per well &#215; 96 = 2,400 &#181;L antigen per 96-well plate; add 100 &#181;L per plate extra due to the usage of a reservoir for the multichannel pipette; a total 2.5 mL of antigen per plate). 
			NOTE: For example, if measuring 100 serum samples then 10 plates are needed (10 samples per plate): 2.5 mL x 10 = 25 mL of antigen solution needed in total.</li>
			<li>Prepare the proper dilution of 4 HA units for the calculated volume using PBS. 
			NOTE: 4 HA units are determined for the HA assay. For the appropriate amount of antigen, divide the calculated volume by the titer corresponding to 4 HA units. For example, 4 HA units correspond to a dilution of 1/64, and we needed 15,000 &#181;L of antigen solution are needed: 15,000/64 = 234.4 &#181;L of the dissolved lyophilized influenza antigen are added.</li>
		</ol>
		</li>
		<li>Preparation of the RBC suspension
		<ol>
			<li>Calculate the volume of RBC suspension needed according to the number of 96-well microtiter plates used (50 &#181;L RBC suspension per well &#215; 96 = 4,800 &#181;L RBC suspension per 96-well plate; add 200 &#181;L per plate extra due to the usage of a reservoir for the multichannel pipette).</li>
			<li>Dilute the RBC stock suspension (normally 10%, v/v; except human type O) with PBS to make the proper concentrations for avian and mammalian RBCs of 0.75% and 1%, respectively.</li>
		</ol>
		</li>
		<li>Preparation of the 96-well microtiter plate
		<ol>
			<li>Label the 96-well microtiter plates (sample ID, positive control, and back titration). Please check the plate orientation in&#160;Figure 5&#160;carefully.</li>
			<li>Add 25 &#181;L of PBS to every well except to the first well of the &#34;back titration&#34; row (Figure 5, 12th&#160;row) using the multichannel pipette. 
			NOTE: A back titration was performed to check if the used antigen dilution equals 4 HA units. An antigen titer of 4 HA units is indicated if hemagglutination occurs in the first three wells of the &#34;back titration&#34; row, but not in the fourth well.</li>
			<li>Add 50 &#181;L of the prepared antigen solution (described in 5.2.1) to the first well of the &#34;back titration&#34; row (12th&#160;row).</li>
			<li>Add 25 &#181;L of the RDE-treated serum samples to the first wells of rows 1 to 10 on each plate, using the multichannel pipette.</li>
			<li>Add 25 &#181;L of the appropriate anti-serum to the first well of the 11th&#160;row as a positive control.</li>
			<li>Perform serial 2-fold dilutions by transferring 25 &#181;L from the first well of each row (1 - 12) to successive wells by using a multichannel pipette. Mix by pipetting up and down 10 - 15 times for each dilution step. The same tips can be used for each dilution step per sample.</li>
			<li>Discard the final 25 &#181;L of the last wells.</li>
			<li>Add 25 &#181;L of the antigen solution by using a multichannel pipette to each well of rows 1 to 11 (serum samples and anti-serum). The same tips can be used if they do not touch the wells.</li>
			<li>Add 25 &#181;L of PBS instead of antigen to each well of the &#34;back titration&#34; row (12th&#160;row).</li>
			<li>Tap the plate carefully 10 times on all four sides to mix.</li>
			<li>Cover the plate with a lid and incubate at room temperature for 30 min. Do not move the plate while incubating.</li>
			<li>Add 50 &#181;L of the RBC suspension to every well.</li>
			<li>Tap the plate carefully 10 times on all 4 sides to mix.</li>
			<li>Cover the plate with a lid and incubate at room temperature for the appropriate amount of time depending on the RBC species used (see&#160;Table 2). Do not move the plate while incubating.</li>
		</ol>
		</li>
		<li>Reading the plate&#160; 
		NOTE: The HI titer is the reciprocal of the last dilution of (anti-) serum that completely inhibits hemagglutination. It is important to consider that the RDE-treated sera were already diluted 1:4 and after the serial dilution step, the sera in the first wells are diluted 1:8, which corresponds to a HI titer of 8.
		<ol>
			<li>Readout of avian RBCs
			<ol>
				<li>Tilt the plate 90&#176; for 25 s.&#160;&#160; 
				NOTE: Tilting the plate is crucial for the differentiation of avian patterns because all three different types of agglutination patterns (completely agglutinated, partially agglutinated, and non-agglutinated) appear as a button when not tilted.</li>
				<li>Mark the results immediately, while the plate is still in a tilted position, on a printed scheme of the 96-well plate. The agglutination patterns of avian RBCs are shown in&#160;Figure 3.</li>
			</ol>
			</li>
		</ol>
		</li>
	</ol>
	</li>
</ol>
Table 1: Influenza antigens and corresponding species of RBCs. According to the manufacturer&#39;s instructions (NIBSC).
<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>Influenza antigen</td>
			<td>A/California/7/09 (H1N1)</td>
			<td>A/Switzerland/9715293/2013 (H3N2)</td>
			<td>A/Texas/50/2012 (H3N2)</td>
			<td>B/Brisbane/60/08</td>
			<td>B/Massachusetts/02/2012</td>
		</tr>
		<tr>
			<td>RBC species</td>
			<td>Chicken</td>
			<td>Guinea Pig</td>
			<td>Guinea Pig</td>
			<td>Turkey</td>
			<td>Turkey</td>
		</tr>
	</tbody>
</table>
Table 2: Assay conditions with different species of RBCs.&#160;According to the WHO protocol. (* flows when tilted).
<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>RBC species</td>
			<td>Chicken</td>
			<td>Turkey</td>
			<td>Guinea pig</td>
			<td>Human type O</td>
		</tr>
		<tr>
			<td>Concentration of RBCs (v/v)</td>
			<td>0.75%</td>
			<td>0.75%</td>
			<td>1%</td>
			<td>1%</td>
		</tr>
		<tr>
			<td>Type of microtiter plate</td>
			<td>V bottom</td>
			<td>V bottom</td>
			<td>U bottom</td>
			<td>U bottom</td>
		</tr>
		<tr>
			<td>Incubation time, RT</td>
			<td>30 min</td>
			<td>30 min</td>
			<td>1 hour</td>
			<td>1 hour</td>
		</tr>
		<tr>
			<td>Appearance of non-agglutinated cells</td>
			<td>Button*</td>
			<td>Button*</td>
			<td>halo</td>
			<td>halo</td>
		</tr>
	</tbody>
</table>

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<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&#39;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&#39;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 &#181;l</td>
			<td>Sigma-Aldrich</td>
			<td>Z683949</td>
			<td></td>
		</tr>
		<tr>
			<td>Eppendorf Multichannel pipette, 8-channel, 10-100 &#181;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&#39;s solution</td>
		</tr>
		<tr>
			<td>Influenza Anti-A/California/7/09 HA serum&#160;</td>
			<td>NIBSC</td>
			<td>14/134&#160;</td>
			<td>Used as positive control at the HI assay</td>
		</tr>
		<tr>
			<td>Influenza Anti-A/Switzerland/9715293/2013-like HA serum&#160;</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&#160;</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&#160;</td>
			<td>NIBSC</td>
			<td>13/254&#160;</td>
			<td>Used as positive control at the HI assay</td>
		</tr>
		<tr>
			<td>Influenza Anti-B/Massachusetts/02/2012 HA serum&#160;</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)&#160;</td>
			<td>NIBSC</td>
			<td>12/168</td>
			<td>Inactivated, partially purified A/California/7/09 (H1N1)(NYMC-X181)&#160; virus (ca. 46&#181;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&#181;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&#181;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&#181;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&#181;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>Turkey RBC (10%)</td>
			<td>Cedarlane</td>
			<td>CLC1180</td>
			<td>10% suspension of turkey red blood cells in Alsever&#39;s solution</td>
		</tr>
		<tr>
			<td>Phosphate Buffered Saline (PBS)</td>
			<td>Gibco</td>
			<td></td>
			<td></td>
		</tr>
	</tbody>
</table>

Source: Kaufmann, L. et al., <a href="https://app.jove.com/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.

<img alt="Figure 1" src="/files/ftp_upload/21787/21787_Figure1.jpg" /> 
Figure 1: Principle of hemagglutination and hemagglutination inhibition.&#160;No hemagglutination occurs in a negative control situation without viruses and antibodies (left column), and erythrocytes hemagglutinate only in the presence of influenza virus (middle column). However, when the hemagglutinin of the influenza virus is blocked by virus-specific antibodies then no hemagglutination can occ...

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

Begin with a V-shaped bottom multi-well plate and add diluted serum from an influenza-vaccinated human containing influenza-specific antibodies, to the test well.
Introduce viral hemagglutinin &#8212; an influenza-specific antigen in the test and control wells.
During incubation, in the test well, influenza-specific antibodies interact with antigens, forming complexes. While in the control well, antigens remain unbound.
Incubate the wells with avian red blood cells or RBCs.
In the control well, free antigens interact with RBC&#39;s surface receptors specific to viral hemagglutinin, causing RBCs to clump, leading to hemagglutination, and forming a compact pattern at the well&#39;s bottom.
However, in the test well, the presence of antigen-antibody complexes and the absence of free antigens inhibit hemagglutination, settling RBCs at the bottom.
Post-assay, tilt the plate. The control well exhibits a small, compact circular pattern with a hazy appearance &#8212; indicative of hemagglutination.
In contrast, the test well shows a tear-like diffused pattern, suggesting hemagglutination inhibition.

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

Video: The Hemagglutination Inhibition Assay to Detect Serum Antibodies Against a Target Antigen - Experiment

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 ...

JoVE Journal

Immunology and Infection

Optimization of a Quantitative Micro-neutralization Assay

The micro-neutralization (MN) assay is a standard technique for measuring the infectivity of the influenza virus and the inhibition of virus replication. In this study, we present the protocol of an imaging-based MN assay to quantify the true antigenic relationships between viruses. Unlike typical plaque reduction assays that rely on visible plaques, this assay quantitates the entire infected cell population of each well. The protocol matches the virus type or subtype with the selection of cell lines to achieve maximum infectivity, which enhances sample contrast during imaging and image processing. The introduction of quantitative titration defines the amount of input viruses of neutralization and enables the results from different experiments to be comparable. The imaging setup with a flatbed scanner and free downloadable software makes the approach high throughput, cost effective, user friendly, and easy to deploy in most laboratories. Our study demonstrates that the improved MN assay works well with the current circulating influenza A(H1N1)pdm09, A(H3N2), and B viruses, without being significantly influenced by amino acid substitutions in the neuraminidase (NA) of A(H3N2) viruses. It is particularly useful for the characterization of viruses that either grow to low HA titer and/or undergo an abortive infection resulting in an inability to form plaques in cultured cells.

This study describes an imaging-based micro-neutralization assay to analyze the antigenic relationships between viruses. The protocol employs a flatbed scanner and has four steps, including titration, titration quantitation, neutralization, and neutralization quantitation. The assay works well with current circulating influenza A(H1N1)pdm09, A(H3N2), and B viruses.

The micro-neutralization (MN) assay is a standard technique for measuring the infectivity of the influenza virus and the inhibition of virus replication. ...

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 ...

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

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