analyzing the antigenic relationship between viruses using an image-based microneutralization assay

Analyzing the Antigenic Relationship between Viruses using an Image-Based Microneutralization Assay

To carry out virus neutralization, prepare the cell monolayer two or three days in advance, then add 50 microliters of VGM to each well of the plate. Use columns 11 and 12 for the virus control and cell control, respectively. Add 50-microliter aliquots of a 1-in-20 dilution of receptor-destroying enzyme or RDE-treated serum to the first row of columns 1 to 10.
Perform twofold serial dilutions by transferring 50 microliters from row A to row H, and discarding 50 microliters from row H. Then, add 50 microliters of diluent to each well of the cell control column. Add 50 microliters of the virus to each well of the plate except for the cell control column. Incubate the plate at 37 degrees Celsius for two to three hours. Then, remove the inoculum and add the overlay to each well. Incubate the plate overnight at 37 degrees Celsius undisturbed. Then, fix and stain the plates.
To quantify neutralization, place a well plate in the scanning area of a flatbed scanner. Scan the plate and run the well plate reader software to calculate the required viral titers.

analyzing-antigenic-relationship-between-viruses-using-an-image-based

Universidad de Cartagena UDEC

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JoVE Encyclopedia of Experiments

Immunology

Immunodiagnostics

Imaging and Visualization Techniques

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1. Virus Titration
NOTE: Depending on the number of viruses and the number of duplicates, a well plate can be set up with the following flexibilities: (1) The viral dilution can be arranged along either the rows or the columns (Figure 1). Each virus should occupy a separate row/column. (2) The viral dilution increment is flexible, but it should start with the highest viral concentration at the top-left corner. (3) There is no restriction on the number of duplicates.
<ol>
	<li>Aliquot sufficient MDCK cells or MDCK-SIAT cells8 into 96-well plates (200 &#181;l/well). Incubate the cells at 37 &#176;C with 5% CO2 for 2 or 3 days to reach confluence. Propagate the cells in DMEM supplemented with 10% heat-inactivated fetal calf serum (FCS) and antibiotics (100 U/ml penicillin and 100 &#181;g/ml streptomycin). Incubate the SIAT cells at 37 &#176;C with 5% CO2 and 1 mg/ml G418 sulfate. 
	NOTE: The dilution factor is dependent upon experience and the cell line used. The cell monolayer must be confluent (i.e., no cell wall or visible outline for MDCK cells and a tightly packed layout for MDCK-SIAT1 cells) at the time of virus inoculation. Examples of dilutions based on the subculture of confluent flasks of MDCK or MDCK-SIAT1 cells are given in Table 1.</li>
	<li>Wash the cells 3 times with 200 &#181;l of virus growth medium (VGM) per well. Sterilize the multiwall plate washer with 70% EtOH for 30 min, and then rinse it in sterile phosphate-buffered saline (PBS) or VGM before the wash.</li>
	<li>Aspirate the VGM and immediately add 50 &#181;l of VGM to each well.</li>
	<li>Add 900 &#181;l of VGM to each of 6 sterile tubes (or fewer, depending upon the number of test viruses and duplicates). Pipette 100 &#181;l of virus into the first tube, mix well, and serially dilute, changing the tips between each dilution. Repeat for each virus to be titrated. 
	NOTE: This will give virus dilutions of 10-1 to 10-6.</li>
	<li>Add 50 &#181;l of each virus dilution, starting at the highest dilution (1 x 10-5 in Figure 1), to duplicated wells (Columns 9 and 10 in Figure 1) of a 96-well plate.</li>
	<li>Place the plate at 37 &#176;C for 2-3 hr to allow the virus to infect the cells. 
	NOTE: A 2 to 3-hour incubation is necessary to ensure that the viruses in the inoculum have enough time to reach the monolayer.</li>
	<li>Prepare the overlay (10 ml/plate) 
	NOTE: The overlay consists of 5 ml of 2x DMEM, Trypsin (2 &#181;g/ml final concentration), 20 &#181;l of 1 mg/ml stock, and 5 ml of cellulose cotton linters (see the Materials List).</li>
	<li>Remove the inoculum.</li>
	<li>Add the overlay (200 &#181;l to each well). Incubate overnight at 37 &#176;C, UNDISTURBED. 
	NOTE: The virus budding takes place after approximately 4 hr, so it is important that the plate is not disturbed after this time.</li>
	<li>Aspirate the overlay from the wells.</li>
	<li>Add ice-cold 4% paraformaldehyde in PBS A (200 &#181;l/well). Place them at 4 &#176;C for 30 min or at room temperature for 20 min. 
	NOTE: PBS A is natural-pH phosphate-buffered saline with 10 g of NaCl, 0.25 g of KCl, 1.437 g of Na2HPO4, 0.25 g of KH2PO4, and 1 L of distilled water (see the Materials List). 
	NOTE: Paraformaldehyde is harmful when inhaled and can cause burns if swallowed. There is also limited evidence of a carcinogenic effect, and it may cause sensitization after skin contact.</li>
	<li>Aspirate the paraformaldehyde and wash the plates twice with PBS A (200 &#181;l/well).</li>
	<li>Store the plates in PBS A at 4 &#176;C for future use, or carry on with the following steps.</li>
	<li>Add permeabilization buffer (100 &#181;l/well). Leave it at room temperature for 30 min.</li>
	<li>Wash the plate twice with PBS A (200 &#181;l/well).</li>
	<li>Add 50 &#181;l of the first antibody, mouse MAb against influenza type A (1:1,000 in ELISA Buffer), per well. Incubate it at room temperature for 1 hr (or 4 &#176;C overnight), with shaking.</li>
	<li>Wash it 3 times in 100 &#181;l of wash buffer (0.05% Tween 80 in PBS A, v/v) per well. Incubate it at room temperature for 5 min between washes. Alternately, wash it 3 times without incubation using 300 &#181;l per well.</li>
	<li>Add 50 &#181;l of the second antibody, goat anti-mouse IgG (H+L) HRP conjugate (1:1,000 in ELISA Buffer), per well. Incubate it at room temperature for 1 hr, with shaking.</li>
	<li>Wash it 3 times as described in step 1.17.</li>
	<li>Add the substrate (50 &#181;l/well). Incubate it at room temperature for 30 min or until the development of a blue color is clearly visible.</li>
	<li>To stop the reaction, wash the plate twice with 200 &#181;l of distilled water per well, incubating at room temperature for 2 -3 min between the washes.</li>
	<li>Air-dry the plate and store it in a dark place (e.g., wrap it in aluminum foil).</li>
</ol>
2. Virus Neutralization
NOTE: Calculate the number of plates required for the neutralization assay. Each plate can accommodate one virus and a number of antisera.
NOTE: Depending on the number of antisera and the number of duplicates, a well plate can be set up with the following flexibilities: (1) The serum dilution can be arranged along either a row or a column (Figure 2). Each serum should occupy a separate row or column. (2) The increment of serum dilution is flexible, but it should start with the highest serum concentration at the top-left corner of a plate. (3) The number of duplicates balances the number of antisera. More duplicates can help to smooth out experimental variations. (4) The number of the VC and the number of the cell control (CC) duplicates are flexible, but they should also be in separate rows or columns. It is expected that the number of VC duplicates is significantly greater than that of the antisera.
<ol>
	<li>Prepare the cell monolayer, as in steps 1.1-1.3, 2 or 3 days in advance.</li>
	<li>Add 50 &#181;l of VGM to each well of the plate.</li>
	<li>Use Columns 11 and 12 for VC and CC, respectively. Add 50 &#181;l aliquots of 1:20 receptor-destroying enzyme (RDE)-treated serum to the first row (A) of Columns 1-10. 
	NOTE: For each virus and serum combination, the assay is performed in duplicate, so one plate may, for example, contain one virus tested against five antisera.</li>
	<li>Perform 2-fold serial dilutions by transferring 50 &#181;l from Row A to Row H (Columns 1-10 in Figure 2) and discarding 50 &#181;l from Row H.</li>
	<li>Add 50 &#181;l of diluent to each well of the CC column (Column 12 in Figure 2).</li>
	<li>Add 50 &#181;l of the virus to each well of the plate, except for the CC column (Columns 1-11, Rows A-H in Figure 2).</li>
	<li>Incubate it at 37 &#176;C for 2-3 hr. Remove the inoculum. Add the overlay (200 &#181;l) to each well. Incubate it overnight at 37 &#176;C, UNDISTURBED. Fix and stain the plates as for the virus titration (steps 1.11-1.22).</li>
</ol>
3. Neutralization Quantification
<ol>
	<li>Place a well plate in the scanning area of a flatbed scanner, as shown in Figure 2a. Use the L-shape position limit to ensure an optimum and repeatable imaging location. 
	NOTE: It is possible to image two well plates in one scan.</li>
	<li>Scan the plate.</li>
	<li>Run the &#34;Wellplate Reader&#34; software to calculate the required viral titers (Figure 3).
	<ol>
		<li>Click the &#34;Load image&#34; button to load an image. Slide the red bar in &#34;Global Threshold&#34; to adjust the sampling threshold. Click the &#34;Update&#34; button to examine the effect.</li>
		<li>Tick the &#34;Calculate Neutralization/Titration&#34; box. Click the &#34;Sampling&#34; button to quantify the ICP. Click the &#34;Save&#34; button to save the sampling results when prompted. 
		NOTE: After the sampling process, a new window called &#34;Neutralization &#38; Titration Calculation&#34; appears automatically if the &#34;Calculate Neutralization/Titration&#34; box is ticked.</li>
		<li>Load or input the well-plate map. Indicate the threshold (e.g., 50%) in &#34;Neutralization: Infection Deduction (%).&#34;</li>
		<li>Select &#34;Neutralization Process&#34; to calculate the titers. Check the titers and click the &#34;Save &#38; Close&#34; button to save the neutralization results. 
		NOTE: Neutralization is reported as the reciprocal of the highest dilution of the serum with the predefined ICP reduction (such as 50% or 80%) against the VC (the mean of Column 11 in Figure 4. A 50% ICP reduction was used in the Representative Results.</li>
	</ol>
	</li>
</ol>

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>VGM</td>
			<td>Sigma</td>
			<td>D6429, P0781</td>
			<td>500 ml DMEM+5 ml Pen/Strepb</td>
		</tr>
		<tr>
			<td>PBS A</td>
			<td></td>
			<td></td>
			<td>Nature pH Phosphate-buffered saline: NaCl 10 g, KCl 0.25 g, Na2HPO4 1.437 g, KH2PO4 0.25 g, and Dist. Water 1 L</td>
		</tr>
		<tr>
			<td>Avicell</td>
			<td>FMC</td>
			<td>RC-581F</td>
			<td>2.4 g in 100 ml distilled water dissolved by agitation on a magnetic stirrer for 1 hr. Sterilize by autoclaving</td>
		</tr>
		<tr>
			<td>2x DMEM</td>
			<td>Gibco</td>
			<td>21935-028</td>
			<td></td>
		</tr>
		<tr>
			<td>Trypsin</td>
			<td>Sigma</td>
			<td>T1426</td>
			<td></td>
		</tr>
		<tr>
			<td>Overlay (10 ml/plate):</td>
			<td></td>
			<td></td>
			<td>5 ml 2x DMEM, Trypsin 2 &#956;g/ml final concentration, Avicell 5 ml</td>
		</tr>
		<tr>
			<td>Triton X- 100 e</td>
			<td>Sigma</td>
			<td>T8787</td>
			<td>Permeabilization buffer: 0.2% in PBS A (v/v)</td>
		</tr>
		<tr>
			<td>Tween 80</td>
			<td>Sigma</td>
			<td>P5188</td>
			<td>Wash Buffer: 0.05% Tween 80 in PBS A (v/v)</td>
		</tr>
		<tr>
			<td>Horse serum</td>
			<td>PAA Labs Ltd</td>
			<td>B15-021</td>
			<td>ELISA Buffer: 10% in PBS A (v/v) + 0.1% Tween 80</td>
		</tr>
		<tr>
			<td>Mouse MAb against influenza type A</td>
			<td>Biorad</td>
			<td>MCA 400</td>
			<td>1 st Antibody: 1:1,000 in ELISA Buffer</td>
		</tr>
		<tr>
			<td>Goat anti-mouse IgG (H+L) HRP conjugate</td>
			<td>Biorad</td>
			<td>172-1011</td>
			<td>2 nd Antibody: 1:1,000 in ELISA Buffer</td>
		</tr>
		<tr>
			<td>True Blu peroxidase substrate</td>
			<td>KPL</td>
			<td>50-78-02</td>
			<td>Substrate: True blue +0.03% H2O2 g (1:1,000 of 30% solution)</td>
		</tr>
		<tr>
			<td>96-well flat-bottom microtitre plates</td>
			<td>Costar</td>
			<td>3596</td>
			<td></td>
		</tr>
		<tr>
			<td>8 channel multiwall-plate washer and manifold</td>
			<td>Sigma</td>
			<td>M2656</td>
			<td></td>
		</tr>
		<tr>
			<td>Perfection Plate Scanner</td>
			<td>Epson</td>
			<td>V750 Pro</td>
			<td>The imaging software can be downloaded freely from http://www.epson.com/cgi-bin/ Store/support/supDetail.jsp? oid=66134&#38;infoType=Downloads.</td>
		</tr>
		<tr>
			<td>Software operational environment: LabVIEW</td>
			<td>National Instruments Corporation</td>
			<td>Window based with NI Vision builder</td>
			<td>Version: LabVIEW2012 or above</td>
		</tr>
	</tbody>
</table>

Source: Lin, Y., et al. <a href="https://appjove.unicartagenaproxy.elogim.com/v/54897">Optimization of a Quantitative Micro-neutralization Assay.</a> J. Vis. Exp. (2016)
This video demonstrates the imaging-based micro-neutralization assay to analyze the antigenic relations between influenza A and B viruses. It provides a quantitative and visual way to assess the effectiveness of antibodies or other treatments in preventing viral infection.

<img alt="Figure 1" src="/files/ftp_upload/21924/21924_Figure1.jpg" /> 
Figure 1: Example of a well-plate setup in a virus titration experiment. Test viruses are assigned to separate rows (A to H). Columns are designed for different viral dilutions (1 to 12). Two columns were used as duplicates for each viral dilution. Scale bar = 10 mm.
<img alt="Figure 2" src="/files/ftp_upload/21924/21924_Figure2.jpg" />...

1. Virus Titration
NOTE: Depending on the number of viruses and the number of duplicates, a well plate can be set up with the following flexibilities: (1) ...

Add media and serially diluted receptor-destroying enzyme-treated antisera onto mammalian cell monolayers, preventing nonspecific virus binding.
Add an influenza virus suspension. Antisera antibodies, specific to viral proteins, bind, neutralizing viruses and preventing cellular entry.
Low neutralizing antibody concentration or non-neutralizing antibodies enable ineffective viral antigen binding, allowing virus attachment to cell receptors.
Discard media. Apply cellulose-containing media, forming a semi-solid overlay.
The virus uses host cell machinery to form viral particles and cause cell lysis.
The overlay restricts virus movement, facilitating localized infection and creating holes in cell monolayers.
Remove the overlay. Fix and permeabilize cells.
Wash with buffer. Add antibodies binding viral antigens within permeabilized cells.
Pipette horseradish peroxidase-conjugated secondary antibodies binding to primary antibodies.
Introduce peroxidase substrates and hydrogen peroxide, resulting in a blue product.
Image wells to quantify virus-infected cell populations against antiserum dilutions.
Identify antisera dilutions indicating a specified reduction in an infected cell population to determine neutralization titers.

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视频: 使用基于图像的微量中和测定分析病毒之间的抗原关系 - 实验

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.

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.

一种优化的血凝抑制 (HI) 测定流感特异抗体滴

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

科研

JoVE Journal

医学

Preparation of Pseudo-Typed H5 Avian Influenza Viruses with Calcium Phosphate Transfection Method and Measurement of Antibody Neutralizing Activity

Since 1996, A/goose/Guangdong/1/96-lineage highly pathogenic avian influenza (HPAI) H5 viruses have been causing flu outbreaks in poultry and wild birds. Occasionally, humans also fall victim to it, which results in high mortality. Nonetheless, HPAI virus research is often hindered, considering that it must be handled within biosafety level 3 laboratories. To address this issue, pseudoviruses are adopted as an alternative to wild-type viruses in some experiments of H5 HPAI studies. Pseudoviruses prove to be the ideal tools to study neutralizing antibodies against H5 HPAI viruses. This protocol describes the procedures and critical steps of H5 HPAI pseudovirus preparations and pseudovirus neutralization assays. Also, it discusses the troubleshooting, limitation, and modifications of these assays.

Here we describe a protocol for pseudovirus packaging and the measurement of antibody neutralizing activity.

磷酸钙转染法制备假型H5禽流感病毒及抗体中和活性测定

Since 1996, A/goose/Guangdong/1/96-lineage highly pathogenic avian influenza (HPAI) H5 viruses have been causing flu outbreaks in poultry and wild birds. ...

免疫与感染

A Step-By-Step Method to Detect Neutralizing Antibodies Against AAV using a Colorimetric Cell-Based Assay

Recombinant adeno-associated viruses (rAAV) have proven to be a safe and successful vector for transferring genetic material to treat various health conditions in both the laboratory and the clinic. However, pre-existing neutralizing antibodies (NAbs) against AAV capsids pose an ongoing challenge for the successful administration of gene therapies in both large animal experimental models and human populations. Preliminary screening for host immunity against AAV is necessary to ensure the efficacy of AAV-based gene therapies as both a research tool and as a clinically viable therapeutic agent. This protocol describes a colorimetric in vitro assay to detect neutralizing factors against AAV serotype 6 (AAV6). The assay utilizes the reaction between an AAV encoding an alkaline phosphatase (AP) reporter gene and its substrate NBT/BCIP, which generates an insoluble quantifiable purple stain upon combination. 
In this protocol, serum samples are combined with an AAV expressing AP and incubated to permit potential neutralizing activity to occur. Virus serum mixture is subsequently added to cells to allow for viral transduction of any AAVs that have not been neutralized. The NBT/BCIP substrate is added and undergoes a chromogenic reaction, corresponding to viral transduction and neutralizing activity. The proportion of area colored is quantitated using a free software tool to generate neutralizing titers. This assay displays a strong positive correlation between coloration and viral concentration. Assessment of serum samples from sheep before and after administration of a recombinant AAV6 led to a dramatic increase in neutralizing activity (125 to &gt;10,000-fold increase). The assay displayed adequate sensitivity to detect neutralizing activity in &gt;1:32,000 serum dilutions. This assay provides a simple, rapid, and cost-effective method to detect NAbs against AAVs.

A comprehensive laboratory protocol and analysis workflow are described for a rapid, cost-effective, and straightforward colorimetric cell-based assay to detect neutralizing elements against AAV6.

使用基于比色细胞的测定法检测针对 AAV 的中和抗体的分步方法

Recombinant adeno-associated viruses (rAAV) have proven to be a safe and successful vector for transferring genetic material to treat various health ...

Analyzing the Antigenic Relationship between Viruses using an Image-Based Microneutralization Assay

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Analyzing the Antigenic Relationship between Viruses using an Image-Based Microneutralization Assay