eoe sub articles

EoE Sub Articles

1. Preparation of target cells for infection
<ol>
	<li>Seed target cells the day prior to performing the experiment in a 96-well plate (the &#39;target&#39; plate).</li>
	<li>Plate sufficient cells per well to ensure an almost confluent (~90%) monolayer. 
	NOTE: This will depend on the relative size of your target cell type. If using human foreskin fibroblasts (HFFs), seed 20,000 cells/well.</li>
	<li>Typically, 21 wells (7 down by 3 across) should be seeded per sample, allowing 4 samples to be analyzed per plate.</li>
	<li>Seed an additional 12 wells per plate (i.e., row 8) to serve as controls.</li>
</ol>
2. Preparation of virus: antibody mixtures
<ol>
	<li>Add 90 &#181;L of media combined with antibody (and well mixed) to create twice the desired initial test antibody concentration into 3 wells of row 1 of a fresh 96 well plate (the &#39;dilution&#39; plate). 
	NOTE: A typical starting concentration of antibody would be 20 &#181;g/mL but should be determined empirically.</li>
	<li>Add 60 &#181;L of media to each of the 18 wells below (i.e., 6 down by 3 across) the 3 wells from step 2.1.</li>
	<li>Add 60 &#181;L of media to 12 additional wells (i.e., row 8).&#160;</li>
	<li>Transfer 30 &#181;L of antibody dilution from row 1 to the corresponding wells in row 2.</li>
	<li>Mix the contents of row 2 by pipetting smoothly up and down 20 times with the multichannel pipette.&#160;</li>
	<li>Transfer 30 &#181;L of antibody dilution from row 2 to row 3.</li>
	<li>Repeat this process of mixing and transferring until the contents of row 7 have been mixed.</li>
	<li>Once row 7 has been mixed, dispose of 30 &#181;L from the wells in row 7.</li>
	<li>Dilute HCMV in a complete medium so that 50 &#181;L contains sufficient infectious particles to result in a MOI=1 infection if added directly to the cells. 
	NOTE: HCMV is a Biosafety Level 2 (BSL-2) category pathogen. Ensure proper safety procedures are in place and use filter tips for all HCMV-exposed media. 
	NOTE: This requires the same number of infectious particles as there are cells/well, which will vary based on cell type and virus tropism. If using HFFs as described above, the required virus concentration is 4x105 pfu/mL. 
	NOTE: Ensure a sufficient excess of diluted virus (e.g., 10% extra) is prepared.</li>
	<li>Add 60 &#181;L of diluted virus stock to all wells containing antibody dilutions, and 6 wells that contain only 60 &#181;L media.</li>
	<li>Add 60 &#181;L of media only to the remaining 6 wells of media only, to serve as uninfected controls.</li>
	<li>Incubate the resulting virus: antibody mixtures at 37oC for 1 h.</li>
</ol>
3. Infection of target cells
<ol>
	<li>Remove all media from target cells.</li>
	<li>Using a multichannel pipette, transfer 100 &#181;L of virus: antibody mixture from the &#8216;dilution&#8217; plate onto the cells in the equivalent position on the &#39;target&#39; plate.</li>
	<li>Incubate the cells at 37 &#176;C, 5% CO2 for 24 h.</li>
</ol>
4. Fixation of cells
<ol>
	<li>Remove all media from cells. 
	NOTE: There is no need to change tips between wells at this point.</li>
	<li>Wash all wells with 200 &#181;L phosphate-buffered saline (PBS) and remove.</li>
	<li>Add 180 &#181;L -20 oC, 100% ethanol to all wells.</li>
	<li>Incubate plate at -20 oC for at least 30 min. 
	NOTE: This step can be extended to at least one month, provided additional ethanol is added as it begins to evaporate. 
	NOTE: The plate is safe to handle under BSL-1 conditions from this point onwards, and non-filter tips can be used.&#160;</li>
</ol>
5. Detection of virally infected cells by immunofluorescence (IF)
<ol>
	<li>Remove ethanol from the plate.</li>
	<li>Wash all wells with 200 &#181;L PBS and remove.</li>
	<li>Add 200 &#181;L PBS to all wells and incubate for 5 min.
	<ol>
		<li>Meanwhile, prepare primary antibody dilution in PBS.</li>
		<li>100 &#181;L of 1:2000 mouse monoclonal anti-IE will be required per well (note dilution is for the specific antibody listed in materials). 
		&#8203;NOTE: Dilution of the antibody used for IF may need optimizing if an alternative is used.</li>
		<li>Vortex mixture to ensure sufficient mixing.</li>
	</ol>
	</li>
	<li>Remove PBS from all wells and add 100 &#181;L primary antibody dilution.</li>
	<li>Incubate at room temperature for 1 h.</li>
	<li>Remove primary antibody from all wells.</li>
	<li>Add 200 &#181;L PBS and incubate for 5 min.
	<ol>
		<li>Meanwhile, prepare secondary antibody and 4&#8242;,6-diamidino-2-phenylindole (DAPI) dilution in PBS.</li>
		<li>100 &#181;L of 1:2000 fluorophore-conjugated anti-Mouse IgG and 1 &#181;g/mL DAPI will be required per well.</li>
		<li>NOTE: Dilution of secondary antibody used for IF may need optimizing if an alternative is used.</li>
		<li>Vortex mixture to ensure sufficient mixing.</li>
	</ol>
	</li>
	<li>Remove PBS from all wells and add 100 &#181;L secondary antibody/DAPI dilution.</li>
	<li>Incubate plate at room temperature for 1 h, protected from light.</li>
	<li>Remove secondary antibody/DAPI from all wells.</li>
	<li>Wash all wells with 200 &#181;L PBS and remove.</li>
	<li>Add 200 &#181;L PBS to all wells and incubate for 5 min.</li>
	<li>Remove PBS and add 100 &#181;L PBS to all wells and store, protected from light, at 4 oC until ready to image. 
	NOTE: Plates can be stored for up to one month with minimal loss in signal/image quality.</li>
</ol>
6. Imaging and counting of infected cells
<ol>
	<li>To quantify infection easily and accurately, image at least 25% of each well, in both the blue (DAPI, nuclei) and secondary antibody (HCMV immediate-early proteins (IE)) channels. 
	NOTE: The signal generated by the IE immunofluorescence should resemble that of the nuclear staining and images should be checked to confirm this.</li>
	<li>This can be performed manually, but the use of an automated microscope is advised. 
	NOTE: Magnification will depend on your instrument, but as large structures (nuclei) are being imaged, 4X/10X objectives are typically sufficient.</li>
	<li>Once images are acquired, process them through the software analysis program of choice.
	<ol>
		<li>First, identify nuclei via the blue channel, to calculate the number of cells present.</li>
		<li>Only these areas then need to be checked for signal in the red channel, as IE is a nuclear-localized protein, to calculate the number of infected cells.</li>
	</ol>
	</li>
	<li>Once the number of infected cells and the total number of cells are calculated, calculate the percentage of infection on a per well basis.</li>
	<li>These values can be corrected for background levels of detection by subtracting the average percentage of infection observed in uninfected wells from all other wells. 
	NOTE: This should be well below 1%. If it is more than 1%, the analysis procedure requires optimization.</li>
	<li>These background-corrected values can now be plotted, interrogated, and analyzed as desired.</li>
</ol>

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>Mouse monoclonal anti-IE antibody clone 6F8.2</td>
			<td>Millipore</td>
			<td>MAB8131</td>
			<td>Primary antibody for immunofluoresence</td>
		</tr>
		<tr>
			<td>Rabbit Anti-Mouse IgG, Alexa fluor 568 conjugated antibody</td>
			<td>Invitrogen</td>
			<td>&#160;A-11061</td>
			<td>Secondary antibody for immunofluoresence</td>
		</tr>
		<tr>
			<td>WiScan&#174; Hermes High Content Imaging System</td>
			<td>Idea Bio-medical</td>
			<td>-</td>
			<td>High throughput, automated fluorescent microscope for quantification</td>
		</tr>
		<tr>
			<td>Metamorph Image Analysis software</td>
			<td>Molecular Devices</td>
			<td>-</td>
			<td>Image analysis software</td>
		</tr>
		<tr>
			<td>Human IgG1 Isotype Control</td>
			<td>Merck</td>
			<td>M5284</td>
			<td>Control antibody for neutralisation</td>
		</tr>
		<tr>
			<td>8F9, anti-HCMV glycoprotein B antibody</td>
			<td>-</td>
			<td>-</td>
			<td>Neutralizing antibody for HCMV</td>
		</tr>
		<tr>
			<td>DAPI</td>
			<td>Thermo Scientific</td>
			<td>62248</td>
			<td>Staining of cellular nuclei</td>
		</tr>
		<tr>
			<td>DMI4000B Inverted Fluorescence microscope</td>
			<td>Leica</td>
			<td>-</td>
			<td>Fluorescent microscope used for representative images</td>
		</tr>
	</tbody>
</table>

an immunofluorescence method to measure the neutralization of human cytomegalovirus

This video demonstrates an immunofluorescence assay designed to measure the neutralization of Human Cytomegalovirus (HCMV). Virus-specific antibodies are mixed with the virus and incubated with human fibroblast cells. The antibodies neutralize the virus, rendering it unable to infect the host cells, while non-neutralized viruses infect the cells. After incubation, the extent of neutralization is determined by quantifying the virus-infected cells using immunofluorescence.

An Immunofluorescence Method to Measure the Neutralization of Human Cytomegalovirus

1. Preparation of target cells for infection

	Seed target cells the day prior to performing the experiment in a 96-well plate (the &#39;target&#39; ...

Take serial dilutions of antibodies specific for human cytomegalovirus, or HCMV.
Upon adding HCMV, the antibodies bind to glycoprotein B on the viral envelope, neutralizing the virus.
Transfer the mixture onto human fibroblasts and incubate.
A trimer glycoprotein complex on the non-neutralized viruses binds to specific cellular receptors, triggering glycoprotein B-mediated virus-host membrane fusion and releasing the genomic DNA-containing nucleocapsid inside.
The released genome enters the nucleus, encoding nuclear-localized immediately early, or IE, proteins.
Antibody-neutralization prevents viral entry into the cells.
Remove the non-internalized viruses. Treat with ethanol at a sub-zero temperature to fix and permeabilize the cells.
Introduce a primary antibody that binds to the IE proteins.
Add a fluorophore-conjugated secondary antibody that binds to the primary antibody and a fluorescent stain that binds to DNA.
Under a microscope, detect dual-stained cells that are virus-infected. Calculate the percentage of infected cells to determine virus neutralization at the different antibody concentrations.

an-immunofluorescence-method-to-measure-neutralization-human

Nanyang Technological University

Research

JoVE Encyclopedia of Experiments

Immunology

Immunodiagnostics

Imaging and Visualization Techniques

97 Views.  This video demonstrates an immunofluorescence assay designed to measure the neutralization of Human Cytomegalovirus (HCMV). Virus-specific antibodies are mixed with the virus and incubated with human fibroblast cells. The antibodies neutralize the virus, rendering it unable to infect the host cells, while non-neutralized viruses infect the cells. After incubation, the extent of neutralization is determined by quantifying the virus-infected cells using immunofluorescence. 

Video: An Immunofluorescence Method to Measure the Neutralization of Human Cytomegalovirus - Concept

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 ...

JoVE Journal

Immunology and Infection

An Improved and High Throughput Respiratory Syncytial Virus (RSV) Micro-neutralization Assay

Respiratory syncytial virus-specific neutralizing antibodies (RSV NAbs) are an important marker of protection against RSV. A number of different assay formats are currently in use worldwide so there is a need for an accurate and high-throughput method for measuring RSV NAbs. We describe here an imaging-based micro-neutralization assay that has been tested on RSV subgroup A and can also be adapted for RSV subgroup B and different sample types. This method is highly reproducible, with inter-assay variations for the reference antiserum being less than 10%. We believe this assay can be readily established in many laboratories worldwide at relatively low cost. Development of an improved, high-throughput assay that measures RSV NAbs represents a significant step forward for the standardization of this method internationally as well as being critical for the evaluation of novel RSV vaccine candidates in the future.

An Improved and High Throughput Respiratory Syncytial Virus RSV Micro-neutralization Assay

This study describes a high throughput, imaging-based micro-neutralization assay to determine the titer of neutralizing antibodies specific for respiratory syncytial virus (RSV). This assay format has been tested on different sample types.

Respiratory syncytial virus-specific neutralizing antibodies (RSV NAbs) are an important marker of protection against RSV. A number of different assay ...

Medicine

Detection of Neutralization-sensitive Epitopes in Antigens Displayed on Virus-Like Particle (VLP)-Based Vaccines Using a Capture Assay

The virus-like particle (VLP) capture assay is an immunoprecipitation method, commonly known as a &#39;pull-down assay&#39; used to purify and isolate antigen-displaying VLPs. Surface antigen-specific antibodies are coupled to, and thus immobilized on a solid and insoluble matrix such as beads. Due to their high affinity to the target antigen, these antibodies can capture VLPs decorated with the cognate antigen anchored in the membrane envelope of the VLPs. This protocol describes the binding of antigen-specific antibodies to protein A- or G-conjugated magnetic beads. In our study, human immunodeficiency virus (HIV)-derived VLPs formed by the group-specific antigen (Gag) viral core precursor protein p55 Gag and displaying the envelope glycoproteins (Env) of HIV are examined. The VLPs are captured utilizing broadly neutralizing antibodies (bNAbs) directed against neutralization-sensitive epitopes in Env. The VLP capture assay outlined here represents a sensitive and easy-to-perform method to demonstrate that (i) the VLPs are decorated with the respective target antigen, (ii) the surface antigen retained its structural integrity as demonstrated by the epitope-specific binding of bNAbs used in the assay and (iii) the structural integrity of the VLPs revealed by the detection of Gag proteins in a subsequent Western blot-analysis. Consequently, the utilization of bNAbs for immunoprecipitation facilitates a prediction of whether VLP vaccines will be able to elicit a neutralizing B cell response in vaccinated humans. We anticipate that this protocol will furnish other researchers with a valuable and straightforward experimental approach to examine potential VLP-based vaccines.

Detection of Neutralization-sensitive Epitopes in Antigens Displayed on Virus-Like Particle VLP-Based Vaccines Using a Capture Assay

Here, we present a protocol to detect neutralization epitopes on antigen-displaying virus-like particles (VLPs). Immunoprecipitation of the human immunodeficiency virus (HIV)-derived VLPs is performed using envelope glycoproteins-specific monoclonal antibodies coupled to protein G-conjugated magnetic beads. Captured VLPs are subsequently subjected to SDS-PAGE and Western blot-analysis employing viral core protein Gag-specific antibodies.

The virus-like particle (VLP) capture assay is an immunoprecipitation method, commonly known as a &#39;pull-down assay&#39; used to purify and isolate ...

An Immunofluorescence Method to Measure the Neutralization of Human Cytomegalovirus

Transcript

An Immunofluorescence Method to Measure the Neutralization of Human Cytomegalovirus

Generation of Escape Variants of Neutralizing Influenza Virus Monoclonal Antibodies

An Improved and High Throughput Respiratory Syncytial Virus (RSV) Micro-neutralization Assay

Detection of Neutralization-sensitive Epitopes in Antigens Displayed on Virus-Like Particle (VLP)-Based Vaccines Using a Capture Assay