an in vitro assay to examine the antibody-dependent enhancement of viral infection

An In Vitro Assay to Examine the Antibody-Dependent Enhancement of Viral Infection

Begin by thawing the serum samples at room temperature. When thawed, transfer 100 microliters of each serum sample to a sterile tube and heat-inactivate for 30 minutes at 56 degrees Celsius.
Next, make 10-fold serial dilutions of the serum sample ranging from 1-to-1 to 1-to-1,000 in cold RPMI-10. Then transfer 10 microliters of each diluted serum sample to the wells of a sterile 96-well V-bottom plate.
Next, thaw dengue 1, 2, 3, and 4 reporter viral particles in a 37 degrees Celsius water bath, ensuring a volume of approximately 170 microliters of each RVP per serum sample. Once thawed, immediately transfer the tubes of RVPs to ice. Then pipette 10 microliters of RVPs into the wells containing serum sample dilutions into the no-serum control wells. Mix thoroughly by pipetting up and down five to 10 times.
Transfer the 96-well V-bottom plate to an incubator, and incubate for an hour at 37 degrees Celsius in the presence of 5% carbon dioxide. While the 96-well V-bottom plate is incubating, clean the surface of the biosafety cabinet with 70% ethanol and run the UV light for 15 minutes.
Then, remove a fully confluent T75 flask of K562 cells from the incubator. Mix the cells well using a sterile 5-milliliter pipette, and transfer 5 milliliters to a sterile 15-milliliter conical tube. Next, remove 10 microliters of cells from the tube. Mix with 10 microliters of trypan blue and load into a hemocytometer. Count the cells and calculate the total number of cells. After centrifuging the cells at 1,200 x g for 10 minutes, decant the supernatant and resuspend the cells in warm RPMI-10 at a concentration of 80,000 cells per 30 microliters of media.
After removing the 96-well V-bottom plate from the incubator, transfer 30 microliters of K562 cells to each well of the 96-well V-bottom plate and mix thoroughly by pipetting up and down five to 10 times. Then, return the plate to the incubator for one hour.
Following the incubation, centrifuge the plate at 1,200 times g for 5 minutes. After centrifugation, decant the media from the wells by turning the plate upside down into a container containing 10% bleach. Then, wash the cells in each well by resuspending them in 125 microliters of warm RPMI-10 and centrifuging as before.
After decanting the last wash, add 100 microliters of warm RPMI 10 to each well. Mix up and down with the pipette, and incubate the plate for 48 hours at 37 degrees Celsius in the presence of 5% carbon dioxide.
Two days, later remove the plate from the incubator and transfer it to a biosafety cabinet. Using a multichannel pipette, mix the contents of each well and transfer to a 96-well U-bottom plate. Rinse each well of the 96-well V-bottom plate with 100 microliters of 1% paraformaldehyde in PBS, and transfer the remaining cells in PBS to the respective wells in the 96-well U-bottom plate. Mix thoroughly using a multichannel pipette.
Cover the plate with aluminum foil and let it sit in the incubator for 30 minutes to fix the cells. Prepare the flow cytometer by running unstained K562 cells to calibrate the side and forward scatter settings using the FL1 channel for GFP fluorescence. Then, acquire 30,000 to 50,000 cells from each sample.

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All procedures involving sample collection have been performed in accordance with the institute&#39;s IRB guidelines.
1. Day 1
NOTE: Perform all the steps described below in a sterile laminar flow biosafety cabinet used for tissue culture in a BSL-2 laboratory.
<ol>
	<li>Thaw the serum samples at room temperature and transfer 100 &#181;L of each serum sample to a sterile tube. Heat inactivate for 30 min at 56&#176;C either in a water bath or a temperature-adjustable thermomixer. Make 10-fold serial dilutions of the serum sample ranging from 1:1 to 1:1,000 using cold RPMI-10 (RPMI with 10% fetal bovine serum).</li>
	<li>Transfer 10 &#181;L of serially diluted serum sample to each well of a sterile 96-well V-bottom plate. Include two sets of control wells, RPMI-10 with RVPs only and without serum sample, and RPMI-10 only without RVPs and without serum sample. Set up each serum sample and RPMI-10 controls in triplicates.</li>
	<li>Remove the Dengue-1, 2, 3 and 4 RVPs from the -80 &#176;C freezer and thaw them in a 37 &#176;C water bath. Transfer the thawed RVPs immediately to ice. Obtain approximately 170 &#181;L RVPs for each serum sample (10 &#181;L of RVPs x 3 wells for each serum dilution (1:1, 1:10, 1:100, 1:1,000) and 10 &#181;L of RVPs x 3 wells for each of the RPMI-10 with RVP only control wells).</li>
	<li>Pipette 10 &#181;L of RVPs into each well of the 96-well V-bottom plate containing the serum sample and to the RPMI-10 with RVPs (no serum) control wells. Do not add RVPs to the RPMI-10 only (no serum sample and no RVP) wells. Mix thoroughly by pipetting up and down 5&#8211;10 times. Add 10 &#181;L cold RPMI-10 in lieu of RVPs to each cold RPMI-10 only (no serum sample and no RVP) control wells.</li>
	<li>Transfer the 96-well V-bottom plate to an incubator and incubate the plate for 1 hour at 37 &#176;C in the presence of 5% CO2. While the 96-well V-bottom plate is incubating, clean the surface of the biosafety cabinet with 70% ethanol and run the UV light for 15 min.</li>
	<li>Remove a fully confluent T75 flask of K562 cells from the incubator, mix the cells well using a sterile 5 mL pipet, and transfer 5 mL of cells to a sterile 15 mL conical tube. 
	NOTE: The K562 cells are maintained in RPMI-10 and subcultured at 1 x 106/mL.</li>
	<li>Count the number of cells by removing 10 &#181;L cells from the sterile 15 mL conical tube and mix with 10 &#181;L tryphan blue. Count the cells using a hemocytometer to determine the total number of cells in the 15 mL conical tube.</li>
	<li>Centrifuge the 15 mL conical with cells at ~1,200 x g for 10 minutes, decant the supernatant, and resuspend the cells in warm RPMI-10 at a concentration of 80,000 cells/30 &#181;L of media (2.66 x 106 cells /mL).</li>
	<li>Remove the 96-well V-bottom plate from the incubator (step 1.6), transfer 30 &#181;L of K562 cells to each well of the 96-well V-bottom plate, and mix thoroughly by pipetting up and down 5&#8211;10 times. Transfer the 96-well V-bottom plate to an incubator and incubate the plate for 1 hr at 37 &#176;C in the presence of 5% CO2.</li>
	<li>After incubating for 1 h, remove the 96-well V-bottom plate from the incubator and centrifuge at ~1,200 x g for 5 minutes. After centrifugation, decant the media from the wells by turning the plate upside down into a container containing 10% bleach.</li>
	<li>Wash the cells in each well by resuspending them in 125 &#181;L of warm RPMI-10, mix thoroughly with a pipette, and centrifuge the plate at ~1,200 x g for 5 min. Decant the media by turning the plate upside down into a container containing 10% bleach. Repeat this wash step two times.</li>
	<li>After washing, add 100 &#181;L of warm RPMI-10 to each well, mix up and down with the pipet, and incubate the plate for 48 h at 37 &#176;C in the presence of 5% CO2.</li>
</ol>
2. Day 3
<ol>
	<li>Remove the 96-well V-bottom plate containing 100 &#181;L of cells and media/well from the incubator and move it to a biosafety cabinet. Using a multichannel pipette, mix the contents of each well and transfer the cells and media to a 96-well U-bottom plate or pre-labeled 5 mL polypropylene tubes.</li>
	<li>Rinse each well in the 96-well V-bottom plate with 100 &#181;L of 1% Paraformaldehyde (PFA) in 1x Phosphate Buffered Saline (PBS) and transfer to the respective wells in the 96-well U-bottom plate or labeled 5 mL polypropylene tubes from step 2.1 to yield a final concentration of 0.5% PFA/well or tube. Mix thoroughly using a multichannel pipette, cover the plate with aluminum foil, and let it sit in the incubator for 30 minutes to fix the cells.</li>
	<li>Prepare the flow cytometer (see Table of Materials for example) by running unstained K562 cells to calibrate the side and forward scatter along with the fluorescence settings. The only fluorescence channel required is FL1, as GFP is the only fluorescence emitted from the cells. Acquire ~30,000&#8211;50,000 cells from each sample. 
	NOTE: Any flow cytometer capable of reading a single fluorescence can be used for acquiring the data, as cells infected with RVPs will only emit green fluorescence due to the presence of GFP, and no other fluorescence channels are needed.</li>
</ol>
3. Data Analysis
<ol>
	<li>Analyze data acquired on the flow cytometer using any flow cytometry analysis software (see Table of Materials).
	<ol>
		<li>Set the first gate based on FSC-A (forward scatter &#8211; area) vs FSC-H (forward scatter &#8211; height) to include single cells and exclude auto-fluorescent doublets from the analysis. Then, gate singlet-gated cells based on SSC-A (side scatter &#8211; area) vs FSC-A to exclude any autofluorescent debris.</li>
		<li>Analyze SSC-A vs FSC-A gated cells for the expression of GFP based on SSC-A vs GFP-A. Determine the percentage of GFP+ cells for each dilution of the serum and control samples by setting a gate around GFP+ cells.</li>
	</ol>
	</li>
	<li>Determine the average percentage of GFP+ cells for each serum sample dilution and control wells by dividing the total percentage of GFP+ cells in the triplicate wells by 3.</li>
	<li>Calculate fold-enhancement of infection by dividing the average percentage of GFP+ cells in the serum samples for each dilution by the average percentage of GFP+ cells in the RPMI-10 with RVPs (no serum sample) control wells.</li>
	<li>Graph fold-enhancement (y-axis) versus dilution (x-axis), and perform statistical analysis using ANOVA followed by Tukey&#39;s post-hoc test for multiple comparisons.</li>
</ol>

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>DENV 1-4 RVP</td>
			<td>Integral Molecular</td>
			<td>RVP-501</td>
			<td></td>
		</tr>
		<tr>
			<td>K562 cells</td>
			<td>ATCC</td>
			<td>CCL-243</td>
			<td></td>
		</tr>
		<tr>
			<td>RPMI</td>
			<td>Corning</td>
			<td>10-040-CV</td>
			<td></td>
		</tr>
		<tr>
			<td>FBS</td>
			<td>GE lifesceinces</td>
			<td>SH 30910.03</td>
			<td>10% FBS in RPMI-10</td>
		</tr>
		<tr>
			<td>Penicillin-Streptomycin</td>
			<td>MP biomedicals</td>
			<td>1670049</td>
			<td>100 IU Pen/mL, 100 ug Strep/mL in RPMI-10</td>
		</tr>
		<tr>
			<td>HEPES</td>
			<td>Cellegro</td>
			<td>25-060-Cl</td>
			<td>0.025M in RPMI-10</td>
		</tr>
		<tr>
			<td>MEM Non-essential Amino Acid Solution (100&#215;)</td>
			<td>Sigma</td>
			<td>M7145</td>
			<td>1x in RPMI-10</td>
		</tr>
		<tr>
			<td>Sodium Pyruvate</td>
			<td>Cellegro</td>
			<td>25-000-Cl</td>
			<td>1mM in RPMI-10</td>
		</tr>
		<tr>
			<td>L-glutamine</td>
			<td>Fisher Scientific</td>
			<td>MT25005CI</td>
			<td></td>
		</tr>
		<tr>
			<td>Sterile V-bottom plates</td>
			<td>Thomas Scientific</td>
			<td>333-8001-01V</td>
			<td></td>
		</tr>
		<tr>
			<td>BD Falcon Polypropylene 5 ml FACS tubes</td>
			<td>VWR</td>
			<td>60819-728</td>
			<td></td>
		</tr>
		<tr>
			<td>Non-sterile U-bottom plates</td>
			<td>Falcon</td>
			<td>Ref 353910</td>
			<td>A high throughput alternative to FACS tubes</td>
		</tr>
		<tr>
			<td>5mL, sterile, serological pipette</td>
			<td>Denville</td>
			<td>P7127</td>
			<td></td>
		</tr>
		<tr>
			<td>200uL sterile pipette tips</td>
			<td>Denville</td>
			<td>P3020 CPS</td>
			<td></td>
		</tr>
		<tr>
			<td>20uL sterile pipette tips</td>
			<td>Denville</td>
			<td>P1121</td>
			<td></td>
		</tr>
		<tr>
			<td>50-200uL multichannel pipette</td>
			<td>Denville</td>
			<td>P3975-8-B</td>
			<td></td>
		</tr>
		<tr>
			<td>5-50uL multichannel pipette</td>
			<td>Denville</td>
			<td>P3975-9-B</td>
			<td></td>
		</tr>
		<tr>
			<td>20% formadehyde</td>
			<td>Tousimis</td>
			<td>#1008B</td>
			<td></td>
		</tr>
		<tr>
			<td>Water Bath</td>
			<td>ThermoFisher</td>
			<td>TSCOL35</td>
			<td></td>
		</tr>
		<tr>
			<td>thermomixer</td>
			<td>Eppendorf</td>
			<td>2231000387</td>
			<td>An alternative to a waterbath for heat inactivation</td>
		</tr>
		<tr>
			<td>CO2 Incubator</td>
			<td>ThermoFisher</td>
			<td>13-998-213</td>
			<td></td>
		</tr>
		<tr>
			<td>Ethanol</td>
			<td>Sigma Aldrich</td>
			<td>E7023</td>
			<td></td>
		</tr>
		<tr>
			<td>Liquid Bleach</td>
			<td>Fisher Scientific</td>
			<td>NC9724348</td>
			<td></td>
		</tr>
		<tr>
			<td>Flowjo 9.8</td>
			<td>TreeStar, Inc.</td>
			<td></td>
			<td>Flow cytometry analysis software</td>
		</tr>
		<tr>
			<td>BD FACSDiva 6.1.2</td>
			<td>Becton Dikinson</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>BD LSR II flow cytometer</td>
			<td>Becton Dikinson</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Liquid Bleach</td>
			<td>Fisher Scientific</td>
			<td>NC9724348</td>
			<td></td>
		</tr>
	</tbody>
</table>

Source: Valiant, W. G., et al. <a href="https://app-jove-com.remotexs.ntu.edu.sg/v/57371">A Simple Flow Cytometry Based Assay to Determine In Vitro Antibody Dependent Enhancement of Dengue Virus Using Zika Virus Convalescent Serum.</a> J. Vis. Exp. (2018).
This video demonstrates an assay to measure antibody-dependent enhancement (ADE) of Dengue virus infection using Zika Virus Convalescent Serum. The assay uses Dengue reporter viral particles (RVPs) encoding a green fluorescent protein. Serum antibodies against the Zika Virus cross-react with RVPs to form a complex. The bound antibodies enhance RVP uptake by cells expressing the Fc receptor, which is detected using flow cytometry.

All procedures involving sample collection have been performed in accordance with the institute&#39;s IRB guidelines.
1. Day 1
NOTE: Perform all the steps ...

Take a microplate with serial dilutions of a serum sample containing antibodies against the Zika virus.
Add Dengue reporter virus particles or RVPs, and incubate.
The engineered RVPs comprise an envelope with structural proteins and a nucleocapsid containing genomic&#160; RNA, encoding a fluorescent protein.
The structural homology of Dengue and Zika viruses&#39; envelope proteins causes the antibodies to cross-react with the RVPs, forming a complex.
Add human leukemia cells impermissible to infection by RVPs.
The cells possess Fc receptors that recognize the RVP-bound antibodies, facilitating RVPs&#39; entry inside endosomes &#8212; termed antibody-dependent enhancement or ADE.
Pellet the cells and discard the supernatant containing non-internalized RVPs. Add a growth media and incubate.
The pH-dependent fusion of RVP and endosomal membranes releases the nucleocapsid, which disassembles to release genomic RNA, enabling fluorescent protein expression.
Fix the cells and analyze them using flow cytometry.
A higher fluorescence at a suitable antibody concentration represents increased RVP internalization, indicating enhanced infection.

34 Görüntüleme. Viral Enfeksiyonun Antikora Bağlı Artışını İncelemek İçin Bir İn Vitro Test

Video: Viral Enfeksiyonun Antikora Bağlı Artışını İncelemek İçin Bir İn Vitro Test - Deney

Zika Virus Specific Diagnostic Epitope Discovery

High-density peptide microarrays allow screening of more than six thousand peptides on a single standard microscopy slide. This method can be applied for drug discovery, therapeutic target identification, and developing of diagnostics. Here, we present a protocol to discover specific Zika virus (ZIKV) diagnostic peptides using a high-density peptide microarray. A human serum sample validated for ZIKV infection was incubated with a high-density peptide microarray containing the entire ZIKV protein translated into 3,423 unique 15 linear amino acid (aa) residues with a 14-aa residue overlap printed in duplicate. Staining with different secondary antibodies within the same array, we detected peptides that bind to Immunoglobulin M (IgM) and Immunoglobulin G (IgG) antibodies present in serum. These peptides were selected for further validation experiments. In this protocol, we describe the strategy followed to design, process, and analyze a high-density peptide microarray.

In this protocol, we describe a technique to discover Zika virus specific diagnostic peptides using a high-density peptide microarray. This protocol can readly be adapted for other emerging infectious diseases.

Zika virüs belirli tanılama Epitope bulma

High-density peptide microarrays allow screening of more than six thousand peptides on a single standard microscopy slide. This method can be applied for ...

JoVE Journal

İmmünoloji ve Enfeksiyon

Quantification of Antibody-dependent Enhancement of the Zika Virus in Primary Human Cells

The recent emergence of the flavivirus Zika and neurological complications, such as Guillain-Barr&#233; syndrome and microcephaly in infants, has brought serious public safety concerns. Among the risk factors, antibody-dependent enhancement (ADE) poses the most significant threat, as the recent re-emergence of the Zika virus (ZIKV) is primarily in areas where the population has been exposed and is in a state of pre-immunity to other closely related flaviviruses, especially dengue virus (DENV). Here, we describe a protocol for quantifying the effect of human serum antibodies against DENV on ZIKV infection in primary human cells or cell lines.

We describe a method to evaluate the effect of pre-existing immunity against dengue virus on the Zika virus infection by using human serum, primary human cells, and infection quantification by quantitative real-time polymerase chain reaction.

Antikor-bağımlı donanýmýn birincil insan hücrelerinde Zika şunun miktar

The recent emergence of the flavivirus Zika and neurological complications, such as Guillain-Barr&#233; syndrome and microcephaly in infants, has brought ...

Targeted Antibody Blocking by a Dual-Functional Conjugate of Antigenic Peptide and Fc-III Mimetics (DCAF)

Elimination of harmful antibodies from organisms is a valuable approach for the intervention of antibody-associated diseases, such as Dengue hemorrhagic fever and autoimmune diseases. Since thousands of antibodies with different epitopes are circulating in blood, no universal method, except for the dual-functional conjugate of antigenic peptide and Fc-III mimetics (DCAF), was reported to target specific harmful antibodies. The development of DCAF molecules makes significant contribution to the progress of targeted therapy, which were demonstrated to eliminate the antibody dependent enhancement (ADE) effect in a Dengue virus (DENV) infection model and to boost the acetylcholine receptor activity in a myasthenia gravis model. Here, we describe a protocol for the synthesis of a DCAF molecule (DCAF1), which can selectively block 4G2 antibody to attenuate ADE effect during Dengue virus infection, and illustrate the binding of DCAF1 to 4G2 antibody by an ELISA assay. In our method, DCAF1 is synthesized by the conjugation of a hydrazine derivative of a Fc-III peptide and a recombinant expressed long &#945;-helix with antigenic sequence through native chemical ligation (NCL). This protocol has been successfully applied to DCAF1 as well as other DCAF molecules for targeting their cognate antibodies.

Targeted Antibody Blocking by a Dual-Functional Conjugate of Antigenic Peptide and Fc-III Mimetics DCAF

Development of a dual-functional conjugate of antigenic peptide and Fc-III mimetics (DCAF) is novel for the elimination of harmful antibodies. Here, we describe a detailed protocol for the synthesis of DCAF1 molecule, which can selectively block 4G2 antibody to eliminate antibody dependent enhancement effect during Dengue virus infection.

Antijenik Peptid ve Fc-III Mimetics (DCAF) Çift Fonksiyonlu Konjuge tarafından Hedefli Antikor Engelleme

Elimination of harmful antibodies from organisms is a valuable approach for the intervention of antibody-associated diseases, such as Dengue hemorrhagic ...

An In Vitro Assay to Examine the Antibody-Dependent Enhancement of Viral Infection

TRANSKRIPT

An In Vitro Assay to Examine the Antibody-Dependent Enhancement of Viral Infection