The authors wish to thank study participants and their parents. We would also like to thank Dr. R&#233;jean Lapointe (CHUM Notre-Dame, Montreal, Canada) for access to his ELISpot reader, Dr. Lubo Alexandrov for statistical analysis, and Denis Blais, Sandra Caron, Silvie Valois and Martine Caty for expert technical assistance. Supported by grants from le Fonds d&#8217;op&#233;ration pour les projets de recherche clinique et d&#8217;&#233;valution des technologies (CHU Sainte-Justine) to H.S. and P.O., by la Fondation Centre de canc&#233;rologie Charles-Bruneau, and by the Leukemia &#38; Lymphoma Society of Canada. I.S.F. was supported by scholarships from la Fondation CHU Sainte-Justine and le Fonds de la recherche du Qu&#233;bec-sant&#233; (FRQS). A.J.G. was the recipient of scholarships from the Department of Microbiology, Infectiology &#38; Immunology, Universit&#233; de Montr&#233;al (Gabriel-Marquis Scholarship), FRQS, and the Canadian Institutes of Health Research (CIHR). N.M. was supported by la Fondation CHU Sainte-Justine, the Cole Foundation, and FRQS.

This research protocol was approved by the Institutional Ethics Review Board of CHU Sainte-Justine, Montreal, Quebec, Canada, where the study was conducted. Informed consent was sought and obtained from all study participants, their parents or legal guardians. All procedures performed on days 1 and 2 must be carried out under sterile conditions (i.e. under a laminar flow hood). Standard safety procedures for handling human blood should be strictly observed.
1. Coating the Plates
<ol>
	<li>Permeabilize the polyvinylidene difluoride (PVDF) membranes at the bottom of 96 well MultiScreen IP white ELISpot plates by adding to each well 20 &#956;l of 35% ethanol for 1 min. Membranes should become slightly translucent.</li>
	<li>Immediately wash the wells 3 times with 200 &#956;l of 1x phosphate buffered saline (PBS; 137 mM NaCl, 2.7 mM KCl, 10 mM Na2HPO4, 2 mM KH2PO4, pH 7.4) using a multichannel pipette or similar device. This step is critical given that ethanol residues can affect cell viability and binding of capture antibody. ELISpot plates are fragile and should be handled carefully: the use of an automated plate washer is not recommended.</li>
	<li>Coat the wells with 10 &#956;l of purified mouse anti-human IFN-&#947; capture antibody diluted in 1x PBS to a final concentration of 10 &#956;g/ml. Cover the plates with regular plastic wrap and incubate overnight at 4 &#176;C.</li>
</ol>
2. Blocking Plates
<ol>
	<li>Empty the wells, tapping them dry, and wash plates 5 times with 200 &#956;l of 1x PBS.</li>
	<li>Fill the wells with 200 &#956;l of complete RPMI 1640 medium and incubate plates for 2 hr at 37 &#176;C (blocking step).</li>
	<li>Wash the plates 5x with 200 &#956;l of 1x PBS and keep the wells full of 1x PBS until cells are ready to be plated.</li>
</ol>
3. Cell Plating and Stimulation
<ol>
	<li>Prepare peripheral blood mononuclear cells (PBMC) from human venous blood samples by centrifugation on Ficoll-Paque gradients using standard procedures. Alternatively, using standard methods, thaw aliquots of PBMC frozen under liquid nitrogen. After the last centrifugation, resuspend cells at a final concentration of 2 x 106 cells/ml and incubate overnight at 37 &#176;C under a 5% CO2 atmosphere in a water jacket incubator.</li>
	<li>Remove PBMC from the incubator and resuspend them in a final volume of 5 ml of complete RPMI 1640 medium.</li>
	<li>Incubate PBMC in the presence of 10 &#956;l of genetically engineered endonuclease from Serratia marcescens for 5 min at room temperature.</li>
	<li>Remove a 50 &#956;l aliquot of PBMC and mix with 50 &#956;l of trypan blue dye. Count cells and estimate % viability using a haemocytometer and microscopic examination (dye exclusion method). Various automated methods for cell counting and assessment of cell viability can also be used. PBMC should be &#62;95% viable. Discard sample when viability is lower than 95%.</li>
	<li>Centrifuge PBMC at 700 x g for 5 min, discard supernatant, and resuspend cells in AIM-V medium supplemented with 2% (v/v) inactivated human serum (HIS) at a final concentration of 2 x 106 cells/ml. Keep at 37 &#176;C until stimulation mixtures are added to the plate.</li>
	<li>Add to the wells, one drop at a time, 100 &#956;l of the appropriate stimulation mixture. Three stimulation mixtures should be used as described below:
	<ol>
		<li>Use AIM-V medium supplemented with 2% (v/v) inactivated human serum (IHS) as negative control.</li>
		<li>Use anti-CD3 monoclonal antibody (clone OKT3) diluted in AIM-V medium supplemented with 2% (v/v) IHS to a final concentration of 0.5 &#956;g/ml as positive control).</li>
		<li>Use VZV antigen, in the form of either &#947;-irradiated (10,000 Gy; 30 min exposure time) live attenuated VZV vaccine (1,350 plaque forming units [PFU]/ml) diluted 1:200 in AIM-V medium supplemented with 2% (v/v) IHS) or 1 &#956;g of an equimolar mixture of 67 peptides (15 amino acid residues) synthesized based on the sequence of the VZV immediately early (IE63) phosphoprotein. Control irradiation efficacy by monitoring the absence of visible signs of cytopathic effects after 4 days in PBMC cultures.</li>
		<li>Prepare all wells in duplicate.</li>
	</ol>
	</li>
	<li>Add to the wells, one drop at a time, 100 &#956;l of cells prepared in step 5.4 to the appropriate wells. Two wells should be left without cells (negative control). Incubate for 20 hr at 37 &#176;C under a 5% CO2 atmosphere in a water jacket incubator. Do not shake, move, or stack the plates on top of one another during incubation.</li>
</ol>
4. Spot Development and Detection
<ol>
	<li>Discard the cells and wash the plates 10x with 1x PBS supplemented with 0.05% (v/v) Tween 20 (PBST). Tween 20 helps detach cells that have adhered to the PVDF membrane following overnight incubation.</li>
	<li>Using a multichannel pipette, add 100 &#956;l of 0.5 mg/ml biotin conjugated anti-IFN-&#947; monoclonal antibody (4S.B3 clone) diluted in 1x PBS containing 0.5% (w/v) bovine serum albumin (BSA). Incubate plates for 2 hr at room temperature.</li>
	<li>Wash the wells 6x with PBST and add each well 100 &#956;l of streptavidin conjugatd with alkaline phosphatase diluted 1:1,000 in 1x PBS containing 0.5% (w/v) BSA. Cover the plates and incubate for 1 hr at room temperature.</li>
	<li>Wash the plates 3x with PBST and 3x with 1x PBS. Add 100 &#956;l of a 5-bromo-4-chloro-3-indolyl phosphate (BCIP)/nitro blue tetrazolium chloride (NBT) substrate solution and incubate 5 min at room temperature.</li>
	<li>Wash the plates under running distilled water. Remove the bottom section of the ELISpot plate and wash both sides of the membrane with distilled water. Air dry the plates.</li>
	<li>Examine the wells and enumerate the spots using a stereoscopic dissection microscope or scan the plates using an automated spot counter. Keep the plates away from light if they cannot be examined on the same day.
	<ol>
		<li>Average the numbers of spots counted in corresponding duplicate wells and subtract the number of spots counted in the negative control wells (no cells) from the number of spots counted in the test wells.</li>
		<li>Express IFN-&#947; production as the number of spot-forming units (SFU) per 106 PBMC.</li>
		<li>Consider that test samples are positive if the number of SFU is &#62;50 per 106 PBMC and 2 standard deviations (SD) above the negative control (cells + AIM-V medium supplemented with 2% (v/v) IHS).</li>
		<li>Use a value of 400 SFU per well when spots are too numerous to be counted.</li>
	</ol>
	</li>
	<li>Test whether ELISpot data are normally distributed or not using the Kolmogorov-Smirnov test. When data are normally distributed, perform statistical comparisons using Student&#8217;s t test (2 groups) or ANOVA and the Bonferroni post-test (multiple comparisons). If data are not normally distributed, use the Mann-Whitney U test (2 groups) or the Kruskal-Wallis test and Dunn&#8217;s post-test (multiple comparisons).</li>
</ol>

The authors declare that they have no competing financial interests.

Modifications and troubleshooting: IFN-&#947; ELISpot assays have been used to examine cell-mediated immune responses directed against a variety of microbial pathogens, including human immunodeficiency virus type 1 (HIV-1)24,25, hepatitis C virus (HCV)26,27, and Mycobacterium tuberculosis28,29, just to name a few. Here we described the development of an IFN-&#947; ELISpot assay to measure cellular immunity against, with the hope of defining correlates of VZV specific immune recon...

First performed in 1989, UCBT is increasingly used as part of the treatment of various neoplastic and nonneoplastic blood disorders in children1. VZV is a cytopathic human alphaherpesvirus which causes two different diseases, varicella (after primary infection) and herpes zoster (after reactivation). Following primary infection, VZV persists throughout the life of the host sheltered within sensory nerves of dorsal root ganglia. One of the most threatening infectious complications following UCBT is associated with VZV2-4. In our clinical center, in absence of VZV prophylaxis, the cumulative incidence of VZV disease VZV disease at 3 years postUCBT was 46%2. In these patients, de novo infection with or reactivation of VZV is often associated with visceral dissemination to the central nervous system, lungs and liver5-7. As a result, acyclovir, valacyclovir or famciclovir prophylaxis is commonly administrated to UBCT recipients8,9. However, this treatment strategy does not take into account the protective potential of VZV specific T lymphocytes or the kinetics of reconstitution of VZV specific T cell responses. Potential problems associated with the expanding use of long term antiherpetic prophylaxis include a) patient overtreatment; b) the development of antiviral drug resistance10,11; and c) impairment of VZV specific immune reconstitution12,13. Because detection of functional VZV specific T lymphocytes correlates with the presence of long term protection from VZV infection and improved clinical outcome4,14,15, monitoring cell mediated immune responses directed against VZV during the posttransplant period might result in a more rational use of antiviral treatment by enabling medical practitioners to distinguish patients who would benefit from VZV prophylaxis from those whose immune system is capable of controlling VZV replication4,13.

The IFN-&#947; ELISpot assay is widely used for the monitoring cell mediated immune responses in a variety of experimental systems and clinical conditions. Spots are generated following the cleavage of a chromogenic substrate, generating a visible and stable precipitate at the site of the reaction. Each individual spot thereby represents the footprint of an individual cytokine-producing cell. IFN-&#947; ELISpot not only measures the ability of individual cells ex vivo to produce IFN-&#947; in response to in vitro stimulation with cognate antigen, but it also provides an estimate of the frequency of responding cells in a given cell population16,17. In addition to its high sensitivity, IFN-&#947; ELISpot is straightforward to perform, making its use possible in the context of personalized clinical protocols aimed at guiding initiation or cessation of antiviral treatment. The procedure detailed below describes an ELISpot assay that is specifically designed to detect and measure the production of IFN-&#947; by peripheral blood mononuclear cells following in vitro stimulation with VZV derived antigens.

<table border="0" cellpadding="0" cellspacing="0" width="857">
	<tbody>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Leucocep tube</td>
			<td style="width:101px;">VWR</td>
			<td style="width:143px;">89048-936/89048-932</td>
			<td style="width:397px;">12 ml or 50 ml tubes may be used depending on the volume of blood.&#160;</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Ficoll-Paque</td>
			<td style="width:101px;">GE Healthcare</td>
			<td style="width:143px;">17-1440-02</td>
			<td>Protect from light.</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Benzonase nuclease</td>
			<td style="width:101px;">Novagen</td>
			<td style="width:143px;">70746-3</td>
			<td>Keep at -20 C.</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">MultiScreenHTS-IP Filter Plate</td>
			<td style="width:101px;">Millipore</td>
			<td style="width:143px;">MSIPS4W10</td>
			<td>Sterile with pore size of 0.45 &#181;m.&#160;</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Mouse anti-human IFN-&#947; capture antibody</td>
			<td style="width:101px;">BD Biosciences</td>
			<td style="width:143px;">551221</td>
			<td>NIB42 clone.&#160;</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Pepmix VZV IE63&#160;</td>
			<td style="width:101px;">JPT Peptide Technologies</td>
			<td style="width:143px;">PM-VZV-IE63</td>
			<td>Dissolve contents of one vial in 40 &#956;L of DMSO. Use within 6 months.</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Biotin-conjugated anti-IFN-&#947; monoclonal antibody</td>
			<td style="width:101px;">BD Biosciences</td>
			<td style="width:143px;">554550</td>
			<td>4SB3 clone.</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Streptavidin conjugated with alkaline phosphatase&#160;</td>
			<td style="width:101px;">Bio-Rad Life Science</td>
			<td style="width:143px;">170-3554</td>
			<td>Dilute for use on the same day.</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">BCIP/NBT</td>
			<td style="width:101px;">Bio-Rad Life Science</td>
			<td style="width:143px;">170-6432</td>
			<td>Protect from light.</td>
		</tr>
	</tbody>
</table>

development of an ifn-&#947; elispot assay to assess varicella-zoster virus-specific cell-mediated immunity following umbilical cord blood transplantation

Varicella zoster virus (VZV) is a significant cause of morbidity and mortality following umbilical cord blood transplantation (UCBT). For this reason, antiherpetic prophylaxis is administrated systematically to pediatric UCBT recipients to prevent complications associated with VZV infection, but there is no strong, evidence based consensus that defines its optimal duration. Because T cell mediated immunity is responsible for the control of VZV infection, assessing the reconstitution of VZV specific T cell responses following UCBT could provide indications as to whether prophylaxis should be maintained or can be discontinued. To this end, a VZV specific gamma interferon (IFN-&#947;) enzyme-linked immunospot (ELISpot) assay was developed to characterize IFN-&#947; production by T lymphocytes in response to in vitro stimulation with irradiated live attenuated VZV vaccine. This assay provides a rapid, reproducible and sensitive measurement of VZV specific cell mediated immunity suitable for monitoring the reconstitution of VZV specific immunity in a clinical setting and assessing immune responsiveness to VZV antigens. &#160;

The IFN-&#947; ELISpot protocol detailed above was developed and optimized in our laboratory to measure the magnitude and the quality of cell mediated immune responses directed against VZV4. Diverse sources of VZV antigen can be used for the stimulation step. These include: a) commercially available detergent inactivated extracts from VZV infected Vero cells18; b) pools of overlapping synthetic peptides from specific VZV encoded proteins, including IE6315 and ORF419; c) live at...

Watch this Scientific Journal Video about Development of an IFN-Î³ ELISpot Assay to Assess Varicella-Zoster Virus-specific Cell-mediated Immunity Following Umbilical Cord Blood Transplantation at Jo

Development of an IFN-&amp;#947; ELISpot Assay to Assess Varicella-Zoster Virus-specific Cell-mediated Immunity Following Umbilical Cord Blood Transplantation

Novel generations of functional assays such as gamma interferon (IFN-&#947;) ELISpot, which detect cytokine production at the single cell level and provide both quantitative and qualitative characterization of T cell responses can be used to assess cell-mediated immune responses directed against varicella zoster virus (VZV).

Development of an IFN-&#947; ELISpot Assay to Assess Varicella-Zoster Virus-specific Cell-mediated Immunity Following Umbilical Cord Blood Transplantation

Varicella zoster virus (VZV) is a significant cause of morbidity and mortality following umbilical cord blood transplantation (UCBT). For this reason, ...

development-an-ifn-elispot-assay-to-assess-varicella-zoster-virus

Research

JoVE Journal

Immunology and Infection

15.5K Views.  Université de Montréal. Novel generations of functional assays such as gamma interferon (IFN-γ) ELISpot, which detect cytokine production at the single cell level and provide both quantitative and qualitative characterization of T cell responses can be used to assess cell-mediated immune responses directed against varicella zoster virus (VZV).

Video: Development of an IFN-&#947; ELISpot Assay to Assess Varicella-Zoster Virus-specific Cell-mediated Immunity Following Umbilical Cord Blood Transplantation