We would like to thank the members of the Inovio Pharmaceuticals R&amp;D department for technical assistance and staff of Acculab for providing excellence husbandry service. In particular we would like to thank Alysha Vu and Joe Agnes for proofreading the manuscript. This work was not funded by any specific grant from funding agencies in the public, commercial, or not-for-profit sectors.

All methods described here have been approved by the Animal Care and Use Committee (ACUC) of Acculab.
NOTE: The protocol requires the use of potential hazardous materials. Please refer to manufacturer&#39;s MSDS and wear proper personal equipment (PPE) throughout the procedure.
1. Preparation of the guinea-pig immunization site, intradermal Mantoux-injection and CELLECTRA-3P-EP-procedure
<ol>
	<li>Preparation of the treatment site on the guinea pig.
	<ol>
		<li>Place guinea pig in an induction chamber and anaesthetize at 5% Isoflurane-vapor.</li>
		<li>Remove animal from the chamber and place nose cone in position, providing 2% Isoflurane vapor to maintain anesthesia throughout the procedure.</li>
		<li>Shave area of approximately 4 cm2 on abdominal flank.</li>
		<li>Disinfect shaved area with Ethanol-swab.</li>
	</ol>
	</li>
	<li>Injection of pDNA-formulation and electroporation procedure.
	<ol>
		<li>Use an Insulin-syringe to inject 100 &#181;l formulation into the dermis by the Mantoux technique. Needle is inserted bevel up at a 10 degree angle with the animal&#8217;s body.</li>
		<li>Remove the needle and immediately insert the CELLECTRA&#174;-3P electrode array across the injection-wheal and apply electrical field.</li>
		<li>Remove animal from anesthesia and monitor its recovery.</li>
	</ol>
	</li>
</ol>
2. Non-terminal bleed
<ol>
	<li>Blood collection from jugular vein of anaesthetized guinea pig.
	<ol>
		<li>Place guinea pig in an induction chamber and anaesthetize animal as described in 1.1.1</li>
		<li>Using a 3 ml syringe with a 27G 1/2inch needle, punctuate the jugular vein of the anaesthetized animal and collect 3 ml of blood.</li>
		<li>Immediately transfer blood into K2EDTA blood collection tube, invert tube several times to mix blood with anti-coagulant and place on ice. 
		NOTE: Avoidance of blood clotting is crucial for successful downstream processing of the blood sample.</li>
		<li>Monitor animal&#8217;s recovery.</li>
	</ol>
	</li>
</ol>
3. Blood sample processing
<ol>
	<li>Separation of PBMCs from whole blood by density-gradient centrifugation
	<ol>
		<li>Dilute blood 1:1 with Hank&#8217;s Balanced Salt Solution (HBSS). 
		NOTE: To avoid contamination all work from here on should be performed in a Biosafety cabinet applying aseptic technique.</li>
		<li>Layer diluted blood gradually over 4.5 ml Ficoll-Paque Plus in a 15 ml-conical tube. Avoid disturbance of the interface. NOTE: To ensure proper separation, Ficoll-Paque must be at Room temperature.</li>
		<li>Centrifuge tubes at 800 rcf for 30 min with brake off. 
		NOTE: As an alternative, SepMate&#8482; tubes (STEMCELL Technologies) can be used for PBMC-separation. HBSS-diluted blood is added to 15 ml SepMate tube filled with 3.5 ml Ficoll-Paque Plus and then centrifuged at 1200 rcf for 10 min (brake on). This time-saving gradient centrifugation technique results in equal viability, cell-yield and spot-formation.</li>
		<li>Harvest Buffy-Coat layer and dilute in R10 medium (10% (v/v) heat-deactivated FBS and 1% (v/v) Pen/Strep in RPMI1640 medium) to a total volume of 15 ml.</li>
		<li>Pellet cells by centrifuging at 450 rcf for 5 min.</li>
		<li>Wash cells twice with R10 medium.</li>
		<li>Re-suspend cells in 1 ml R10 and pass cell-suspension through 70 &#181;m cell-strainer in to a new tube.</li>
		<li>Count cells and determine number of live cells by Trypan-blue staining.</li>
		<li>Dilute cells with R10 medium to a concentration of 1x10^6 live cells per ml.</li>
	</ol>
	</li>
</ol>
4. Preparation of ELISpot-plates and cell-stimulation
<ol>
	<li>Coat and block wells of a 96-well ELISpot PVDF-membrane plate before seeding the PBMCs.
	<ol>
		<li>Plates should be pre-treated for 60 seconds. Ethanol pre-treatment will result in less unspecific spot-formation and spots with improved definition. 
		NOTE: Take extra care to ensure the complete membrane comes into contact with 15 &#181;l 35% Ethanol.</li>
		<li>After 60 seconds add 150 &#181;l 1x PBS per well, and then empty out wells by inverting the plates. 
		NOTE: The Ethanol pre-treatment is very time-sensitive. Do not incubate the well-membrane longer than 60 sec. Membranes must not dry out during the following steps of the procedure.</li>
		<li>Wash plates three times with 250 &#181;l 1x PBS per well.</li>
		<li>Coat with 100 &#181;l/well of 5 &#181;g/ml capture anti-guinea pig IFN-&#947; antibody V-E4 in PBS.</li>
		<li>Incubate plates for at least 12 hrs at 4&#176; C.</li>
		<li>Wash plates by adding 250 &#181;l/well PBS three times.</li>
		<li>Add 200 &#181;l/well blocking buffer (10% (w/v) Sucrose and 2% (w/v) BSA in PBS) and incubate for 2 hours at room temperature.</li>
		<li>Wash plates with 250 &#181;l/well PBS three times.</li>
	</ol>
	</li>
	<li>Plate PBMCs with antigen-specific peptides, positive and negative controls.
	<ol>
		<li>Dilute protein-peptide pool in R10 to desired final peptide concentration.</li>
		<li>Assay the sample PBMCs in triplicates. 
		NOTE: Add medium with stimulants to the ELISpot plates first and add the PBMC suspension second.</li>
		<li>Add 50 &#181;l of peptide-R10 medium to indicated wells.</li>
		<li>For negative control add 50 &#181;l empty peptide formulation in R10 into indicated wells.</li>
		<li>For positive control add 5 &#181;g/ml Concanavalin A (ConA) in R10 medium into indicated wells.</li>
		<li>Add 100 &#181;l of PBMC cell-suspension to all wells</li>
		<li>Incubate plates in humidified 5% CO2 atmosphere at 37&#176; Celsius for 18 hrs. 
		NOTE: Place ELISpot plates on an even surface/rack in the incubator and avoid any disturbance of the plates during the incubation time.</li>
	</ol>
	</li>
</ol>
5. Detection of Interferon-gamma positive spots
<ol>
	<li>Incubation with detection Antibody and plate development. 
	NOTE: For all steps in this section no aseptic technique is required.
	<ol>
		<li>Carefully remove plates from incubator, and safely empty wells. Wash Plates by adding 250 &#181;l/well PBS for three times.</li>
		<li>Add 100 &#181;l/well of 2 &#181;g/ml biotinylated detection anti-guinea pig IFN-&#947; antibody N-G3 diluted in blocking buffer. 
		NOTE: Filter (22 &#181;m) antibody solution to reduce background.</li>
		<li>Incubate with detection antibody for 2 hrs at room temperature.</li>
		<li>Discard supernatant and wash plates by adding 250 &#181;l/well PBS three times.</li>
		<li>Add 100 &#181;l/well of alkaline phosphatase (ALP)-conjugated streptavidin diluted in blocking buffer. 
		NOTE: Filter (22 &#181;m) solution to reduce background.</li>
		<li>Incubate with ALP-Streptavidin conjugate for 1 hr at room temperature.</li>
		<li>Discard ALP-Streptavidin solution and wash plates by adding 250 &#181;l/well PBS two times, and remove excess PBS by inverting the plate and blotting it against a clean paper towel.</li>
		<li>Wash plates by adding 250 &#181;l/well UltraPure DI water one time, and remove excess water by inverting the plate and blotting it against a clean paper towel.</li>
		<li>Add 100 &#181;l of BCIP/NBT (CAUTION) substrate solution into each well. Caution: BCIP/NBT is highly flammable and toxic if swallowed, in contact with skin, or inhaled. Before use refer to the MSDS.</li>
		<li>Incubate for 20 min at room temperature protected from light.</li>
		<li>Rinse the plate 4 times with deionized water. Invert plate and tap to remove excess water.</li>
		<li>Remove plastic drainage from bottom of ELISpot plates and allow the plates to dry.</li>
		<li>Quantify spots manually or by using an automated ELISpot reader, for example the CTL-Immunospot S6 plate reader.</li>
	</ol>
	</li>
</ol>

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T., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Prophylactic+Herpes+Simplex+Virus+2+(HSV-2)+Vaccines+Adjuvanted+with+Stable+Emulsion+and+Toll-Like+Receptor+9+Agonist+Induce+a+Robust+HSV-2-Specific+Cell-Mediated+Immune+Response,+Protect+against+Symptomatic+Disease,+and+Reduce+the+Latent+Viral+Reservoir.">Prophylactic Herpes Simplex Virus 2 (HSV-2) Vaccines Adjuvanted with Stable Emulsion and Toll-Like Receptor 9 Agonist Induce a Robust HSV-2-Specific Cell-Mediated Immune Response, Protect against Symptomatic Disease, and Reduce the Latent Viral Reservoir.</a> Journal of Virology. 91 (9), 02257 (2017).</li></ol>

The authors Katherine Schultheis, Holly M. Pugh, Bryan S. Yung, Janet Oh, Holly M. Pugh, Jacklyn Nguyen, Laurent Humeau, Kate E. Broderick, and Trevor R.F. Smithare employees of Inovio Pharmaceuticals and as such receive salary and benefits, including ownership of stock and stock option, from the company.

The guinea pig is a valuable animal model for the pre-clinical development of vaccines and intradermal delivery strategies. The above protocol describes the methodology to measure antigen-specific T cell responses in this highly relevant model. The assay provides a clear enumeration of peripheral T-cells producing Interferon-gamma upon stimulation with antigen-specific peptides. The kinetics of the immune response can be monitored by non-terminal blood sampling.
We optimized the protocol and identified critical aspects to obtain optimal results using this assay. For example, the formation of blood clots in the collection tube will result in suboptimal PBMC recovery and viability. Guinea pig blood clots very rapidly18. It&#39;s crucial to perform the blood draw quickly. Au-Birck et al. provide a comprehensive guide for bleeding techniques in the guinea-pig model16. Since the blood collection requires general anesthesia, it is recommended to review applicable standard operating procedures of this aspect of the procedure19. Insufficiently anaesthetized guinea pigs will react by moving their legs or vocalization after a brief pinch of the tissue between their toes with your fingernails or by flinching their ears and moving their whiskers forward after pinching their ear. The immediate transfer of the blood into a tube with anticoagulant and mix thoroughly by rolling or inverting the tube several times is an essential step in this protocol. For the here described protocol EDTA-tubes were used, and no other anticoagulants were tested. Please note that EDTA is hypertonic, tubes should ideally be filled more than half full, and therefore the appropriate tube size for the sample volume must be used.
When performed correctly, density gradient centrifugation consistently results in clean PBMC preparation. The density-gradient medium must be at room temperature when layering the gradient before centrifugation, and brakes should not be used for stopping the centrifuge when using regular tubes. A significant improvement in terms of practicality of the PBMC-processing was the combination of density gradient centrifugation with PBMC-isolation devices. This allows for a reduction in centrifugation time. Density gradient centrifugation in PBMC-isolation devices and regular tubes results in similar viability, live cell counts of processed PBMCs, and spot formation. Independent of the type of tube used for the separation, the buffy coat harvest should follow immediately. Over an extended period of time contact with the density gradient medium is cytotoxic to the PBMCs.
Accurate counting of viable PBMCs is important to seed consistently equal numbers of cells into the assay-plate wells. Some method of live/dead discrimination should be applied. In our lab, we use the trypan-blue exclusion assay and an automated cell-counter.
Spot quality, defined as sharp-edged and high contrasted spots, will result in improved accuracy and consistent spot counting, especially when using an automated counting system. In line with the manufacturer&#39;s recommendations, we observed ethanol pre-treatments of the ELISpot plates to be a crucial step to reduce the number of background spots and improve the definition of the spots. The use of a plate reader to image the ELISpot assay-plates at the end of the protocol is highly recommended. Original images of each well can be easily and efficiently obtained and stored. Using an image analysis software digital images also allow for objective analysis and spot counting compared to manual counting by individual operators.
An absolute necessity for the development of the assay described here was the generation of a suitable anti-guinea pig Interferon-gamma antibody pair. Mouse monoclonal antibodies V-E4 and N-G3 were developed by hybridoma-technique with B-cell clones from mice which were immunized with recombinant guinea-pig interferon gamma20. V-E4, which is IgG1 isotype, and N-G3, which is IgG2a, are reported to bind both to the recombinant and native antigen. Assigning V-E4 as the capture antibody and biotinylated N-G3 as the detection antibody resulted in high sensitivity for both recombinant and native protein while retaining low background signal in a sandwich-ELISA format. Both antibodies are available to the scientific community through a manufacturing agreement led by the laboratory of Dr. Hubert Schaefer, who is co-author of this publication. Requests will be received by Dr. Schaefer&#39;s lab and then manufactured by a commercial partner.
Interferon-gamma is reported to be unstable in regular buffers or media21. Schaefer et al.20 reported only a moderate decrease of recovery rate when using degraded IFN-y, which had lost biological functionality, in an ELISA format using antibodies V-E4 and N-G3. However, increasing the incubation time of peptide stimulation of PBMCs over 18 h should be carefully tested.
The advantages of using PBMCs has been discussed. However, the here described assay only reflects antigen-specific responses of circulating T-cells in the periphery. Tissue-infiltrating T-cells or cells isolated from lymphatic organs, such as spleen and lymph nodes, may exhibit different properties22,23,24. No significant differences in cellular responses were observed upon comparison of interferon-gamma spots from PBMCs and splenocytes from guinea pigs that were immunized with the same pNP influenza vaccine14.
In this protocol, we described an intradermal vaccination procedure. A principal rationale for ID immunization is targeting the high density of dendritic cells present in the skin25. We and others have shown that these cells can be specifically targeted by adapting delivery-method26, formulation27, or drug-design28. Activated professional antigen-presenting cells may migrate to a draining lymph node and activate the adaptive immune system29,30,31,32. Dendritic cells are the essential antigen presentation cell-type for priming productive cellular immune responses.
For this study animals were immunized with plasmid DNA encoding the nucleoprotein of influenza H1N1 strain A/PuertoRico/8. Skin delivery of this pDNA vaccine (pNP) in combination with electroporation had been shown to elicit antigen-specific humoral responses in guinea pigs33, ferrets, and non-human primates (NHPs)1,34. Additionally, this vaccine elicited robust T-cell responses in mice after delivery into the epidermis35, which could be attributed to CD4 and CD8 T-cells by stimulating with peptides representing specific epitopes for these cell-populations. The pNP vaccine was also immunogenic after mucosal delivery as demonstrated by generation of humoral responses in rabbits and guinea pigs, as well as cellular and humoral responses in mice36. Most recently, our group was able to demonstrate the generation of IFN-&#947;T-cell responses in the rabbit after intra-muscular delivery (unpublished data).
Currently, the observed Interferon-gamma spots in the guinea pig ELISpot cannot be allocated to a CD4+ or CD8+ T-cell subset. Especially for the design and development of immune therapies targeting the skin, it would be very useful to determine whether observed interferon-gamma spot frequencies are caused by expansion of one particular subset or a balanced response of both in order to evaluate the effectiveness of the vaccination to trigger cross-presentation. This limitation might be overcome by negative cell-sorting for CD4 or CD8 prior to seeding the cells on the plate or by identification of MHC class II (for CD4 responses) or MHC class I (for CD8 responses) restricted epitopes.
The here described Interferon-gamma ELISpot assay using guinea pig PBMCs addresses the need to assess the course of cellular responses in the guinea pig laboratory model. This will refine the development of vaccines and skin delivery protocols. We believe that it allows for the employment of this relevant animal model to study diseases with important T-cell components such as TB37, Ebola38, HSV39, and others. It will reduce the use of less relevant animal models.

The protocol described here permits the detection of interferon-gamma (IFN-&#947;) secreting cells after antigen recall in a peripheral blood mononuclear cell (PBMC) population harvested from Hartley guinea pigs. We have applied the assay to characterize the kinetics and magnitudes of antigen-specific T cell responses to an Influenza vaccination-regimen in the guinea pig. We believe this protocol will significantly propel the pre-clinical development of vaccination programs in this highly relevant animal model.
T cells elicited by vaccines play an essential role in the protection against infectious agents and immunotherapeutic pathways associated with other diseases. The importance of T cells has been highlighted in multiple vaccine studies. Immunization of ferrets and mice with a plasmid DNA (pDNA) encoding for H5 hemagglutinin and N1 neuraminidase provided protection from morbidity and mortality in an influenza virus challenge in the absence of neutralizing antibodies, indicating the importance of T cell immunity1. In addition to strong neutralizing humoral response, T-cells play a crucial role for not only viral clearance2 but also protection from infection with respiratory syncytial virus (RSV) in mice3. In humans, pre-existing CD8+ T cells were associated with decreased disease severity during the H1N1 pandemic in 20094. CD4+ T cell counts together with certain cytokine plasma concentration are correlated with disease severity in RSV-infected children5.
The guinea pig has gained prominence as a laboratory model for research and development in various areas of medicine such as skin sensitization, nutritional research, studies of the auditory system and, most relevant for this work, infectious diseases. It was crucial for the discovery of vaccines against tuberculosis and diphtheria. More recently the guinea pig is used as a model for Influenza6 and Ebola7. Furthermore, possessing physiological similarities to human skin8, the guinea pig offers an accessible small animal model for dermal drug delivery methods. In contrast to its importance as a laboratory model, the availability of guinea pig specific assays and probes to characterize immune responses remains limited9. Basic cellular assays such as the IFN-&#947; enzyme-linked immunospot (ELISpot-assay) that is routinely used in pre-clinical and clinical research to enumerate T cell responses have not been available.
The first solid-phase enzyme-linked immunospot assays were used to determine the number of specific antibody-secreting cells in a diverse B cell-population10,11. The format has advanced to detect cells secreting cytokines including IL-1, IL-2, IL-4, IL-5, IL-6, IL-10, GM-CSF, TNF-alpha, TNF-beta, granzyme B, and IFN-&#947;. The ELISpot assay possesses high sensitivity; potentially each cytokine-producing cell can be detected. The limit of detection for ELISpot assays has been reported to be lower than 10 spots per 100,000 PBMCs12. Recently a guinea-pig IFN-&#947; specific antibody pair was made available13, and this has opened up the opportunity to address this deficiency in the field.
IFN-&#947; is considered an important effector molecule in the adaptive immune response; it can be produced and secreted by both CD4 and CD8 T cells upon activation. IFN-&#947; has a wide range of biological functions in the context of an immune response to a virus infection, such as the activation of macrophages and the up regulation of major histocompatibility complex (MHC) I and MHCII expression. It also promotes B-cell differentiation, inhibits T-helper-2 cell growth, and activates natural killer cells. IFN-&#947; blocks viral replication in infected somatic cells and upregulates expression of MHC molecules. Thus, the production of IFN-&#947; is a very important indication of the quality of the T cell response to a vaccine or pathogen.
An important aspect of the here presented IFN-&#947; ELISpot is the use of PBMCs rather than splenocytes13. PBMCs can be obtained by processing a non-terminal collected blood sample, whereas the collection of splenocytes requires animal euthanization before harvesting of the spleen. The use of PBMCs enables IFN-&#947; T-cell responses to be monitored over a period of time and evaluation of the effects on the T-cell responses such as prime-boost regimens14.
For researchers who are currently using the guinea pig as an animal model, the IFN-&#947; ELISpot method will broaden the range of scientific data they can gain. Its availability may now reduce the necessity to conduct studies with less relevant animal models for the target disease, which were previously chosen due to reagent availability for examination of cellular responses. The use of PBMCs rather than spleen or lymph nodes allows for non-terminal experiments and continuous monitoring of individual animals.
Below we provide a detailed description of the steps involved in the detection of an IFN-&#947; cellular response in guinea pigs to pDNA vaccine. We outline our specific delivery procedure and describe the general ELISpot assay encompassing blood sampling, blood processing, PBMC harvest, assay procedure, and data analysis. A schematic of the ELISpot assay is depicted in Figure 1.
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/58595/58595fig1.jpg" /> 
Figure 1: Schematic overview of the guinea pig ELISpot protocol. <a href="https://www-jove-com.bdigitaluss.remotexs.co/files/ftp_upload/58595/58595fig1large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>

<table><tbody><tr><td>Cellectra-3P</td><td>Inovio Pharmaceuticals</td><td></td><td>ID-Electroporatio device/ non-commercial, R&amp;amp;D-only</td></tr><tr><td>K&lt;sub&gt;2&lt;/sub&gt;EDTA blood collection tube</td><td>BD</td><td>367844</td><td></td></tr><tr><td>Hank&amp;rsquo;s Balanced Salt Solution</td><td>Gibco&amp;nbsp;</td><td>14175-079</td><td></td></tr><tr><td>Ficoll-Paque Plus</td><td>GE Healthacre&amp;nbsp;</td><td>17144003</td><td>density gradient medium</td></tr><tr><td>SepMate tube</td><td>STEMCELL Technologies</td><td>85415</td><td>PBMC-isolation device</td></tr><tr><td>RPMI&amp;nbsp;</td><td>Thermo Fisher</td><td>11875-135</td><td></td></tr><tr><td>heat-deactivated FBS&amp;nbsp;</td><td>VWR</td><td>97068-085</td><td></td></tr><tr><td>Pen/Strep&amp;nbsp;</td><td>Gibco&amp;nbsp;</td><td>15140-122</td><td></td></tr><tr><td>&amp;nbsp;&amp;beta;-Mercaptoethanol</td><td>Gibco&amp;nbsp;</td><td>21985-023</td><td></td></tr><tr><td>96-well ELISpot PVDF-membrane&amp;nbsp;</td><td>Millipore</td><td>MSIPS4W10</td><td>ELISpot assay plate</td></tr><tr><td>Ethanol&amp;nbsp;</td><td>Sigma</td><td>459844-1L</td><td></td></tr><tr><td>UPDI</td><td>Invitrogen</td><td>10977-015</td><td>Ultra-Pure DI water</td></tr><tr><td>Sucrose&amp;nbsp;</td><td>Sigma</td><td>S7903</td><td></td></tr><tr><td>Bovine Serum Albumin</td><td>Sigma</td><td>A7906</td><td></td></tr><tr><td>10x PBS</td><td>HyClone</td><td>SH30378.03</td><td></td></tr><tr><td>Concanavalin A (ConA)&amp;nbsp;</td><td>Sigma</td><td>C5275-5MG</td><td></td></tr><tr><td>alkaline phosphatase (ALP)-conjugated streptavidin&amp;nbsp;</td><td>R&amp;amp;D Systems Inc.&amp;nbsp;</td><td>SEL002</td><td></td></tr><tr><td>BCIP/NBT&amp;nbsp;</td><td>R&amp;amp;D Systems Inc.&amp;nbsp;</td><td>SEL002</td><td></td></tr><tr><td>capture anti-guinea pig IFN-&amp;gamma; antibody V-E4</td><td>GenScript</td><td>Order #:U5472CE080-3</td><td>LOT # A217071153</td></tr><tr><td>biotinylated detection anti-guinea pig IFN-&amp;gamma; antibody N-G3&amp;nbsp;</td><td>GenScript</td><td>Order #:U5472CE080-1</td><td>LOT # A21700598</td></tr><tr><td>CTL S6 Micro Analyzer</td><td>ImmunoSpot</td><td>#S6MIC12</td><td>automated ELISpot plate reader</td></tr><tr><td>Vi-CELL XR</td><td>Beckman-Coulter</td><td>731050</td><td>automated cell counter</td></tr><tr><td>ImmunoSpot 5.1</td><td>ImmunoSpot</td><td></td><td>assay analysis software</td></tr><tr><td>ESCO Class II Type A2</td><td>ESCO</td><td></td><td>Biosafety cabinet</td></tr><tr><td>Falcon cell strainer</td><td>Corning, Inc.</td><td>VWR cat# 21008-952</td><td></td></tr><tr><td>Exel Comfort Point Insulin syringe</td><td>MedLab Supply&amp;nbsp;</td><td>26028</td><td></td></tr><tr><td>Rotanta 460R</td><td>Hettich</td><td></td><td>centrifuge</td></tr><tr><td>Vi-CELL XR Quad Pak Reagent Kit</td><td>Beckman Coulter</td><td>383722</td><td></td></tr><tr><td>Incu Safe</td><td>Panasonic-Healthcare</td><td></td><td>incubator</td></tr><tr><td>22 &amp;micro;m Filter</td><td>ThermoScientific&amp;nbsp;</td><td>VWR act# 597-4520</td><td>22 &amp;micro;m Filter</td></tr><tr><td>KimWipes</td><td>Kimberly-Clark Professional&amp;nbsp;</td><td>VWR cat# 21903-005&amp;nbsp;</td><td>paper towels</td></tr></tbody></table>

optimized interferon-gamma elispot assay to measure t cell responses in the guinea pig model after vaccination

The guinea pig has played a pivotal role as a relevant small animal model in the development of vaccines for infectious diseases such as tuberculosis, influenza, diphtheria, and viral hemorrhagic fevers. We have demonstrated that plasmid-DNA (pDNA) vaccine delivery into the skin elicits robust humoral responses in the guinea pig. However, the use of this animal to model immune responses was somewhat limited in the past due to the lack of available reagents and protocols to study T cell responses. T cells play a pivotal role in both immunoprophylactic and immunotherapeutic mechanisms. Understanding T cell responses is crucial for the development of infectious disease and oncology vaccines and accommodating delivery devices. Here we describe an interferon-gamma (IFN-&#947;) enzyme-linked immunospot (ELISpot) assay for guinea pig peripheral blood mononuclear cells (PBMCs). The assay enables researchers to characterize vaccine-specific T-cell responses in this important rodent model. The ability to assay cells isolated from the peripheral blood provides the opportunity to track immunogenicity in individual animals.

The results presented here serve as a reference for expected outcomes following the use of this protocol, emphasizing the importance of crucial steps and confirming the benefits of described optimizations.
After density-gradient centrifugation, as described in step 3.1 of the protocol, the red viscous liquid at the bottom of the tube will contain most of the red blood cells. As shown in Figure 2A, above the red blood cells is a layer of the density-medium. Between a layer of clear or yellow plasma on top and the density gradient medium is the white or light brown buffy coat layer, which contains most of the white blood cells and platelets. Incomplete separation would manifest as the presence of red blood cells on top of the density-gradient medium. The red coloration of the plasma in Figure 2A is most probably due to hemolysis, as this blood sample was stored in the collection tube 1.5 h before being processed. Figure 2B shows the gradient before (left) and after (right) centrifugation in PBMC-isolation device. Blood sample in Figure 2B was processed with minimal delay after blood collection, and the plasma coloration is yellow.
The effect of pre-wetting the membranes with ethanol (see step 4.1 of the protocol) on spot-development is shown in Figure 3. Spots in pre-treated wells display improved definition, and there is a reduction in the number of spots in medium/DMSO negative control wells (Figure 3A). Typical viability of processed guinea pig PBMCs ranges around 90% and is similar for both regular 15 mL tubes and PBMC-isolation devices (Figure 3B). Yield of viable cells from a 3 mL blood sample typically ranges between 3 x 106 and 5 x 106.
Animals were immunized with plasmid DNA encoding the nucleoprotein (NP) of H1N1 Influenza strain A/PuertoRico8. Figure 4 shows enumeration of the T-cell IFN-&#947; responses presented as spot-forming units (SFU) generated across the duration of a vaccination regimen. ELISpot assay with PBMCs harvested from non-immunized animals results in negligible spot counts (no tx). Fourteen days after the first immunization (prime), an average of 970 IFN-&#947; spots per million PBMCs were counted after immunogenic stimulation. Seven days after the second immunization (boost), T-cell responses against all three pools were expanded, reaching an average of 5020 IFN-&#947; SFUs/106 PBMCs. Forty-six days after the second immunization (memory), an average total of 6310 IFN-&#947; SFUs/106 PBMCs were counted in peripheral blood.
FIGURE LEGENDS:
<img alt="Figure 2" class="xfigimg" src="/files/ftp_upload/58595/58595fig2.jpg" /> 
Figure 2: Representative images of layers after density-gradient centrifugation. (A) Blood sample before (left) and after (right) centrifugation in regular tubes. (B) Blood sample before (left) and after (right) centrifugation in PBMC-isolation devices. <a href="https://www-jove-com.bdigitaluss.remotexs.co/files/ftp_upload/58595/58595fig2large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 3" class="xfigimg" src="/files/ftp_upload/58595/58595fig3.jpg" /> 
Figure 3: Optimization of the protocol. (A) Typical appearance of IFN-&#947; positive spots in an ELISpot assay plate well developed according to the protocol. Example triplicate wells are shown after pre-treatment with ethanol (right) or without pre-treatment (left). Cells were incubated overnight either with DMSO (top) as no-stimulus control, antigen-matched peptides (middle), or the mitogen ConA (bottom). PBMCs originated from the same individual animal. Wells were imaged using an automated plate-scanner. (B) Comparison of different density-gradient tubes on processed cells. 3 mL of blood samples from 3 individual guinea pigs were collected. 1.5 mL of each sample were processed using either regular tubes (density gradient medium) or PBMC-isolation devices. Viability (left, % &#177; SEM) and number of live cells (right, x 106 &#177; SEM) were determined using an automatic cell counting system and trypan-blue exclusion assay. <a href="https://www-jove-com.bdigitaluss.remotexs.co/files/ftp_upload/58595/58595fig3large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 4" class="xfigimg" src="/files/ftp_upload/58595/58595fig4.jpg" /> 
Figure 4: Detection of robust cellular immune responses over the course of a vaccination regimen. On days 1 and 15, 30 &#181;g of pDNA encoding influenza A nucleoprotein (NP) was delivered intra-dermally to abdominal flank of Hartley guinea pigs immediately followed by skin-electroporation. IFN-&#947; ELISpot response was measured 14 days after the first immunization (prime), and 7 days (boost) and 46 days (memory) after the second immunization. Mean SFUs + SEM were plotted for 5 treated guinea pigs and 2 untreated guinea pigs (no tx). <a href="https://www-jove-com.bdigitaluss.remotexs.co/files/ftp_upload/58595/58595fig4large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>

Watch this Scientific Journal Video about Optimized Interferon-gamma ELISpot Assay to Measure T Cell Responses in the Guinea Pig Model after Vaccination at JoVE.com

Optimized Interferon-gamma ELISpot Assay to Measure T Cell Responses in the Guinea Pig Model after Vaccination

The development of the interferon-gamma ELISpot assay for guinea pig PBMCs allows the characterization of T-cell responses in this highly relevant model for studying infectious diseases. We have applied the assay to measure T cell responses associated with intradermal delivery of DNA vaccines.

The guinea pig has played a pivotal role as a relevant small animal model in the development of vaccines for infectious diseases such as tuberculosis, ...

optimized-interferon-gamma-elispot-assay-to-measure-t-cell-responses

Universidad San Sebastian

Research

JoVE Journal

Immunology and Infection

21.4K Views.  Inovio Pharmaceuticals, Inc. The development of the interferon-gamma ELISpot assay for guinea pig PBMCs allows the characterization of T-cell responses in this highly relevant model for studying infectious diseases. We have applied the assay to measure T cell responses associated with intradermal delivery of DNA vaccines.

Video: Optimized Interferon-gamma ELISpot Assay to Measure T Cell Responses in the Guinea Pig Model after Vaccination

Enzyme-linked Immunospot Assay (ELISPOT):  Quantification of Th-1 Cellular Immune Responses Against Microbial Antigens

Adaptive immunity is an important component to clearance of intracellular pathogens. The ability to detect and quantify these responses in humans is an important diagnostic tool. The enzyme-linked immunospot assay (ELISPOT) is gaining popularity for its ability to identify cellular immune responses against microbial antigens, including immunosuppressed populations such as those with HIV infection, transplantation, and steroid use. This assay has the capacity to quantify the immune responses against specific microbial antigens, as well as distinguish if these responses are Th1 or Th2 in character. ELISPOT is not limited to the site of inflammation. It is versatile in its ability to assess for immune responses within peripheral blood, as well as sites of active involvement such as bronchoalveolar lavage, cerebral spinal fluid, and ascites. Detection of immune responses against a single or multiple antigens is possible, as well as specific epitopes within microbial proteins. This assay facilitates detection of immune responses over time, as well as distinctions in antigens recognized by host T cells. Dual color ELISPOT assays are available for detection of simultaneous expression of two cytokines. Recent applications for this technique include diagnosis of extrapulmonary tuberculosis, as well as investigation of the contribution of infectious antigens to autoimmune diseases.

Enzyme-linked Immunospot Assay ELISPOT:  Quantification of Th-1 Cellular Immune Responses Against Microbial Antigens

Identification of microbial targets of adaptive immunity in idiopathic diseases can be accomplished by the use of the enzyme-linked immunospot assay.

Adaptive immunity is an important component to clearance of intracellular pathogens.  The ability to detect and quantify these responses in humans is an ...

Retroviral Transduction of T-cell Receptors in Mouse T-cells

T-cell receptors (TCRs) play a central role in the immune system. TCRs on T-cell surfaces can specifically recognize peptide antigens presented by antigen presenting cells (APCs)1. This recognition leads to the activation of T-cells and a series of functional outcomes (e.g. cytokine production, killing of the target cells). Understanding the functional role of TCRs is critical to harness the power of the immune system to treat a variety of immunology related diseases (e.g. cancer or autoimmunity).
It is convenient to study TCRs in mouse models, which can be accomplished in several ways. Making TCR transgenic mouse models is costly and time-consuming and currently there are only a limited number of them available2-4. Alternatively, mice with antigen-specific T-cells can be generated by bone marrow chimera. This method also takes several weeks and requires expertise5. Retroviral transduction of TCRs into in vitro activated mouse T-cells is a quick and relatively easy method to obtain T-cells of desired peptide-MHC specificity. Antigen-specific T-cells can be generated in one week and used in any downstream applications. Studying transduced T-cells also has direct application to human immunotherapy, as adoptive transfer of human T-cells transduced with antigen-specific TCRs is an emerging strategy for cancer treatment6.
Here we present a protocol to retrovirally transduce TCRs into in vitro activated mouse T-cells. Both human and mouse TCR genes can be used. Retroviruses carrying specific TCR genes are generated and used to infect mouse T-cells activated with anti-CD3 and anti-CD28 antibodies. After in vitro expansion, transduced T-cells are analyzed by flow cytometry.

We present a protocol to produce antigen-specific mouse T-cells using retroviral transduction

T-cell receptors (TCRs) play a central role in the immune system. TCRs on T-cell surfaces can specifically recognize peptide antigens presented by antigen ...

Generation of Multivirus-specific T Cells to Prevent/treat Viral Infections after Allogeneic Hematopoietic Stem Cell Transplant

Viral infections cause morbidity and mortality in allogeneic hematopoietic stem cell transplant (HSCT) recipients. We and others have successfully generated and infused T-cells specific for Epstein Barr virus (EBV), cytomegalovirus (CMV) and Adenovirus (Adv) using monocytes and EBV-transformed lymphoblastoid cell (EBV-LCL) gene-modified with an adenovirus vector as antigen presenting cells (APCs). As few as 2x105/kg trivirus-specific cytotoxic T lymphocytes (CTL) proliferated by several logs after infusion and appeared to prevent and treat even severe viral disease resistant to other available therapies. The broader implementation of this encouraging approach is limited by high production costs, complexity of manufacture and the prolonged time (4-6 weeks for EBV-LCL generation, and 4-8 weeks for CTL manufacture &#x2013; total 10-14 weeks) for preparation. To overcome these limitations we have developed a new, GMP-compliant CTL production protocol. First, in place of adenovectors to stimulate T-cells we use dendritic cells (DCs) nucleofected with DNA plasmids encoding LMP2, EBNA1 and BZLF1 (EBV), Hexon and Penton (Adv), and pp65 and IE1 (CMV) as antigen-presenting cells. These APCs reactivate T cells specific for all the stimulating antigens. Second, culture of activated T-cells in the presence of IL-4 (1,000U/ml) and IL-7 (10ng/ml) increases and sustains the repertoire and frequency of specific T cells in our lines. Third, we have used a new, gas permeable culture device (G-Rex) that promotes the expansion and survival of large cell numbers after a single stimulation, thus removing the requirement for EBV-LCLs and reducing technician intervention. By implementing these changes we can now produce multispecific CTL targeting EBV, CMV, and Adv at a cost per 106 cells that is reduced by &gt;90%, and in just 10 days rather than 10 weeks using an approach that may be extended to additional protective viral antigens. Our FDA-approved approach should be of value for prophylactic and treatment applications for high risk allogeneic HSCT recipients.

A rapid, simple and cost-effective protocol for the generation of donor-derived multivirus-specific CTLs (rCTL) for infusion to allogeneic hematopoietic stem cell transplant (HSCT) recipients at risk of developing CMV, Adv or EBV infections. This manufacturing process is GMP-compliant and should ensure the broader implementation of T-cell immunotherapy beyond specialized centers.

Viral infections cause morbidity and mortality in allogeneic hematopoietic stem cell transplant (HSCT) recipients. We and others have successfully ...

Use of Interferon-&gamma; Enzyme-linked Immunospot Assay to Characterize Novel T-cell Epitopes of Human Papillomavirus

A protocol has been developed to overcome the difficulties of isolating and characterizing rare T cells specific for pathogens, such as human papillomavirus (HPV), that cause localized infections. The steps involved are identifying region(s) of HPV proteins that contain T-cell epitope(s) from a subject, selecting for the peptide-specific T cells based on interferon-&gamma; (IFN-&gamma;) secretion, and growing and characterizing the T-cell clones (Fig. 1). Subject 1 was a patient who was recently diagnosed with a high-grade squamous intraepithelial lesion by biopsy and underwent loop electrical excision procedure for treatment on the day the T cells were collected1. A region within the human papillomavirus type 16 (HPV 16) E6 and E7 proteins which contained a T-cell epitope was identified using an IFN- g enzyme-linked immunospot (ELISPOT) assay performed with overlapping synthetic peptides (Fig. 2). The data from this assay were used not only to identify a region containing a T-cell epitope, but also to estimate the number of epitope specific T cells and to isolate them on the basis of IFN- &gamma; secretion using commercially available magnetic beads (CD8 T-cell isolation kit, Miltenyi Biotec, Auburn CA). The selected IFN-&gamma; secreting T cells were diluted and grown singly in the presence of an irradiated feeder cell mixture in order to support the growth of a single T-cell per well. These T-cell clones were screened using an IFN- &gamma; ELISPOT assay in the presence of peptides covering the identified region and autologous Epstein-Barr virus transformed B-lymphoblastoid cells (LCLs, obtained how described by Walls and Crawford)2 in order to minimize the number of T-cell clone cells needed. Instead of using 1 x 105 cells per well typically used in ELISPOT assays1,3, 1,000 T-cell clone cells in the presence of 1 x 105 autologous LCLs were used, dramatically reducing the number of T-cell clone cells needed. The autologous LCLs served not only to present peptide antigens to the T-cell clone cells, but also to keep a high cell density in the wells allowing the epitope-specific T-cell clone cells to secrete IFN-&gamma;. This assures successful performance of IFN-&gamma; ELISPOT assay. Similarly, IFN- &gamma; ELISPOT assays were utilized to characterize the minimal and optimal amino acid sequence of the CD8 T-cell epitope (HPV 16 E6 52-61 FAFRDLCIVY) and its HLA class I restriction element (B58). The IFN- &gamma; ELISPOT assay was also performed using autologous LCLs infected with vaccinia virus expressing HPV 16 E6 or E7 protein. The result demonstrated that the E6 T-cell epitope was endogenously processed. The cross-recognition of homologous T-cell epitope of other high-risk HPV types was shown. This method can also be used to describe CD4 T-cell epitopes4.

Use of Interferon-&#947; Enzyme-linked Immunospot Assay to Characterize Novel T-cell Epitopes of Human Papillomavirus

Characterizing T-cell epitopes of pathogens that cause localized infections such as human papillomavirus is a challenge because of limited number of T cells in circulation. A method is described in which rare T cells were isolated and were characterized starting with a very small number of cells.

A protocol has been developed to overcome the difficulties of isolating and characterizing rare T cells specific for pathogens, such as human ...

In Vitro Assay to Evaluate the Impact of Immunoregulatory Pathways on HIV-specific CD4 T Cell Effector Function

T cell exhaustion is a major factor in failed pathogen clearance during chronic viral infections. Immunoregulatory pathways, such as PD-1 and IL-10, are upregulated upon this ongoing antigen exposure and contribute to loss of proliferation, reduced cytolytic function, and impaired cytokine production by CD4 and CD8 T cells. In the murine model of LCMV infection, administration of blocking antibodies against these two pathways augmented T cell responses. However, there is currently no in vitro assay to measure the impact of such blockade on cytokine secretion in cells from human samples. Our protocol and experimental approach enable us to accurately and efficiently quantify the restoration of cytokine production by HIV-specific CD4 T cells from HIV infected subjects.
Here, we depict an in vitro experimental design that enables measurements of cytokine secretion by HIV-specific CD4 T cells and their impact on other cell subsets. CD8 T cells were depleted from whole blood and remaining PBMCs were isolated via Ficoll separation method. CD8-depleted PBMCs were then incubated with blocking antibodies against PD-L1 and/or IL-10R&#945; and, after stimulation with an HIV-1 Gag peptide pool, cells were incubated at 37 &#176;C, 5% CO2. After 48 hr, supernatant was collected for cytokine analysis by beads arrays and cell pellets were collected for either phenotypic analysis using flow cytometry or transcriptional analysis using qRT-PCR. For more detailed analysis, different cell populations were obtained by selective subset depletion from PBMCs or by sorting using flow cytometry before being assessed in the same assays. These methods provide a highly sensitive and specific approach to determine the modulation of cytokine production by antigen-specific T-helper cells and to determine functional interactions between different populations of immune cells.

In Vitro Assay to Evaluate the Impact of Immunoregulatory Pathways on HIV-specific CD4 T Cell Effector Function

We developed an in vitro assay to investigate the role of immunoregulatory pathways in the regulation of cytokine secretion by HIV-specific CD4 T cells.

T cell exhaustion is a major factor in failed pathogen clearance during chronic viral infections. Immunoregulatory pathways, such as PD-1 and IL-10, are ...

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;

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).

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

Application of Long-term cultured Interferon-&#947; Enzyme-linked Immunospot Assay for Assessing Effector and Memory T Cell Responses in Cattle

Effector and memory T cells are generated through developmental programing of na&#239;ve cells following antigen recognition. If the infection is controlled up to 95 % of the T cells generated during the expansion phase are eliminated (i.e., contraction phase) and memory T cells remain, sometimes for a lifetime. In humans, two functionally distinct subsets of memory T cells have been described based on the expression of lymph node homing receptors. Central memory T cells express C-C chemokine receptor 7 and CD45RO and are mainly located in T-cell areas of secondary lymphoid organs. Effector memory T cells express CD45RO, lack CCR7 and display receptors associated with lymphocyte homing to peripheral or inflamed tissues. Effector T cells do not express either CCR7 or CD45RO but upon encounter with antigen produce effector cytokines, such as interferon-&#947;. Interferon-&#947; release assays are used for the diagnosis of bovine and human tuberculosis and detect primarily effector and effector memory T cell responses. Central memory T cell responses by CD4+ T cells to vaccination, on the other hand, may be used to predict vaccine efficacy, as demonstrated with simian immunodeficiency virus infection of non-human primates, tuberculosis in mice, and malaria in humans. Several studies with mice and humans as well as unpublished data on cattle, have demonstrated that interferon-&#947; ELISPOT assays measure central memory T cell responses. With this assay, peripheral blood mononuclear cells are cultured in decreasing concentration of antigen for 10 to 14 days (long-term culture), allowing effector responses to peak and wane; facilitating central memory T cells to differentiate and expand within the culture.

Long-term cultured interferon-&#947; enzyme-linked immunospot assay is used as a measure of central memory responses and correlates with protective anti-mycobacterial vaccine responses. With this assay, peripheral blood mononuclear cells are stimulated with mycobacterial antigens and interleukin-2 for 14 days, enabling differentiation and expansion of central memory T cells.

Effector and memory T cells are generated through developmental programing of na&#239;ve cells following antigen recognition. If the infection is ...

In Vivo Assay for Detection of Antigen-specific T-cell Cytolytic Function Using a Vaccination Model

Current methodologies for antigen-specific killing are limited to in vitro use or utilized in infectious disease models. However, there is not a protocol specifically intended to measure antigen-specific killing without an infection. This protocol is designed and describes methods to overcome these limitations by allowing for the detection of antigen-specific killing of a target cell by CD8+ T cells in vivo. This is accomplished by merging a vaccination model with a traditional CFSE-labeled target killing assay. This combination allows the researcher to assess the antigen-specific CTL potential directly and quickly as the assay is not dependent upon tumor growth or infection. In addition, the readout is based on flow cytometry and so should be readily accessible to most researchers. The major limitation of the study is identifying the timeline in vivo that is appropriate to the hypothesis being tested. Variations in antigen strength and mutations in the T cells that may result in differential cytolytic function need to be carefully assessed to determine the optimal time for cell harvest and assessment. The appropriate concentration of peptide for vaccination has been optimized for hgp10025-33 and OVA257-264, but further validation would be needed for other peptides that may be more appropriate to a given study. Overall, this protocol allows a quick assessment of killing function in vivo and can be adapted to any given antigen.

In Vivo Assay for Detection of Antigen-specific T-cell Cytolytic Function Using a Vaccination Model

The goal of this protocol is to allow for detection of in vivo antigen-specific killing of a target cell in a murine model.

Current methodologies for antigen-specific killing are limited to in vitro use or utilized in infectious disease models. However, there is not a protocol ...

ELISPOT Assay: Detection of IFN-&#947; Secreting Splenocytes

Source: Tonya J. Webb1 
1 Department of Microbiology and Immunology, University of Maryland School of Medicine and the Marlene and Stewart Greenebaum Comprehensive Cancer Center, Baltimore, Maryland 21201
ELISPOT is a standardized, reproducible assay used to detect cellular immune responses. The assay utilizes an enzyme-linked immunosorbent assay (ELISA)- based method to detect single-cell immune responses which can be visualized by spots, hence the name ELISPOT. ELISPOT was first described in 1983, by Czerkinsky, as a method of enumerating the number of B cell hybridomas producing antigen-specific immunoglobulins (1). The same group further developed the assay to measure the frequency of cytokine producing T lymphocytes. Now ELISPOT has become a gold standard for measuring antigen-specific T cell immunity in clinical trials and vaccine candidates. For example, after vaccination or during an infection, plasma cells and memory B cells secrete antibodies that provide protection. Typically, these B cell responses are assessed by measuring serum titers of antigen-specific antibodies. However, this type of analysis, typically measured by ELISA, may not include memory B cells, which can be present even in the absence of detectable serum antibody levels. Furthermore, it has been well-established that circulating memory B cells are important for the rapid and protective antibody response observed following pathogen re-exposure, thus it is critical to be able to detect these cells. Therefore, to clearly assess antigen-specific memory B-cell responses, both ELISA and ELISPOT should be used (2).
ELISPOT assay uses a plate containing membrane-lined wells that are coated with antibodies in order to capture secreted proteins of interest. Then, the plate is loaded with cells and stimuli to induce protein production. The secreted proteins are captured by the antibodies coated on the surface. After appropriate incubation time, cells are removed and the secreted molecule is detected by using a biotinylated antibody that is specific for a different epitope, as compared to the capture antibody. Next, streptavidin peroxidase is added, followed by the addition of a substrate that allows the detection of the spots (Figure 1). The strength of this assay is that it allows one to quantitate the number of cells producing the protein of interest. Importantly, one can assess if there are changes in the total number of cells producing a specific protein or if individual cells within a population are producing more protein. Moreover, it can provide information regarding kinetics and can be used to assess overall immune activation (mitogen stimulation) relative to antigen-specific responses (antigen simulation). The ELISPOT assay will permit the detection one activated cell amongst 300,000 cells following mitogenic or antigen-specific activation.
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/10497/10497fig1.jpg" /> 
Figure 1: ELISPOT protocol overview.
The major advantages of this assay are its- a. Simplicity- the protocol is relatively simple and straightforward. It does not require technical expertise, b. Sensitivity- it permits the detection of immune cells at the single cell level and requires very few cells compared to other methods such as flow cytometry, c. Functionality- it provides quantitative data regarding immune function.
This lab exercise demonstrates the ELISPOT protocol for detection of IFN-&#947; secreting-splenocytes, but as mentioned above this assay can also be used to assess antibody secretion by B cells (3).

ELISPOT Assay: Detection of IFN-&amp;#947; Secreting Splenocytes

Source: Tonya J. Webb1
1 Department of Microbiology and Immunology, University of Maryland School of Medicine and the Marlene and Stewart Greenebaum ...

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Assay for the Identification of Interferon-Gamma Secretion in Murine Lymphoid Organs

Source: Mazet, J. M., et al. <a href="https://app-jove-com.bdigitaluss.remotexs.co/v/59819">Imaging of In Situ Interferon Gamma Production in the Mouse Spleen following Listeria monocytogenes Infection.</a> J. Vis. Exp. (2019).
This video demonstrates an imaging method to visualize in situ interferon-gamma (IFN-&#947;) secretion in the mouse spleen. Following bacterial infection and the induction of IFN-&#947; accumulation inside T cells and natural killer cells, the spleen is harvested from the mouse. Upon obtaining tissue sections, immunostaining is performed to visualize IFN-&#947; inside the cells.

Source: Mazet, J. M., et al. Imaging of In Situ Interferon Gamma Production in the Mouse Spleen following Listeria monocytogenes Infection. J. Vis. Exp. ...

Optimized Interferon-gamma ELISpot Assay to Measure T Cell Responses in the Guinea Pig Model after Vaccination

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Chapters in this video

Enzyme-linked Immunospot Assay (ELISPOT): Quantification of Th-1 Cellular Immune Responses Against Microbial Antigens

Retroviral Transduction of T-cell Receptors in Mouse T-cells

Generation of Multivirus-specific T Cells to Prevent/treat Viral Infections after Allogeneic Hematopoietic Stem Cell Transplant

Use of Interferon-γ Enzyme-linked Immunospot Assay to Characterize Novel T-cell Epitopes of Human Papillomavirus

In Vitro Assay to Evaluate the Impact of Immunoregulatory Pathways on HIV-specific CD4 T Cell Effector Function

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

Application of Long-term cultured Interferon-γ Enzyme-linked Immunospot Assay for Assessing Effector and Memory T Cell Responses in Cattle

In Vivo Assay for Detection of Antigen-specific T-cell Cytolytic Function Using a Vaccination Model

ELISPOT Assay: Detection of IFN-γ Secreting Splenocytes

Assay for the Identification of Interferon-Gamma Secretion in Murine Lymphoid Organs