Name: Enzyme-linked Immunospot Assay (ELISPOT): Quantification of Th-1 Cellular Immune Responses Against Microbial Antigens (Video) | JoVE Journal
Uploaded: 2010-11-23T17:00:00.000+00:00
Duration: 6 min 13 s
Description: Learn ELISPOT to detect and quantify Th-1 cellular immune responses against microbial antigens. This method enables researchers to analyze T-cell cytokine production through spot forming units.

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5 minutes

Supported in part by Vanderbilt CTSA grant 1 UL1 RR024975 from the National Center for Research Resources, National Institutes of Health. This work was funded by NIH R01 HL 83839; R01 AI 65744.

For the following protocol, we use peripheral blood mononuclear cells (PBMC) as the cells of interest. However, this protocol may be used with other cell types. Perform the assay in a tissue culture hood using sterile technique.
 Day 1: Preparation of Plate and Cells
<ol>
 <li> Take out an ELISPOT plate and open it in the hood.</li>
 <li> Wash the plate 3 times with 150 μL of 1X PBS using a multichannel pipettor, if available, otherwise single well pipetting is acceptable.</li>
 <li> Take out a package of reservoirs and open it in the hood.</li>
 <li> Add 2 μg/mL of the capture antibody, anti-human Interferon-γ, to 11 mLs of 1X PBS. Vortex well. Transfer antibody mix to a reservoir.</li>
 <li> Place 100 μL of the coating solution into each well using the multichannel.</li>
 <li> Wrap the plate in parafilm.</li>
 <li> Place the plate in the refrigerator overnight. The plates are good for weeks. The rule of thumb is that if there is still liquid left in the wells, the plate can be used.</li>
</ol>
 Cell Preparation
 If the PBMCs are fresh then count the cells and incubate overnight at 37°C with 5% CO2. For PBMC stored in liquid nitrogen, thawing of cells is necessary. The protocol is as follows:
<ol>
 <li> Thaw the cryovial of cells in 37°C water bath until almost completely thawed.</li>
 <li> Immediately resuspend in 5 mL of R10 media.</li>
 <li> Spin for 5 minutes at 225 x g.</li>
 <li> Resuspend again in 5 mL of R10 media and count the viable cells using trypan blue.</li>
 <li> Bring concentration to 2 x 106 cells/mL in R10 media.</li>
 <li> Rest overnight at 37°C with 5% CO2.</li>
</ol>
 Day 2: Setting-Up the Plate
<ol>
 <li> Label plate lid with specimen identification number, well conditions, and date.</li>
 <li> Wash the plate 6 times with 150 μL of sterile 1x PBS using the dump and blot method. Be careful not to splash the plate while dumping. This could cause the wells of the plate to turn purple.</li>
 <li> Add 100 μL of R20 to each well.</li>
 <li> Incubate the plate at 37°C for 1 hour.</li>
 <li> While the plate is incubating, count the cells that were rested overnight.</li>
 <li> Spin the cell suspension at 1500 rpm for 5 minutes. Decant the supernatant and resuspend the pellet so that you have a final concentration of 1X106 cells per 1mL in R10 media.</li>
 <li> After the 1 hour incubation, remove the R20 using the dump and blot method. Be careful not to splash the plate while dumping.</li>
 <li> Add 100 μL of cell solution (105 cells) to each well. Add appropriate peptides and/or antigens to the corresponding wells in duplicate. Use a new pipette tip every time you go into the peptide working solution. Normal peptide concentration is 10-40 μg/mL. Since peptides vary, each investigator should titrate peptides to obtain optimal results.</li>
 <li> In the negative control wells, only add cells (no peptides)</li>
 <li> In the positive control wells, add phytohemagglutinin (PHA; 10 μg/mL; Sigma).</li>
 <li> Incubate overnight at 37°C with 5% CO2 (~18 hours).</li>
</ol>
 Day 3: Developing the Plate
<ol>
 <li> Wash the plate 6 times with 150 μL 1xPBS using the dump and blot method.</li>
 <li> Add 100 μL PBS to each well of the plate using the multichannel pipettor.</li>
 <li> Put the plate in the refrigerator for 15 minutes.</li>
 <li> Take the plate out of the refrigerator and place it in the tissue culture hood.</li>
 <li> Prepare the biotin antibody solution
 <ul>
 <li> Add 0.5 μg/mL of biotin antibody (mAB 7-B6-1) to 11mL PBS.</li>
 <li> Vortex the solution to mix well.</li>
 <li> If planning to add the solution using a multichannel, pour it into a reservoir.</li>
 </ul>
 </li>
 <li> Discard the PBS by flicking it into the wastebasket. Be careful not to splash the plate while dumping.</li>
 <li> Place 100 μL of the solution into each well.</li>
 <li> Incubate the plate in the tissue culture hood at room temperature for one hour.</li>
 <li> Wash plate 6 times with 150μL 1xPBS using the dump and blot method. On the 6th wash, leave the PBS on the plate until you have prepared the Streptavidin antibody.</li>
 <li> Prepare the Streptavidin antibody.
 <ul>
 <li> Add 5.5 μ l Streptavidin antibody (Streptavidin-ALP) to 11mL PBS (1:2000 dilution).</li>
 <li> Vortex the solution to mix well.</li>
 <li> If planning to add the solution using a multichannel, pour it into a reservoir.</li>
 </ul>
 </li>
 <li> Discard the PBS remaining in the wells and blot the plate.</li>
 <li> Add 100 μL of Streptavidin solution to each well.</li>
 <li> Incubate the plate in the tissue culture hood at room temperature for 1 hour.</li>
 <li> Wash plates 6 times with PBS using the dump and blot method. On the 6th wash, leave the PBS on the plate until you have made the color solution.</li>
 <li> Work in the dark. Prepare the alkaline phosphatase substrate solution (Alkaline Phosphatase Substrate Kit IV BCIP/NBT):
 <ul>
 <li> Add 4 drops of substrate reagent 1 to 11 mL Tris buffer, vortex well. Add 4 drops of substrate reagent 2 to Tris buffer, vortex well. Add 4 drops of substrate reagent 3 to Tris buffer, vortex well.</li>
 </ul>
 </li>
 <li> Discard the remaining PBS and blot the plate.</li>
 <li> Add 100 μL of the color solution to each well of the plate using the multichannel.</li>
 <li> Turn on the light after 5 to 10 minutes of color developing.</li>
 <li> Allow the color reagents to remain on the plate until the spots begin to turn purple and are quite dark. This should take anywhere from 5 to 20 minutes. Be careful not to leave the substrate solution on too long because doing so could cause high background.</li>
 <li> Wash the ELISPOT plate 3 times with tap water.</li>
 <li> Leave to dry for about an hour or overnight.</li>
 <li> Read plate either manually or with ELISPOT plate reader.</li>
 <li> Store the plate in a box away from light exposure.</li>
</ol>
 Representative Results:
 ELISPOT is designed to quantitate immune responses against microbial antigens. Each spot indicates a single responsive cell, expressing the cytokine of interest. The spots can be counted manually or using an ELISPOT plate reader.
 Assessment that the ELISPOT has been performed correctly should begin with investigation of the negative control for background production of the cytokine of interest. In the negative control wells one would expect to see no spots, although a little background (purple color) may be present. Typically, there should be less than an average of five spots per well. The positive control wells confirm that the reaction has been performed correctly, and that the cells are responsive to antigenic stimulation. In the positive control wells you would expect to see a deep purple background due to a confluence of spots (Fig1). There should be little to no white background. If the positive control and negative control samples are appropriate, one can then count the wells containing the antigen of interest. A "suboptimal" result would be reflected by high background (the wells are purple), no spots or very few spots, blank center, poorly defined spots or confluent spots. A list of troubleshooting tips is provided (Table 1).
 <img alt="ELISPOT assay result; immunology experiment; purple spots indicate cytokine-producing cells." src="/files/ftp_upload/2221/2221fig1.jpg" data-altupdatedat="1788235205000" data-alt="Figure 1"> 
 Figure 1. A representative of ELISPOT plate. Each plate should contain a positive control, negative control and peptide of interest conducted at the minimum in two wells. C9, C10, D9 and D10 are the positive control wells, as you can see there is a deep purple color, confluent cells and hardly any white background. The negative control wells are G11, G12, H11 and H12. These wells have no purple background and no spots as expected. The spots G7, G8, H7 and H8, illustrate cellular responses to microbial peptides; the purple spots represent a single responsive cell expressing the cytokine of interest.
 Table 1
<table border="1">
 <tbody>
 <tr>
 <td> Observation</td>
 <td> Possible Problem</td>
 <td> Possible Solution</td>
 </tr>
 <tr>
 <td> High background</td>
 <td>1. Both sides of membrane not washed properly 
 2. Too many cells secreting cytokine 
 3. Plate not dried properly 
 4. Over developed plate</td>
 <td>1. Wash both sides of the membrane with distilled water before and after color development. Reagents may leak through the membrane into the base of the plate, and these can cause high background if not washed away. 
 2. Reduce the number of cells per well, this will require optimization 
 3. Dry the plate longer before reading 
 4. Reduce developing time</td>
 </tr>
 <tr>
 <td> No spots/Very few Spots</td>
 <td>1. Not enough cells secreting cytokine/protein of interest 
 2. Ensure the cells are stimulated correctly 
 3. Cells not incubated for long enough or may take time to respond to stimulant 
 4. Inadequate color development 
 5. Not enough primary or secondary antibody</td>
 <td>1. Increase the number of cells. This will require optimization 
 2. Also use a positive stimulation control - a stimulant that you know will induce expression of your cytokine/protein of interest 
 3. Increase the cell incubation time or use indirect method (pre-treat cells with stimulant) 
 4. Monitor color development with an overhead microscope and ensure that the developing reagents have been stored correctly and have not lost activity 
 5. Concentration of the primary and/or secondary antibody will need to be increased. This will require optimization.</td>
 </tr>
 <tr>
 <td> Blank Areas</td>
 <td>1. Membrane not pre-treated 
 2. Membrane has dried out at some stage 
 3. Cells unevenly distributed</td>
 <td>1. Ensure membrane is adequately pretreated with 70% ethanol (all membranes don't need this so check with your supplier). Wash well with PBS 3X afterwards 
 2. Ensure membrane does not dry 
 3. Ensure you mix the cells gently to have a good homologous cell suspension before pipetting out into the wells</td>
 </tr>
 <tr>
 <td> Blank Center</td>
 <td>1. Damage from washing</td>
 <td>1. Flow rate on automated washer (or pipetting) may be too high. Need a more gentle washing procedure</td>
 </tr>
 <tr>
 <td> False Positives</td>
 <td>1. Secondary antibody aggregates 
 2. Cells still on the membrane 
 3. Cell culture contamination</td>
 <td>1. Filter the secondary antibody 
 2. Ensure all the cells are washed from the membrane with PBS Tween 20 before secondary antibody incubation. Cells left on the membrane will give irregular shaped spots 
 3. Keep reagents as sterile and clean as possible. Ensure your cell culture technique is aseptic. Check for false positives by running a media negative control. For plate movement, <a href="http://www.abcam.com/index.html?pageconfig=resource&amp;rid=11457#7">see poorly defined spots</a></td>
 </tr>
 <tr>
 <td> Confluent Spots</td>
 <td>1. Poor coating, too much antibody 
 2. Prolonged cell culture 
 3. Cells over-stimulated</td>
 <td>1. Reduce primary antibody concentration 
 2. The longer the cells are incubated, the more cytokine/protein they will secrete. This will result in larger spots that will start to merge and become indistinguishable. 
 3. Reduce cell culture step incubation time. Over-stimulation will result in a lot of cytokine/protein being secreted by the cell. This will produce spots that will start to merge and become indistinguishable. Reduce the amount of stimulant in the culture media or culture for a shorter amount of time</td>
 </tr>
 <tr>
 <td> Poorly Defined Spots</td>
 <td>1. Membrane not pre-treated (if your membrane requires this, check with supplier) 
 2. Plate movement during cell incubation</td>
 <td>1. The membrane must be pre-treated with ethanol or this can result in fuzzy, poorly defined spots. It will be difficult for the reader to distinguish these 
 2. Do not allow the plate to move during cell incubation as cells that have moved will create more than one spot. If possible use a dedicated incubator that will not be opened during the incubation. Do not tap the plate after adding cells</td>
 </tr>
 <tr>
 <td> Positive Control wells is low</td>
 <td>1. Underdevelopment - may be a result of using Streptavidin-ALP and/or BCIP/NBT solutions that have not been brought to room temperature</td>
 <td>1. Bring reagents to room temperature before adding to the wells</td>
 </tr>
 <tr>
 <td> Density of the spots makes it difficult to quantify them</td>
 <td>1. Too many cells were added to the wells</td>
 <td>1. Make dilutions of cells (i.e., 1 x 106, 5 x 105, 1 x 105, 5 x 104, 1 x 104 cells per well) to determine the optimal number of cells that will result in formation of distinct spots</td>
 </tr>
 </tbody>
</table>

<ol><li>Dosanjh, D. P., Hinks, T. S., Innes, J. A., Deeks, J. J., Pasvol, G., Hackforth, S., Varia, H., Millington, K. A., Gunatheesan, R., Guyot-Revol, V., Lalvani, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Improved+diagnostic+evaluation+of+suspected+tuberculosis%5BTitle%5D)+AND+%22Ann+Intern+Med%22%5BJournal%5D)">Improved diagnostic evaluation of suspected tuberculosis.</a> Ann Intern Med. 148, 325-336 (2008).</li>
<li>Sung, H. K. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Diagnosis+of+abdominal+tuberculosis+by+T-cell+based+assays+on+peripheral+blood+and+peritoneal+fluid+mononuclear+cells%5BTitle%5D)+AND+%22Journal+of+Infection%22%5BJournal%5D)">Diagnosis of abdominal tuberculosis by T-cell based assays on peripheral blood and peritoneal fluid mononuclear cells.</a> Journal of Infection. 59, 409-415 (2009).</li>
<li>Kim, S. -H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Diagnosis+of+Central+Nervous+System+Tuberculosis+by+T-Cell-Based+Assays+on+Peripheral+Blood+and+Cerebrospinal+Fluid+Mononuclear+Cells%5BTitle%5D)+AND+%22Clinical+and+Vaccine+Immunology%22%5BJournal%5D)">Diagnosis of Central Nervous System Tuberculosis by T-Cell-Based Assays on Peripheral Blood and Cerebrospinal Fluid Mononuclear Cells.</a> Clinical and Vaccine Immunology. , 1356-1362 (2008).</li>
<li>Drake, W. P., Dhason, M. S., Nadaf, M., Shepherd, B. E., Vadivelu, S., Hajizadeh, R., Newman, L. S., Kalams, S. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Cellular+recognition+of+Mycobacterium+tuberculosis+ESAT-6+and+KatG+peptides+in+systemic+sarcoidosis%5BTitle%5D)+AND+%22Infect+Immun%22%5BJournal%5D)">Cellular recognition of Mycobacterium tuberculosis ESAT-6 and KatG peptides in systemic sarcoidosis.</a> Infect Immun. 75, 527-530 (2007).</li>
<li>Allen, S. S., Evans, W., Carlisle, J., Hajizadeh, R., Nadaf, M., Shepherd, B. E., Pride, D. T., Johnson, J. E., Drake, W. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Superoxide+dismutase+A+antigens+derived+from+molecular+analysis+of+sarcoidosis+granulomas+elicit+systemic+Th-1+immune+responses%5BTitle%5D)+AND+%22Respir+Res%22%5BJournal%5D)">Superoxide dismutase A antigens derived from molecular analysis of sarcoidosis granulomas elicit systemic Th-1 immune responses.</a> Respir Res. 25, 36-36 (2008).</li>
<li>Mutsvangwa, J., Millington, K. A., Chaka, K., Mavhudzi, T., Cheung, Y. B., Mason, P. R., Butterworth, A. E., Corbett, E. L., Lalvani, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Identifying+recent+Mycobacterium+tuberculosis+transmission+in+the+setting+of+high+HIV+and+TB+burden%5BTitle%5D)+AND+%22Thorax%22%5BJournal%5D)">Identifying recent Mycobacterium tuberculosis transmission in the setting of high HIV and TB burden.</a> Thorax. 65, 315-320 (2010).</li>
<li>Streeck, H., Frahm, N., Walker, B. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((The+role+of+IFN-gamma+Elispot+assay+in+HIV+vaccine+research%5BTitle%5D)+AND+%22Nat+Protoc%22%5BJournal%5D)">The role of IFN-gamma Elispot assay in HIV vaccine research.</a> Nat Protoc. 4, 461-469 (2009).</li>
<li>Chaudhary, N., Staab, J. F., Marr, K. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Healthy+human+T-Cell+Responses+to+Aspergillus+fumigatus+antigens%5BTitle%5D)+AND+%22PLoS+One%22%5BJournal%5D)">Healthy human T-Cell Responses to Aspergillus fumigatus antigens.</a> PLoS One. 5, e9036-e9036 (2010).</li>
<li>Manigold, T., Mori, A., Graumann, R., Llop, E., Simon, V., Ferr, S. M., Valdivieso, F., Castillo, C., Hjelle, B., Vial, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Highly+differentiated%2C+resting+gn-specific+memory+CD8%2B+T+cells+persist+years+after+infection+by+andes+hantavirus%5BTitle%5D)+AND+%22PLoS+Pathog%22%5BJournal%5D)">Highly differentiated, resting gn-specific memory CD8+ T cells persist years after infection by andes hantavirus.</a> PLoS Pathog. 6, e1000779-e1000779 (2010).</li>
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No conflicts of interest declared.

The ELISPOT assay has emerged as one of the most important and widely used assays to monitor immune responses in humans and a variety of other species. With the ELISPOT assay, immune cell frequencies can be measured at the single cell level without expansion or manipulation of cell populations. ELISPOT assay has been widely applied to investigate antigen specific immune responses in various diseases including infections, cancers, allergies and autoimmune diseases. The application of this assay has been largely applied t...

<table><tbody><tr><td colspan="4">&lt;p class=&quot;jove_title&quot;&gt;A. Preparation of Media&lt;/p&gt;</td></tr><tr><td colspan="4">&lt;img src=&quot;/files/ftp_upload/2221/2221materialsA.jpg&quot; alt=&quot;Materials A&quot;/&gt;</td></tr><tr><td colspan="4">&lt;p class=&quot;jove_title&quot;&gt;B. Preparation of Tris Buffer&lt;/p&gt;</td></tr><tr><td colspan="4">&lt;ol&gt; &lt;li&gt; Add 800 ml of ddH&lt;sub&gt;2&lt;/sub&gt;O to 1 L beaker.&lt;/li&gt; &lt;li&gt; Measure 12 grams of Tris and add to the beaker.&lt;/li&gt; &lt;li&gt; Add 0.12 grams of magnesium chloride hexahydrate (MgCl&lt;sub&gt;2&lt;/sub&gt;:6H&lt;sub&gt;2&lt;/sub&gt;O) into the beaker.&lt;/li&gt; &lt;li&gt; Stir with magnetic stirring rod on automatic stirring plate. &lt;/li&gt; &lt;li&gt; Once dissolved, pH the solution to 9.5.&lt;/li&gt; &lt;li&gt; Add 200 mL of ddH&lt;sub&gt;2&lt;/sub&gt;O to the beaker, bringing it up to 1000mL.&lt;/li&gt; &lt;li&gt; Filter the solution with a 0.22 &amp;mu;m filter.&lt;/li&gt; &lt;/ol&gt;</td></tr><tr><td colspan="4">&lt;p class=&quot;jove_title&quot;&gt;C. Materials necessary for ELISPOT analysis:&lt;/p&gt;</td></tr><tr><td colspan="4">&lt;img src=&quot;/files/ftp_upload/2221/2221materialsb.jpg&quot; alt=&quot;Materials B&quot;/&gt;</td></tr></tbody></table>

enzyme-linked immunospot assay (elispot):  quantification of th-1 cellular immune responses against microbial antigens

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.

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 ...

The overall goal of this procedure is to detect T-cell responses to antigens of interest. This is accomplished by first coding with capture antibodies. The second step of the procedure is to add cells and antigens of interest and then incubate overnight.The third step of the procedure is to add detection antibodies. The final step of the procedure is developing the plate with substrate reagents and analyzing spots. Ultimately, results can be obtained that chose cytokines of interest through analysis of spot forming units.Hi, my name is Dr.Ki Richter and I'm a research instructor in Dr.Wonder Drake's Laboratory at Vanderbilt University in the division of Infectious Diseases. Today we are demonstrating the Ellie Spot technique, also known as the enzyme linked immuno spot assay. Dr.Isfahan chambers and Tiffany Cone will be demonstrating the technique for you today.In order to maintain cell culture standards, perform manipulations under sterile conditions in the hood, open an ally spot plate and wash three times with PBS. Prepare a coating solution of the capture antibody in PBS. Mix well by vortexing for convenience.Transfer the antibody solution to a reservoir for the multichannel, pipetter and aliquot 100 microliters of coating solution into each. Well seal the plate with perfil and place in a refrigerator overnight. These antibody coated plates are good for weeks.The rule of thumb dictates that a plate can be used if there is still liquid left in the wells. To prepare the cells for this assay stain freshly isolated peripheral blood mononuclear cells with trien blue for viability and count them Plate the pbmc at 2 million cells per milliliter in our 10 media and incubate overnight at 37 degrees Celsius with 5%carbon dioxide. In order to keep track of the experiment, label the Ali spot plate lid with the specimen identification number, well conditions and date.Wash the plate six times with sterile PBS using the dump and blot method. Be careful not to splash the plate while dumping, as this could cause the wells of the plate to turn purple. Add our 20 media to each well and incubate the plate at 37 degrees Celsius for one hour.While the plate is incubating, count the cells that were rested overnight. Harvest the pbmc by centrifugation. Decant the supernatant and resuspend the pellet in our 10 media at a concentration of 1 million cells per milliliter.After the one hour incubation of the ally spot plate, remove the R 20 media using the dump and blot method. Be careful not to splash the plate while dumping. Add 100 microliters of cell suspension to each.Well follow the experimental design with addition of peptides and antigens to the corresponding test wells. Always include a negative control of cells with no peptides and also positive control wells with added phyto hemagglutinin. Incubate the plate overnight at 37 degrees Celsius with 5%carbon dioxide.Wash the plate six times with 150 microliters of one XPBS using the dump and blot method. Add 100 microliters of PBS to each well of the plate and place the plate in the refrigerator or at room temperature for 15 minutes. Meanwhile, prepare the biotin antibody solution in PBS.Take the ALI spot plate out of the refrigerator and discard the PBS by flicking it into the waste basket. Be careful not to splash the plate while dumping a aliquot the biotin antibody solution into each well and incubate the plate in the tissue culture hood at room temperature for one hour. Wash the plate six times with PBS using the dump and blot method on the last wash.Leave the PBS on the plate now. Prepare the STREPTAVIDIN antibody solution in PBS, making sure to vortex the solution to mix. Well discard the last PBS wash from the wells and plot the ALI spot plate.Add streptavidin antibody solution to each well and incubate the plate in the tissue culture hood at room temperature for one hour. In order to remove excess trevain antibody, wash the plate six times with PBS using the dump and blot method on the last wash, lead the PBS on the plate. Given the light sensitivity of the B-C-I-P-M-B-T alkaline phosphatase substrate solution.Work in the dark when preparing the stalk solution as described by manufacturers in the alkaline phosphatase substrate kit instructions discard the remaining PBS from the wells and blot the ly spot plate. Add substrate solution to each well and turn on the light. After five to 10 minutes of color developing, allow the reagents to remain on the plate until the spots begin to turn purple and are quite dark.This takes up to 20 minutes. Wash the Ali spot plate three times with tap water and allow to air dry. Each plate contains a positive control, negative control and peptide of interest conducted at the minimum in duplicate.The positive control wells are a deep purple color reflect a confluent layer of cells and hardly any white background. The negative control wells have no purple background and no spots as expected. The test sample wells illustrate cellular responses to microbial peptides here.The purple spots represent a single responsive cell expressing the cytokine of interest When conducting the LA spot technique. Please be careful to not puncture the membrane with your pipette tips or to allow your plate to dry out because this will lead to high background.

enzyme-linked-immunospot-assay-elispot-quantification-th-1-cellular

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

This video demonstrates the use of the ELISpot assay to assess the function of antibody-secreting B cells, ASCs. The antibodies secreted by the ASCs interact with the membrane-bound polyclonal antibody fragments, which are detected by the enzyme-conjugated IgG or IgM antibodies. The colored spot produced in each well indicates the presence of the specific antibody's isotype, which correlates with the B cells' secretory activity.

Functional Assessment of Antibody-Secreting B Cells Using ELISpot Assay

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).

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

Long-term cultured interferon-γ 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.

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

Human peripheral blood is commonly used for the assessment of the humoral immune response. Here, the methods for isolating human B cells from peripheral blood, differentiating human B cells into antibody (Ab)-secreting B cells (ASCs) in culture, and enumerating the total IgM- and IgG-ASCs via an ELISpot assay are described.

The Isolation, Differentiation, and Quantification of Human Antibody-secreting B Cells from Blood: ELISpot as a Functional Readout of Humoral Immunity

Qa-1 (HLA-E in human) belongs to a group of non-classical major histocompatibility complex 1b molecules. Immunization with Qa-1-binding epitopes has been shown to augment tissue-specific immune regulation and ameliorate several autoimmune diseases. Herein we describe an overlapping peptide library strategy for the identification of Qa-1 epitopes in a protein.

Overlapping Peptide Library to Map Qa-1 Epitopes in a Protein

In this paper, we describe a protocol to characterize T-dependent and T-independent immunoglobulin (Ig) isotype responses in mice using ELISA. This method used alone or in combination with flow cytometry will allow researchers to identify differences in B cell-mediated Ig isotype responses in mice following T-dependent and T-independent antigen immunization.

Characterization of Thymus-dependent and Thymus-independent Immunoglobulin Isotype Responses in Mice Using Enzyme-linked Immunosorbent Assay

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.

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

Here, we present a protocol for the ex vivo qualitative detection of antigen-specific CD8+ T cells. Analysis is possible with single cell suspensions from organs or from small amounts of blood. A broad range of studies require the analysis of cytotoxic T cell responses (vaccination and cancer immunotherapy studies).

Simultaneous Quantification of Anti-vector and Anti-transgene-Specific CD8+ T Cells Via MHC I Tetramer Staining After Vaccination with a Viral Vector

The protocol presents detailed methods for evaluating whether the nanoemulsion ophiopogonin D adjuvant promotes effective cellular immune responses.

In Vitro Cellular Activity Evaluation of the Nanoemulsion Vaccine Adjuvant Ophiopogonin D

Learn ELISPOT to detect and quantify Th-1 cellular immune responses against microbial antigens. This method enables researchers to analyze T-cell cytokine production through spot forming units.

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

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