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 &mu;L 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 &mu;g/mL of the capture antibody, anti-human Interferon-&gamma;, to 11 mLs of 1X PBS. Vortex well. Transfer antibody mix to a reservoir.</li>
 <li> Place 100 &mu;L 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&deg;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&deg;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&deg;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 &mu;L 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 &mu;L of R20 to each well.</li>
 <li> Incubate the plate at 37&deg;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 &mu;L 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 &mu;g/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 &mu;g/mL; Sigma).</li>
 <li> Incubate overnight at 37&deg;C with 5% CO2 (~18 hours).</li>
</ol>
 Day 3: Developing the Plate
<ol>
 <li> Wash the plate 6 times with 150 &mu;L 1xPBS using the dump and blot method.</li>
 <li> Add 100 &mu;L 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 &mu;g/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 &mu;L 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 150&mu;L 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 &mu; 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 &mu;L 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 &mu;L 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 &quot;suboptimal&quot; 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="Figure 1" src="/files/ftp_upload/2221/2221fig1.jpg" /> 
 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&#39;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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Improved+diagnostic+evaluation+of+suspected+tuberculosis.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Diagnosis+of+abdominal+tuberculosis+by+T-cell+based+assays+on+peripheral+blood+and+peritoneal+fluid+mononuclear+cells.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Diagnosis+of+Central+Nervous+System+Tuberculosis+by+T-Cell-Based+Assays+on+Peripheral+Blood+and+Cerebrospinal+Fluid+Mononuclear+Cells.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cellular+recognition+of+Mycobacterium+tuberculosis+ESAT-6+and+KatG+peptides+in+systemic+sarcoidosis.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Superoxide+dismutase+A+antigens+derived+from+molecular+analysis+of+sarcoidosis+granulomas+elicit+systemic+Th-1+immune+responses.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Identifying+recent+Mycobacterium+tuberculosis+transmission+in+the+setting+of+high+HIV+and+TB+burden.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+role+of+IFN-gamma+Elispot+assay+in+HIV+vaccine+research.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Healthy+human+T-Cell+Responses+to+Aspergillus+fumigatus+antigens.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Highly+differentiated,+resting+gn-specific+memory+CD8++T+cells+persist+years+after+infection+by+andes+hantavirus.">Highly differentiated, resting gn-specific memory CD8+ T cells persist years after infection by andes hantavirus.</a> PLoS Pathog. 6, e1000779-e1000779 (2010).</li><li>Ndongala, M. L., Kamya, P., Boulet, S., Peretz, Y., Rouleau, D., Tremblay, C., Leblanc, R., C&#244;t&#233;, P., Baril, J. G., Thomas, R., V&#233;zina, S., Boulassel, M. R., Routy, J. P., S&#233;kaly, R. P., Bernard, N. F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Changes+in+function+of+HIV-specific+T-cell+responses+with+increasing+time+from+infection.">Changes in function of HIV-specific T-cell responses with increasing time from infection.</a> Viral Immunol. 2, 159-168 (2010).</li></ol>

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 to studies where the quantification of the antigen-specific cells is critical or comparison of the immune response to specific antigen in various disease states, such as tuberculosis before or after antibiotic therapy 1. However, recent reports have shown that this assay can be used to detect frequencies of antigen-specific T cells within isolated cell material such as the ascitic fluid, cerebral spinal fluid or bronchoalveolar lavage. Sung-Han Kim et. al. has shown that by using the ELISPOT assay on PBMCs from patients suspected of abdominal tuberculosis, there is a sensitivity of 89% for diagnosing active abdominal TB 2. It has also been shown that the T-cell based ELISPOT assay had a 91% sensitivity for diagnosis of active TB in patients with suspected of central nervous system involvement by tuberculosis using peripheral blood and cerebrospinal fluid mononuclear cells 3. Our laboratory utilizes the ELISPOT assay to assess cellular recognition of mycobacterial antigen ESAT-6 in PBMC from sarcoidosis patients. This report is important because it shows how the ELISPOT assay can be used also as a diagnostic method to delineate a role for infectious agents in idiopathic diseases 4,5. Another benefit of this assay is it also allows the identification of tuberculosis in patients who have undergone BCG vaccination. The assay is not limited by immunosuppressive conditions such as steroid use and HIV infection 6. It has also facilitated quantification and characterization of the human immunodeficiency virus (HIV)-specific CD8+ T cell responses 7. Responses can even be detected among healthy subjects to microbial antigens, such as Aspergillus fumigatus 8. ELISPOT has also been used to identify immunodominant antigens from survivors of Hantavirus cardiopulmonary syndrome 9, as well as to assess T cell function during chronic HIV infection 10. Because of the short-term in vitro culture, the measured response closely mirrors the in vivo T cell frequency. The ELISPOT assay has the lowest detection threshold among the 'standard' T cell assays such as the lymphoproliferation assay (LPA) and cytotoxic T lymphocyte (CTL) assay. This summary provides only a limited view of the vast possibilities of the relatively simple, inexpensive assay. The possible applications of this assay continue to expand.

A.	Preparation of Media
<img src="/files/ftp_upload/2221/2221materialsA.jpg" alt="Materials A" />
B.	Preparation of Tris Buffer
<ol>
<li>	Add 800 ml of ddH2O to 1 L beaker.</li>
<li>	Measure 12 grams of Tris and add to the beaker.</li>
<li>	Add 0.12 grams of magnesium chloride hexahydrate (MgCl2:6H2O) into the beaker.</li>
<li>	Stir with magnetic stirring rod on automatic stirring plate. </li>
<li>	 Once dissolved, pH the solution to 9.5.</li>
<li>	 Add 200 mL of ddH2O to the beaker, bringing it up to 1000mL.</li>
<li>	Filter the solution with a 0.22 &mu;m filter.</li>
</ol>
C.	Materials necessary for ELISPOT analysis:
<img src="/files/ftp_upload/2221/2221materialsb.jpg" alt="Materials B" />

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.

Watch this Scientific Journal Video about Enzyme-linked Immunospot Assay ELISPOT:  Quantification of Th-1 Cellular Immune Responses Against Microbial Antigens at JoVE.com

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.

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 ...

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

Nanyang Technological University

Research

JoVE Journal

Immunology and Infection

34.7K Views.  Vanderbilt University School of Medicine. Identification of microbial targets of adaptive immunity in idiopathic diseases can be accomplished by the use of the enzyme-linked immunospot assay.

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

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 ...

Concurrent Quantification of Cellular and Extracellular Components of Biofilms

Confocal laser scanning microscopy (CLSM) is a powerful tool for investigation of biofilms. Very few investigations have successfully quantified concurrent distribution of more than two components within biofilms because: 1)&#160;selection of fluorescent dyes having minimal spectral overlap is complicated, and 2)&#160;quantification of multiple fluorochromes poses a multifactorial problem. Objectives: Report a methodology to quantify and compare concurrent 3-dimensional distributions of three cellular/extracellular components of biofilms grown on relevant substrates. Methods: The method consists of distinct, interconnected steps involving biofilm growth, staining, CLSM imaging, biofilm structural analysis and visualization, and statistical analysis of structural parameters. Biofilms of Streptococcus mutans (strain UA159) were grown for 48 hr on sterile specimens of Point 4 and TPH3 resin composites. Specimens were subsequently immersed for 60 sec in either Biot&#232;ne PBF (BIO) or Listerine Total Care (LTO) mouthwashes, or water (control group; n=5/group). Biofilms were stained with fluorochromes for extracellular polymeric substances, proteins and nucleic acids before imaging with CLSM. Biofilm structural parameters calculated using ISA3D image analysis software were biovolume and mean biofilm thickness. Mixed models statistical analyses compared structural parameters between mouthwash and control groups (SAS software; &#945;=0.05). Volocity software permitted visualization of 3D distributions of overlaid biofilm components (fluorochromes). Results: Mouthwash BIO produced biofilm structures that differed significantly from the control (p&#60;0.05) on both resin composites, whereas LTO did not produce differences (p&#62;0.05) on either product. Conclusions: This methodology efficiently and successfully quantified and compared concurrent 3D distributions of three major components within S. mutans biofilms on relevant substrates, thus overcoming two challenges to simultaneous assessment of biofilm components. This method can also be used to determine the efficacy of antibacterial/antifouling agents against multiple biofilm components, as shown using mouthwashes. Furthermore, this method has broad application because it facilitates comparison of 3D structures/architecture of biofilms in a variety of disciplines.

A protocol for the concurrent quantification and comparison of three cellular and extracellular components within biofilms is presented. The methodology involves the use of confocal laser scanning microscopy, biofilm structural analysis and visualization software, and statistical analysis software.

Confocal laser scanning microscopy (CLSM) is a powerful tool for investigation of biofilms. Very few investigations have successfully quantified ...

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 ...

Measuring Influenza Neuraminidase Inhibition Antibody Titers by Enzyme-linked Lectin Assay

Antibodies to neuraminidase (NA), the second most abundant surface protein on influenza virus, contribute toward protection against influenza. Traditional methods to measure NA inhibiting (NI) antibody titers are not practical for routine serology. This protocol describes the enzyme-linked lectin assay (ELLA), a practical alternative method to measure NI titers that is performed in 96 well plates coated with a large glycoprotein substrate, fetuin. NA cleaves terminal sialic acids from fetuin, exposing the penultimate sugar, galactose. Peanut agglutinin (PNA) is a lectin with specificity for galactose and therefore the extent of desialylation can be quantified using a PNA-horseradish peroxidase conjugate, followed by addition of a chromogenic peroxidase substrate. The optical density that is measured is proportional to NA activity. To measure NI antibody titers, serial dilutions of sera are incubated at 37 &#176;C O/N on fetuin-coated plates with a fixed amount of NA. The reciprocal of the highest serum dilution that results in &#8805;50% inhibition of NA activity is designated as the NI antibody titer. The ELLA provides a practical format for routine evaluation of human antibody responses following influenza infection or vaccination.

We describe the enzyme-linked lectin assay (ELLA) for measuring influenza neuraminidase (NA)-inhibition antibody titers in sera. The assay uses peanut agglutinin to quantify galactose residues that become accessible when NA removes sialic acid from fetuin-coated, 96-well plates.

Antibodies to neuraminidase (NA), the second most abundant surface protein on influenza virus, contribute toward protection against influenza. Traditional ...

Isolation of Salmonella typhimurium-containing Phagosomes from Macrophages

Salmonella typhimurium is a facultative intracellular bacterium that causes gastroenteritis in humans. After invasion of the lamina propria, S. typhimurium bacteria are quickly detected and phagocytized by macrophages, and contained in vesicles known as phagosomes in order to be degraded. Isolation of S. typhimurium-containing phagosomes have been widely used to study how S. typhimurium infection alters the process of phagosome maturation to prevent bacterial degradation. Classically, the isolation of bacteria-containing phagosomes has been performed by sucrose gradient centrifugation. However, this process is time-consuming, and requires specialized equipment and a certain degree of dexterity. Described here is a simple and quick method for the isolation of S. typhimurium-containing phagosomes from macrophages by coating the bacteria with biotin-streptavidin-conjugated magnetic beads. Phagosomes obtained by this method can be suspended in any buffer of choice, allowing the utilization of isolated phagosomes for a broad range of assays, such as protein, metabolite, and lipid analysis. In summary, this method for the isolation of S. typhimurium-containing phagosomes is specific, efficient, rapid, requires minimum equipment, and is more versatile than the classical method of isolation by sucrose gradient-ultracentrifugation.

Isolation of Salmonella typhimurium-containing Phagosomes from Macrophages

We describe here a simple and quick method for the isolation of Salmonella typhimurium-containing phagosomes from macrophages by coating the bacteria with biotin and streptavidin.

Salmonella typhimurium is a facultative intracellular bacterium that causes gastroenteritis in humans. After invasion of the lamina propria, S. ...

Development and Validation of an Ultrasensitive Single Molecule Array Digital Enzyme-linked Immunosorbent Assay for Human Interferon-&#945;

The main aim of this protocol is to describe the development and validation of an interferon (IFN)-&#945; single molecule array digital Enzyme-Linked ImmunoSorbent Assay (ELISA) assay. This system enables the quantification of human IFN-&#945; protein with unprecedented sensitivity, and with no cross-reactivity for other species of IFN.
The first key step of the protocol is the choice of the antibody pair, followed by the conjugation of the capture antibody to paramagnetic beads, and biotinylation of the detection antibody. Following this step, different parameters such as assay configuration, detector antibody concentration, and buffer composition can be modified until optimum sensitivity is achieved. Finally, specificity and reproducibility of the method are assessed to ensure confidence in the results. Here, we developed an IFN-&#945; single molecule array assay with a limit of detection of 0.69 fg/mL using high-affinity autoantibodies isolated from patients with biallelic mutations in the autoimmune regulator (AIRE) protein causing autoimmune polyendocrinopathy syndrome type 1/autoimmune polyendocrinopathy-candidiasis-ectodermal dystrophy (APS1/APECED). Importantly, these antibodies enabled detection of all 13 IFN-&#945; subtypes.
This new methodology allows the detection and quantification of IFN-&#945; protein in human biological samples at attomolar concentrations for the first time. Such a tool will be highly useful in monitoring the levels of this cytokine in human health and disease states, most particularly infection, autoimmunity, and autoinflammation.

Here we present a protocol to describe the development and validation of a single molecule array digital ELISA assay, which enables the ultra-sensitive detection of all IFN-&#945; subtypes in human samples.

The main aim of this protocol is to describe the development and validation of an interferon (IFN)-&#945; single molecule array digital Enzyme-Linked ...

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

Antibodies, also termed as immunoglobulins (Ig), secreted by differentiated B lymphocytes, plasmablasts/plasma cells, in humoral immunity provide a formidable defense against invading pathogens via diverse mechanisms. One major goal of vaccination is to induce protective antigen-specific antibodies to prevent life-threatening infections. Both thymus-dependent (TD) and thymus-independent (TI) antigens can elicit robust antigen-specific IgM responses and can also induce the production of isotype-switched antibodies (IgG, IgA and IgE) as well as the generation of memory B cells with the help provided by antigen presenting cells (APCs). Here, we describe a protocol to characterize TD and TI Ig isotype responses in mice using enzyme-linked immunosorbent assay (ELISA). In this protocol, TD and TI Ig responses are elicited in mice by intraperitoneal (i.p.) immunization with hapten-conjugated model antigens TNP-KLH (in alum) and TNP-polysaccharide (in PBS), respectively. To induce TD memory response, a booster immunization of TNP-KLH in alum is given at 3 weeks after the first immunization with the same antigen/adjuvant. Mouse sera are harvested at different time points before and after immunization. Total serum Ig levels and TNP-specific antibodies are subsequently quantified using Ig isotype-specific Sandwich and indirect ELISA, respectively. In order to correctly quantify the serum concentration of each Ig isotype, the samples need to be appropriately diluted to fit within the linear range of the standard curves. Using this protocol, we have consistently obtained reliable results with high specificity and sensitivity. When used in combination with other complementary methods such as flow cytometry, in vitro culture of splenic B cells and immunohistochemical staining (IHC), this protocol will allow researchers to gain a comprehensive understanding of antibody responses in a given experimental setting.

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.

Antibodies, also termed as immunoglobulins (Ig), secreted by differentiated B lymphocytes, plasmablasts/plasma cells, in humoral immunity provide a ...

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 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, ...

Evaluation of Host-Pathogen Responses and Vaccine Efficacy in Mice

Vaccines are a 20th century medical marvel. They have dramatically reduced the morbidity and mortality caused by infectious diseases and contributed to a striking increase in life expectancy around the globe. Nonetheless, determining vaccine efficacy remains a challenge. Emerging evidence suggests that the current acellular vaccine (aPV) for Bordetella pertussis (B. pertussis) induces suboptimal immunity. Therefore, a major challenge is designing a next-generation vaccine that induces protective immunity without the adverse side effects of a whole-cell vaccine (wPV). Here we describe a protocol that we used to test the efficacy of a promising, novel adjuvant that skews immune responses to a protective Th1/Th17 phenotype and promotes a better clearance of a B. pertussis challenge from the murine respiratory tract. This article describes the protocol for mouse immunization, bacterial inoculation, tissue harvesting, and analysis of immune responses. Using this method, within our model, we have successfully elucidated crucial mechanisms elicited by a promising, next-generation acellular pertussis vaccine. This method can be applied to any infectious disease model in order to determine vaccine efficacy.

Here we present an elegant protocol for in vivo evaluation of vaccine effectiveness and host immune responses. This protocol can be adapted for vaccine models that study viral, bacterial, or parasitic pathogens.

Vaccines are a 20th century medical marvel. They have dramatically reduced the morbidity and mortality caused by infectious diseases and contributed to a ...

Opsonophagocytic Killing Assay to Assess Immunological Responses Against Bacterial Pathogens

A key aspect of the immune response to bacterial colonization of the host is phagocytosis. An opsonophagocytic killing assay (OPKA) is an experimental procedure in which phagocytic cells are co-cultured with bacterial units. The immune cells will phagocytose and kill the bacterial cultures in a complement-dependent manner. The efficiency of the immune-mediated cell killing is dependent on a number of factors and can be used to determine how different bacterial cultures compare with regard to resistance to cell death. In this way, the efficacy of potential immune-based therapeutics can be assessed against specific bacterial strains and/or serotypes. In this protocol, we describe a simplified OPKA that utilizes basic culture conditions and cell counting to determine bacterial cell viability after co-culture with treatment conditions and HL-60 immune cells. This method has been successfully utilized with a number of different pneumococcal serotypes, capsular and acapsular strains, and other bacterial species. The advantages of this OPKA protocol are its simplicity, versatility (as this assay is not limited to antibody treatments as opsonins), and minimization of time and reagents to assess basic experimental groups.

This opsonophagocytic killing assay is used to compare the ability of phagocytic immune cells to respond to and kill bacteria based on different treatments and/or conditions. Classically, this assay serves as the gold&#160;standard for assessing effector functions of antibodies raised against a bacterium as opsonin.

A key aspect of the immune response to bacterial colonization of the host is phagocytosis. An opsonophagocytic killing assay (OPKA) is an experimental ...