Name: application of long-term cultured interferon-&#947; enzyme-linked immunospot assay for assessing effector and memory t cell responses in cattle
Uploaded: 2015-07-11T13:00:00
Duration: 15 min 57 s
Description: Watch this Scientific Journal Video about Application of Long-term cultured Interferon-Î³ Enzyme-linked Immunospot Assay for Assessing Effector and Memory T Cell Responses in Cattle at JoVE.com

Research was supported by USDA, ARS Cris: 3625-32000-104 and Agriculture and Food Research Initiative Competitive Grant no. 2011-67015-30736 from the USDA National Institute of Food and Agriculture. We thank Jessica Pollock, Emma Frimml-Morgan, Shelly Zimmerman, Kristin Bass, Bruce Pesch, Molly Stafne, Allen Jensen, and Tracy Porter for their excellent technical assistance as well as Rebecca Madison, Doug Ewing, Katie Pille, Jay Steffen, David Lubbers, Robin Zeisness, and David Panthen for the excellent care and handling of animals.

1. Prepare the Following Solutions
<ol>
	<li>Prepare complete RPMI (cRMPI) by adding fetal bovine serum (FBS) to a concentration of 10 % (volume / volume), glutamine (2 &#181;M), sodium pyruvate (1 &#181;M), non-essential amino acids (0.1 &#181;M), penicillin&#8211;streptomycin (100 units / ml penicillin and 0.1 mg / ml streptomycin) and 2-mercaptoethanol (50 mM) into RPMI 1640.</li>
	<li>Prepare 2 X acid citrate dextrose, by mixing sodium citrate (77 &#181;M), citric acid (38&#160;&#181;M), and dextrose (122&#160;&#181;M), in distilled water.</li>
	<li>Prepare Ca2+ Mg2+ free phosphate buffered saline (PBS) pH 7.2: NaCl (137 mM), KCl (2.7 mM), Na2HPO4 (dibasic, 10 mM), KH2PO4 (monobasic, 2 mM) in distilled water; adjust the pH to 7.2.</li>
	<li>Prepare PBS 1% (PBS 1 % BSA) by adding bovine serum albumin into PBS (volume / weight, 1g / 100 ml&#160;of PBS).</li>
	<li>Prepare PBS + 0.05 % tween 20 (PBST) by adding 0.05 % tween 20 into PBS (volume / volume, 500 &#956;l of tween 20 / 1 L of PBS).</li>
	<li>Prepare 100 mM Tris HCL pH 8.2 buffer by mixing Tris (100 mM), HCl (0.50 mM) in distilled water.</li>
	<li>Prepare 35 % ethanol solution by diluting ethyl alcohol (purity &#8805;99.5%) in distilled water (volume / volume, 35 ml of ethyl alcohol / 100 ml of distilled water).</li>
	<li>Filter sterilize cRPMI, 2 x acid citrate dextrose, PBS, PBS 1 % BSA using 0.22 &#956;m filters.</li>
</ol>
2. Long-term Cultured Cells (14 Day Protocol)
(Day one)
<ol>
	<li>Prepare antigen solutions at twice the final concentration in cRPMI. Antigen choice is critical and should be tailored to the purpose of the study.</li>
	<li>Dilute recombinant M. tuberculosis antigens TB10.4 and antigen Ag85A to 2 &#956;g / ml (of each antigen; the final concentration is 1 &#956;g / ml).</li>
	<li>Dilute M. bovis purified protein derivative (PPD-B, Prionics Ag) to 10 &#956;g / ml (the final concentration is 5 &#956;g / ml).</li>
	<li>Dilute the recombinant early secretory antigenic target 6 (ESAT-6) and culture filtrate protein 10 (CFP-10) fusion protein (rESAT-6:CFP10) to 2 &#956;g / ml (final concentration of 1 &#956;g / ml). 
	NOTE: These antigens are immunodominant IFN-&#947; inducing antigens of tuberculous mycobacteria and may be included to stimulate PBMC from M. bovis infected animals. BCG or M. bovis &#916;RD1 vaccinated animals do not respond to rESAT-6:CFP10, as the region encoding these antigens (i.e., RD1) is deleted in these strains. rESAT-6:CFP10 used in these study was a kind gift from Dr. C. Minion, Iowa State University. TB10.4 and antigen Ag85A are immunodominant antigens present in virulent and vaccine M. bovis strains 37. PPDB, as a purified protein derivative, is a complex of several antigens including antigens shared with non-tuberculous mycobacteria. Infected animals will potentially respond to all antigens (namely: TB10.4, Ag 85A, rESAT-6:CFP10 and PPDb), while vaccinated animals should not respond to rESAT-6:CFP10 11.</li>
	<li>Plate 500 &#956;l / well of antigen solution in quadruplicates for each animal in a 24 well plate. For this protocol, use antigens as a pool; thus use all wells contain all the antigens and no controls (such as, null or mitogen) this step. Incubate plate at 39 &#176;C / 5 % CO2. The normal temperature of cattle is 39 &#176;C; thus, some investigators utilize 39 &#176;C for culture of bovine cells. Also, in certain instances with human cells, cell culture at 39 &#176;C provides added benefit26,30.</li>
	<li>Pre-load syringes coupled with 16 or 18 G&#160;hypodermic needles with 6 ml of 2 X acid-citrate-dextrose; and collect 60 ml of bovine blood by jugular venipuncture.</li>
	<li>Isolate PBMC by standard density gradient centrifugation of the peripheral blood buffy coat fractions adjusting cell concentration to 4 X 106 cells / ml as described by Maue et al. 27</li>
	<li>Add cells (500 &#956;l / well) into antigen preloaded 24 well plate, in quadruplicate for each animal (step 2.5). Incubate plate at 39 &#176;C / 5 % CO2.</li>
</ol>
(Days three and seven)
<ol start="9">
	<li>Using sterile technique, carefully remove 500 &#956;l of the supernatant from each well without disturbing the cell layer.</li>
	<li>Replenish well volume by adding 500 &#956;l / well of cRPMI containing IL-2 (30 U / &#956;l, i.e., 15 U / well; recombinant human IL-2 Sigma I7908). Incubate plate at 39 &#176;C / 5 % CO2.</li>
</ol>
(Days 10 and 12)
<ol start="11">
	<li>Using sterile technique, remove 750 &#956;l of supernatant from each well. Replenish the volume with 750 &#956;l / well of cRPMI ( without IL-2). Incubate plate at 39 &#176;C / 5 % CO2.</li>
</ol>
3. ELISPOT Assay (Three Day Protocol)
(Day one (12 th day of Long-term culture protocol))
<ol>
	<li>Label the ELISPOT plate properly for each set of samples, to avoid mistakes when plating cells: long-term cells plus antigen presenting cells (APCs), long-term cells without APCs and ex vivo / short-term cells (Figure 1). Replicates for each treatment (i.e. antigenic stimulation) should be done at least in duplicate.</li>
	<li>Prepare capture antibody solution by diluting mouse anti-bovine IFN-&#947; antibody (MCA2112, clone CC330) in PBS (8 &#956;g / ml).</li>
	<li>Using a multichannel pipettor pre-wet the wells of the ELISPOT plate with 15 &#956;l / well of 35 % ethanol for 1 min. To prevent membrane damage, do not touch the bottom of wells with pipette tips at any point during the assay.</li>
	<li>Wash plate six times with 300 &#956;l / well of PBS. Wash fluid should be discarded by plate inversion. Washes should be done rapidly, not allowing plate wells to dry.</li>
	<li>Pipette 100 &#956;l / well of capture anti-IFN-&#947; antibody (from step 1 above). Incubate at 4 &#176;C O/N (keep the plate inside a zippered storage bag).</li>
</ol>
(Day two (13 th day of the long-term culture protocol))
<ol start="6">
	<li>Prepare antigen solutions at twice the final concentration. Treatments are: no stimulation (cRPMI), PPD-B (20 &#956;g / ml, final concentration: 10 &#956;g / ml), protein cocktail of TB10.4 and Ag85A (2 &#956;g / ml of each protein, final concentration: 1 &#956;g / ml of each protein), rESAT-6:CFP10 (for M. bovis infection, 2 &#181;g / ml, final concentration: 1 &#956;g / ml), and a positive control, such as pokeweed (20 &#956;g / ml, final concentration: 10 &#956;g / ml). Other mitogen may be used instead of PWM (e.g., Concanavalin A). 
	NOTE: for this step antigens compose four different treatments and are not used as a pool (as in step 2.5). The four treatments are: NS, PPDb, protein cocktail (TB10.4 and Ag85A constitute one treatment) and PWM.</li>
</ol>
4. Short-term Cell Culture and Adherent Cell (APCs) Isolation
<ol>
	<li>Collect 60 ml blood from the same animals bled on day 1 (under: long-term culture, 14 day protocol) and isolate PBMCs as before.</li>
	<li>Adjust cell concentration to 2 X 106 cells / ml. Label tubes clearly to prevent misallocation when plating cells. These cells will be used for APCs isolation and also as short-term cell culture (step 6.5). Label tubes with both the animal number and the type of culture (in this case: ex vivo, short-term or fresh cells).</li>
	<li>Remove excess capture antibody from the ELISPOT by plate inversion. Wash plates 6 times with 300 &#956;l PBST.</li>
	<li>Remove as much PBST as possible. Pipette 50 &#956;l of cell suspensions to the wells labeled as long-term cells plus APCs. Block other wells with 200 &#956;l / well of cRPMI. Incubate 90 min at or 39 &#176;C. Pre-warm cRPMI to or 39 &#176;C (necessary for subsequent steps). Fresh PBMCs not used for adherent cell (APCs) isolation will be needed in subsequent steps and should be stored.</li>
</ol>
5. Cultured Cells
<ol>
	<li>During the 90 min incubation (step 4.4, short-term culture and adherent cell isolation step), harvest long-term cultured cells.</li>
	<li>Using 5 or 10 &#956;l pipettes, pipette media with cells up and down to detach cells, combine the quadruplicate replicates from each animal into a single 15 ml tube. Centrifuge the tubes for 5 min at 400 g.</li>
	<li>After centrifugation, discard the supernatant by tube inversion. Dislodge cell pellet. Add 5 ml of PBS, gently re-suspend pellet, if necessary. Repeat the centrifugation. Repeat the washing step twice. Discard supernatant by tube inversion and gently re-suspend cells in 1ml cRPMI. Adjust cell concentration to 2 X 105 cells / ml.</li>
</ol>
6. Plating Fresh and Cultured PBMCs
<ol>
	<li>After the 90 min incubation, shake plates on plate shaker for 30 sec. Remove non-adherent cells by plate inversion. Pipette 150 &#956;l / well of warm cRPMI (from step 4, under: Short-term cells and adherent cell (APCs) isolation step).</li>
	<li>Shake plates and discard wash fluid. Repeat wash 3 times. Add 100 &#956;l / well of each antigen into appropriate wells (pre-labeled).</li>
	<li>To plate cells, pipette 100 &#956;l / well of long-term cultured cell suspension (2 x 105 cells / ml) into the wells labeled as long-term cells plus APCs. Ensure that the long-term cells added in this step are from the same animal as the APC&#8217;s already in the well. Do not mix APC&#8217;s and long-term cultured cells from different animals.</li>
	<li>Pipette 100 &#956;l / well of the long-term cultured cell suspension (2 x 105 cells / ml) into wells without APCs for assessment of APC requirement to the long-term culture response.</li>
	<li>Add 100 &#956;l / well of short-term cells (freshly isolated and adjusted to 2 x 106 cells / ml) for ex vivo response assessment. Incubate plates O/N at 39 &#176;C / 5 % CO2 incubator. Ensure that plates are lying flat and do not stack plates. 
	NOTE: If analysis of cell phenotype is desired, flow cytometry can be performed as an ancillary assay. Cells are plated in 96 well U bottom plates (instead of ELISPOT plates) as described for ELISPOT assay. Capture antibody adsorption (steps 3.4 to 3.5) should be skipped. Cells are then incubated O/N at 39 &#176;C / 5 % CO2 and a standard cell staining protocol performed. Cell staining reagents are included in the reagents table.</li>
</ol>
Day three (14 th day of the Long-term culture protocol)
<ol start="6">
	<li>Dilute detection antibody (mouse anti-bovine IFN-&#947;, clone CC302) to 5 &#956;g / ml in PBS 1 % BSA. Discard fluids from wells and wash plates: six times with PBST (300 &#956;l / well), placing on shaker each time for 10 sec before and in-between washes, once with dH2O (300 &#956;l / well), an additional 6 times with PBST (300 &#956;l / well) and two times with PBS (300 &#956;l / well). After cells are removed from the plates, and if no biosafety concerns apply, procedures may be performed outside biological safety cabinets.</li>
	<li>Discard wash fluid. Remove as much wash fluid as possible by tapping plate on paper towels. Add detection antibody (100 &#956;l / well).</li>
	<li>Incubate plates for 120 min at or 39 &#176;C. During this incubation step, prepare alkaline phosphatase solution following the manufacturer&#39;s instructions. Thirty min before use (i.e., 90 min after addition of detection antibody to wells) dilute the reagent in PBST. Invert the tube gently to mix reagents and incubated at RT.</li>
	<li>After the 120 min incubation, discard excess detection antibody by plate inversion. Wash plate 6 times with PBST (300 &#956;l / well). Remove as much wash fluid as possible by tapping plate on paper towels.</li>
	<li>Add 100 &#956;l / well of alkaline phosphatase solution (from step 3 above). Incubate plates for 45 min at RT. Discard fluid and wash each plate 6 times with PBST (300 &#956;l / well).</li>
	<li>Prepare the substrate solution following the manufacturers&#39; instructions (Vector Blue AP substrate III): Dilute reagents in 100 mM Tris HCL pH 8.2 buffer. Pipette 50 &#956;l / well of the substrate solution.</li>
	<li>Incubate at RT until blue color starts to develop (approximately 30 min). Discard fluid and wash plates with copious amounts of dH2O. Remove bottom plate to expose membrane, wash back of wells and allow plate to air dry.</li>
	<li>Read plates on immunospot image analyzer or ELISPOT reader (Table 1), or manually, using a stereomicroscope. Alternatively, keep plates in dark at RT until reading. Because of the high stability of the reaction substrate, quality is preserved for several years. 
	NOTE: Cells and antigen concentrations were optimized to avoid wells with uncountable spots 23,24,27,37. It is possible that uncountable spots formation occur in different settings or due to biological variation. If that is the case, cell concentration should be optimized so a readable spot counting response is obtained.</li>
</ol>
7. Plates Reading and Data Analysis
<ol>
	<li>Perform analysis as per the Cellular Technology Limited (CTL) ELISPOT plate reader guide (Table 1). After the spots counts are obtained for each of the wells, calculate the average between duplicates. The specific immune response to each antigenic stimulus is calculated by subtracting the average number of spots in non-stimulated wells from that of antigen-stimulated wells.</li>
</ol>

The authors declare that they have no competing financial interests.

IFN-&#947; production in long-term cultured ELISPOT assays is generally due to Tcm in humans, but how this response develops in culture is poorly understood. Whether Tcm are present in circulation and expand in vitro, or if the Tcm responses result from differentiation of effector and Tem cells into Tcm during culture is not known. However, studies with samples from humans have found that CD4+ T cells from ex vivo and long-term cultured IFN-&#947; ELISPOT assays have unrelated epitope specificities, implying that Tcm responses do not develop from effector cells28. Obviously, the duration of the response may vary, but if pathogen clearance is achieved, eventually both ex vivo and Tcm responses decline. Also, responses of long-term cultures measured early and long after antigenic priming (when the effector response is lower) is shown to correlate, indicating that circulating Tcm populations early and long after antigenic priming remains related in vivo. On the other hand, the magnitude of the ex vivo response does not appear to be related to the magnitude of the memory response29. These results suggest that long-term cultured IFN-&#947; ELISPOT responses result from in vitro expansion of Tcm and an associated waning of effector responses in the sample, rather than the differentiation of effector cells into Tcm phenotype.
With cattle, the relative contribution of effector and memory subsets to responses detected by the long-term IFN-&#947; ELISPOT assays is not known. Recent studies indicate a correlation between vaccine elicited long-term cultured IFN-&#947; ELISPOT responses and protection against subsequent experimental infection with M. bovis23-24. It has been reported that weak long-term IFN-&#947; ELISPOT responses to vaccination are associated with the absence of protection30. Both live and killed vaccines induce similar ex vivo IFN-&#947; ELISPOT responses, but vaccination with live BCG elicits stronger long-term cultured IFN-&#947; ELISPOT responses than do killed vaccine preparations. Interestingly, live vaccines are protective while killed formulations fail to provide protection against challenge with virulent tuberculous mycobacteria 31-32, 33.
Assessment of Tcm responses is also feasible by sorting these cells from PBMC. Direct enrichment of Tcm from PBMC, however, requires expensive devices, highly trained personal and is difficult as these cells are not numerous in the blood stream. Long-term culture of PBMC provides enrichment of Tcm over Tem and effector cells without expensive devices; however, because these T cell populations are expanded in vitro they may be less representative of in vivo memory responses. It is possible to access IFN-&#947; memory responses (using the long-term culture or Tcm sorting strategies) by assays other than the ELISPOT such as: cytokine ELISAs34, cytokine bead arrays (CBA), intracellular staining (ICS), or cytokine protein arrays (CPA). These methods; however, are generally less sensitive than ELISPOT assays35.
An advantage of ELISPOT is its ability to detect the immediate capture of the cytokine shortly after its release preventing dilution in the supernatant and degradation by enzymatic cleavage or cytokine uptake by other cells. ELISPOT assays detect single cells producing cytokines providing precise results even in low signal to noise scenarios (i.e., low specific responses)35. Also, with ICS, detection of cytokines prior to release may result in false identification of cells producing cytokines (e.g., due to post-translational modulation before or during the secretory process)34. The transport inhibitors employed with ICS assays to minimize cytokine secretion during antigen stimulation (known as Golgi stop proteins) limit the duration of cell stimulation due to the cell toxicity of these proteins ultimately impacting cytokine production35.
With that said - CBA, CPA and ICS are useful techniques for measuring multiple cytokines simultaneously and / or for determining cell surface marker expression (i.e., with ICS). Therefore, these methods may be used in combination with the IFN-&#947; ELISPOT assay36. While previous bovine TB vaccine efficacy studies measured Tcm responses via ELISPOT assay, the detection of Tcm responses by other techniques will likely yield comparable results. Importantly, the ELISPOT assay is less expensive and simpler to perform than CBA, CPA and ICS analysis techniques34. Manual counting of the SFC is an alternative to automated counting, and ELISPOT plates can be stored at RT for long period of time with minimal quality loss, before or after spot counting37.
The long-term cultured IFN-&#947; ELISPOT response has been applied to assess memory responses by human, and cattle when evaluating tuberculosis vaccine responses14-16,31-32,33. Tcm responses are also assumed to play significant roles in host responses to several other infectious agents of cattle, such as: Mycoplasma mycoides subsp. mycoides42, Anaplasma marginale38 and bovine respiratory syncytial virus39. Potentially, the long-term ELISPOT assay might be adapted for other animal species, cytokines and infections. These broader applications will be especially useful for veterinary immunology applications due to current limited availability of specific reagents.
Tcm responses are crucial for protection against several infections, but measuring them early after infection / immunization (for disease outcome prediction or vaccine efficacy) may be cumbersome. Analysis of the immune response under ex vivo and short antigenic stimulation conditions will more often represent effector responses, due to the overlap of memory and effector responses, especially during the beginning of immunological memory formation. In the context of chronic diseases in which the antigen load is continual, ex vivo responses will assess effector cell responses or a combination of memory and effector cell responses. The long-term cultured IFN-&#947; ELISPOT assay, described here, likely enables the measurement of Tcm responses rather than effector T cell or combined CD4+ T cell responses. In summary, the long-term cultured IFN-&#947; ELISPOT assay provides a valuable approach for estimating T cell memory responses and has been employed successfully to evaluate memory responses of several species to a variety of infections agents12-16, 20-25,29,31,33,34,38,39.

Effector and memory T cells are generated through developmental programing of na&#239;ve CD4+ T cells after antigen recognition. Differentiation of na&#239;ve CD4+ T cells into cytokine producing cells requires pathogen recognition by innate immune cells, antigen presentation to T cells, co-stimulation and transcriptional changes resulting in polarized cytokine production. For example: antigen presenting cells produce interleukin (IL)-12 in response to intracellular pathogens, which, together with antigen recognition, promotes differentiation of T cells into T helper 1 (Th1) cells1,2 by signaling through signal transducer and activator of transcription 4 (STAT4) and T-box expressed in T cells (T-bet), leading to cell activation, IL-2 production, clonal expansion and interferon (IFN)-&#947; production3,4. 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 lifetime5. Sallusto et al.6, revealed two functionally distinct subsets of memory T cells in humans based on the expression of lymph node homing receptors. Central memory T cells (Tcm) express C-C chemokine receptor (CCR)-7 and CD45RO and are mainly located in T-cell areas of secondary lymphoid organs. Tcm have limited effector function, a low activation threshold and retain high IL-2 production and proliferative capacity. Effector memory T cells (Tem) express CD45RO, lack CCR7 and display receptors for homing to peripheral or inflamed tissues. Effector cells do not express either CCR7 or CD45RO but promptly produce effector cytokines, such as IFN-&#947;, upon antigen recognition.
IFN-&#947; release assays (IGRA) are used for the diagnosis of bovine and human tuberculosis7. Mycobacterium bovis is the principal agent of bovine tuberculosis (bTB) while human cases of tuberculosis are caused mainly by Mycobacterium tuberculosis. With these tests, whole blood or peripheral blood mononuclear cells (PBMC) are stimulated with mycobacterial antigens for 16 to 24 hr and IFN-&#947; production within the supernatant is measured by ELISA or through detection of cells producing IFN-&#947; using ELISPOT techniques. As a result of the brief stimulation period (i.e., 16 to 24 h) and rapid cytokine production, ex vivo assays detect primarily effector and Tem responses. This has been confirmed by flow cytometric analysis of cell populations in these cultures 8-10.
Ex vivo IFN-&#947; responses are routinely included within the immune response panel evaluation of tuberculosis vaccines, including those used to evaluate responses by cattle. Most effective bovine tuberculosis vaccines elicit specific IFN-&#947; responses, but not all vaccines that induce IFN-&#947; responses are protective. Also, levels of IFN-&#947; elicited by vaccination, as measured before infection, do not necessarily correlate with protection. For instance, different BCG strains may have different capacities to induce ex vivo IFN-&#947; response, in spite of similar protection levels11. Thus, ex vivo IGRAs are valuable for tuberculosis diagnosis and for accessing vaccine immunogenicity; however, their use as predictors of vaccine efficacy is limited. Tcm responses to vaccination, on the other hand, may be used to predict vaccine efficacy, as demonstrated with simian immunodeficiency virus (SIV) infection in non-human primates12,13, tuberculosis in mice14 and malaria in humans15,16.
After an effective immune response resulting in pathogen clearance, Tcm are maintained and provide protection to an eventual second infection by the same agent. A notable exception to this scenario is M. tuberculosis infection of humans in which patients receiving curative anti-mycobacterial therapy are susceptible to re-infection17,18. Additionally, events governing immunological memory during chronic infections, wherein the antigenic stimulation persists, are not well understood19. During chronic infections, such as with human immunodeficiency virus (HIV) and tuberculosis, a significant Tcm response is associated with a favorable outcome (e.g., latency with tuberculosis and subclinical disease with HIV)20,21. Several studies with mice and humans have demonstrated that long-term cultured IFN-&#947; ELISPOT assays measure Tcm responses16,20-22. With this assay, PBMCs are cultured in decreasing concentration of antigen for 10 to 14 days, allowing effector responses to peak and wane; facilitating Tcm to differentiate and expand within the culture.
Long-term cultured IFN-&#947; ELISPOT assays have also been used in veterinary research, yet the phenotype of responding cells has been difficult to assess due to a lack of critical reagents, especially an antibody to CCR7. Effective bovine tuberculosis vaccines [e.g. using a single dose of M. bovis Bacille Calmette Guerin (BCG), BCG followed by viral-vectored Antigen 85A subunit vaccine, or attenuated M. bovis&#916;RD1] elicit long-term cultured IFN-&#947; ELISPOT responses following vaccination that correlate with protection (i.e., lower mycobacterial burden and decreased TB-associated pathology) against subsequent challenge with virulent M. bovis23,24. Furthermore, the numbers of antigen-specific IFN-&#947;-secreting cells within long-term PBMC cultures are higher at 12 months but decrease at 24 months after BCG vaccination of neonatal calves, correlating with the degree of protection detectable post M. bovis challenge25. In this scenario, the cultured IFN-&#947; ELISPOT is an important tool for predicting vaccine efficacy, providing a means to prioritize vaccine candidates for high cost efficacy trials. Additionally, cultured ELISPOT techniques can be adapted for various hosts, pathogens and cytokines by altering antigens or antibodies for a variety of purposes in different research fields.

<table border="0" cellpadding="0" cellspacing="0">
	<tbody>
		<tr>
			<td rowspan="2">Sodium citrate (dihydrate)</td>
			<td rowspan="2">Various</td>
			<td rowspan="2"></td>
			<td rowspan="2"></td>
		</tr>
		<tr>
		</tr>
		<tr>
			<td rowspan="2">Citric acid (monohydrate)</td>
			<td rowspan="2">Various</td>
			<td rowspan="2"></td>
			<td rowspan="2"></td>
		</tr>
		<tr>
		</tr>
		<tr>
			<td rowspan="2">Dextrose</td>
			<td rowspan="2">Various</td>
			<td rowspan="2"></td>
			<td rowspan="2"></td>
		</tr>
		<tr>
		</tr>
		<tr>
			<td>ELISPOT PVDF plate</td>
			<td>Various</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Vectastain ABC &#8211; AP KIT Standard</td>
			<td>Vector Laboratories</td>
			<td>AK-5000</td>
			<td></td>
		</tr>
		<tr>
			<td>Vector Blue Alkaline Phosphatase Substrate Kit</td>
			<td>Vector Laboratories</td>
			<td>SK-5300</td>
			<td></td>
		</tr>
		<tr>
			<td>Ag85A</td>
			<td>Lionex</td>
			<td>LRP-0004.3</td>
			<td>23, 24, 25, 37</td>
		</tr>
		<tr>
			<td>TB 10.4</td>
			<td>Lionex</td>
			<td>LRP-0061.6</td>
			<td>23, 24, 25, 37</td>
		</tr>
		<tr>
			<td>PPDb</td>
			<td>Prionics AG</td>
			<td>7600055</td>
			<td></td>
		</tr>
		<tr>
			<td>rESAT-6:CFP10</td>
			<td>Kind gift Dr. Minion, Iastate</td>
			<td></td>
			<td>23, 24, 25, 27,&#160; 37</td>
		</tr>
		<tr>
			<td>Mouse anti-bovine IFN-&#947; Clone CC302</td>
			<td>Serotec</td>
			<td>MCA1783B</td>
			<td>23, 24, 25, 27,&#160; 37</td>
		</tr>
		<tr>
			<td>Mouse anti-bovine IFN-&#947; Biotinylated Clone CC330</td>
			<td>Serotec</td>
			<td>MCA2112</td>
			<td>23, 24, 25, 27,&#160; 37</td>
		</tr>
		<tr>
			<td>Mouse anti-bovine CD4 Clone CC8</td>
			<td>Serotec</td>
			<td>MCA1653GA</td>
			<td></td>
		</tr>
		<tr>
			<td>Mouse anti-bovine CD45RO Clone IL116A</td>
			<td>Serotec</td>
			<td>MCA2434GA</td>
			<td></td>
		</tr>
		<tr>
			<td>Rat anti-human CCR7 Clone 3D12</td>
			<td>Abcam</td>
			<td>ab95665</td>
			<td></td>
		</tr>
		<tr>
			<td>Mouse anti-bovine IFN-&#947;-Pe Clone CC302</td>
			<td>Serotec</td>
			<td>MCA1783PE</td>
			<td></td>
		</tr>
		<tr>
			<td>Goat anti- mouse IgG2a- Alexa Fluor 350</td>
			<td>Invitrogen</td>
			<td>A-21130</td>
			<td></td>
		</tr>
		<tr>
			<td>Goat anti-mouse IgG3 APC-CY7</td>
			<td>SouthernBiotechnology</td>
			<td>1080-193</td>
			<td></td>
		</tr>
		<tr>
			<td>Goat anti-rat IgG-APC</td>
			<td>Invitrogen</td>
			<td>A10540</td>
			<td></td>
		</tr>
		<tr>
			<td>BD Cytofix/Cytoperm&#8482;</td>
			<td>BD Biosciences</td>
			<td>554714</td>
			<td></td>
		</tr>
		<tr>
			<td>BrefeldinA</td>
			<td>Sigma-Aldrich</td>
			<td>B7651</td>
			<td></td>
		</tr>
		<tr>
			<td>Pokeweed Mitogen</td>
			<td>Sigma-Aldrich</td>
			<td>L8777</td>
			<td></td>
		</tr>
		<tr>
			<td>Recombinat human Interleukin 2</td>
			<td>Sigma-Aldrich</td>
			<td>I7908</td>
			<td></td>
		</tr>
	</tbody>
</table>

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.

Approximately one-month post-aerosol infection with M. bovis (104 colony-forming units), PBMCs from infected (n=8) and control animals (n=8) were cultured in the presence of antigens and IL-2 for 13 days. Development of Tcm responses after infection was determined using IFN-&#947; ELISPOT assay. Representative Tcm (in the presence or absence of APCs) and exvivo IFN-&#947; ELISPOT responses from infected animals (Three animals) and a non-infected animal (one animal) are shown in Figure 1. A successful long-term IFN-&#947; ELISPOT assay results in cells producing IFN-&#947; (Spot-Forming Cells, SFC) under stimulated conditions and near absence of a response from non-infected animals and under non-stimulated conditions. Also, a strong T cell response should occur in response to PWM. Specific responses to M. bovis were assessed by PPD-B or rESAT-6:CFP10 antigenic stimulation. As shown in Figure 1, robust Tcm and ex vivo responses to PPD-B and rESAT-6:CFP10 were detected from all three M. bovis-infected animals. Minimal to no Tcm and ex vivo responses were detected from the control animal. Also, the presence of APCs was required for optimal Tcm responses by infected animals, as demonstrated by greatly reduced responses by long-term cells cultured in the absence of autologous APCs. IFN-&#947; response by PBMCs from M. bovis infected and non-infected animals are presented in Figure 2. The number of SFCs from each animal was calculated as the average number of SFCs in duplicate samples in response to PPD-B minus the respective response to media alone. Responses were estimated for 106 cells. Responses differed (P &#60; 0.05) based upon infection status, presence or absence of APCs and culture duration (i.e., long-term culture versus ex vivo culture).
<table border="0" cellpadding="0" cellspacing="0">
	<colgroup>
		<col />
		<col />
	</colgroup>
	<tbody>
		<tr>
			<td colspan="2">Acquiring image of plates</td>
		</tr>
		<tr>
			<td>1.</td>
			<td>Turn on machine</td>
		</tr>
		<tr>
			<td>2.</td>
			<td>Open &#8220;Immuno Capture&#8221; Version 6.3 software.</td>
		</tr>
		<tr>
			<td>3.</td>
			<td>Step 1: select plate type.</td>
		</tr>
		<tr>
			<td>4.</td>
			<td>Step 2: load plate.</td>
		</tr>
		<tr>
			<td>5.</td>
			<td>Step 3: select scanning options.</td>
		</tr>
		<tr>
			<td>6.</td>
			<td>Step 4: start scanning.</td>
		</tr>
		<tr>
			<td>7.</td>
			<td>Obtain overview image of plate.</td>
		</tr>
		<tr>
			<td>8.</td>
			<td>Eject plate.</td>
		</tr>
		<tr>
			<td>9.</td>
			<td>Quit &#8220;Immuno Capture&#8221; software.</td>
		</tr>
		<tr>
			<td colspan="2">Counting the spot forming units</td>
		</tr>
		<tr>
			<td>1.</td>
			<td>Open &#8220;Immuno Spot Capture&#8221; Version 5.0 software.</td>
		</tr>
		<tr>
			<td>2.</td>
			<td>Select object type: normal.</td>
		</tr>
		<tr>
			<td>3.</td>
			<td>Select counting module: smart count.</td>
		</tr>
		<tr>
			<td>4.</td>
			<td>Step 1: load plate.</td>
		</tr>
		<tr>
			<td>5.</td>
			<td>Step 2: define counting parameters: test accuracy of spot recognition on wells with different spots.</td>
		</tr>
		<tr>
			<td>6.</td>
			<td>Start auto count.</td>
		</tr>
		<tr>
			<td colspan="2">Quality control</td>
		</tr>
		<tr>
			<td>1.</td>
			<td>Open &#8220;Immunospot Capture&#8221; Version 5.0 software.</td>
		</tr>
		<tr>
			<td>2.</td>
			<td>Select counting module: quality control.</td>
		</tr>
		<tr>
			<td>3.</td>
			<td>Step 1: load plate.</td>
		</tr>
		<tr>
			<td>4.</td>
			<td>Step 2: Analyze highlighted wells individually.</td>
		</tr>
		<tr>
			<td>5.</td>
			<td>Finish quality control.</td>
		</tr>
	</tbody>
</table>
Table 1 - AutoImmun Diagnostika ELISPOT reader image acquisition and cell counting procedure.
<img alt="Figure 1" src="/files/ftp_upload/52833/52833fig1.jpg" /> 
Figure 1.&#160;Image of wells from a long-term cultured IFN-&#947; ELISPOT assay.&#160;Cultured IFN-&#947; ELISPOT assay was performed approximatelyone month after challenge with virulent M. bovis (three animals). Non-infected animals were included as controls (one animal). Long-term cell lines were generated by stimulating PBMC with a cocktail of recombinant Ag85A (1 &#181;g / ml), TB10.4 (1 &#181;g / ml), rESAT-6:CFP10 (1 &#181;g / ml) and PPD-B (5 &#181;g / ml) for 13 days followed by transfer to ELISPOT plates in the presence or absence of autologous APC. Short-term cells consisted of PBMC isolated on day 13 and plated directly into ELISPOT plates. Long-term and short-term cells were stimulated with PPD-B (10 &#181;g / ml), rESAT-6:CFP10 (1 &#181;g / ml), medium alone or pokeweed mitogen (5 &#956;g / ml) for 24 h.
<img alt="Figure 2" src="/files/ftp_upload/52833/52833fig2.jpg" /> 
Figure 2.&#160;Representative results from a long-term cultured IFN-&#947; ELISPOT response to M. bovis purified protein derivative (PPD-B).&#160;Cultured IFN-&#947;ELISPOT assay was performed approximatelyone month after challenge with virulentM. bovis (eight animals). Non-infected animals were included as controls (eight animals Long-term cells were generated by stimulating PBMC with a cocktail of recombinant Ag85A (1 &#181;g / ml), TB10.4 (1 &#181;g / ml), rESAT-6:CFP10 (1 &#181;g / ml) and PPD-B (5 &#181;g / ml) for 13 days followed by transfer to ELISPOT plates in the presence or absence of autologous APCs. Short-term cells consisted of PBMC isolated on day 13 and plated directly into the ELISPOT plate. Long-term and short-term cells were stimulated with PPD-B (10 &#956;g / ml) or medium alone for 24 h. Specific responses from each animal (SFC / 106 cells) are presented as response to PPD-B (average of duplicate samples) minus the response to media alone.

Watch this Scientific Journal Video about Application of Long-term cultured Interferon-Î³ Enzyme-linked Immunospot Assay for Assessing Effector and Memory T Cell Responses in Cattle at JoVE.com

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

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.

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 ...

application-long-term-cultured-interferon-enzyme-linked-immunospot

Research

JoVE Journal

Immunology and Infection

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 ...

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-&amp;#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 ...

The Use of Fluorescent Target Arrays for Assessment of T Cell Responses In vivo

The ability to monitor T cell responses in vivo is important for the development of our understanding of the immune response and the design of immunotherapies. Here we describe the use of fluorescent target array (FTA) technology, which utilizes vital dyes such as carboxyfluorescein succinimidyl ester (CFSE), violet laser excitable dyes (CellTrace Violet: CTV) and red laser excitable dyes (Cell Proliferation Dye eFluor 670: CPD) to combinatorially label mouse lymphocytes into &#62;250 discernable fluorescent cell clusters. Cell clusters within these FTAs can be pulsed with major histocompatibility (MHC) class-I and MHC class-II binding peptides and thereby act as target cells for CD8+ and CD4+ T cells, respectively. These FTA cells remain viable and fully functional, and can therefore be administered into mice to allow assessment of CD8+ T cell-mediated killing of FTA target cells and CD4+ T cell-meditated help of FTA B cell target cells in real time in vivo by flow cytometry. Since &#62;250 target cells can be assessed at once, the technique allows the monitoring of T cell responses against several antigen epitopes at several concentrations and in multiple replicates. As such, the technique can measure T cell responses at both a quantitative (e.g.&#160;the cumulative magnitude of the response) and a qualitative (e.g.&#160;functional avidity and epitope-cross reactivity of the response) level. Herein, we describe how these FTAs are constructed and give an example of how they can be applied to assess T cell responses induced by a recombinant pox virus vaccine.

The Use of Fluorescent Target Arrays for Assessment of T Cell Responses In vivo

The ability to monitor T cell responses in detail in vivo is important for the development of our understanding of the immune response. Here we describe the use of fluorescent target arrays (FTAs) in an in vivo T cell assay that assesses &#62;250 parameters simultaneously by flow cytometry.

The ability to monitor T cell responses in vivo is important for the development of our understanding of the immune response and the design of ...

Induction of Murine Intestinal Inflammation by Adoptive Transfer of Effector CD4+CD45RBhigh T Cells into Immunodeficient Mice

There are many different animal models available for studying the pathogenesis of human inflammatory bowel diseases (IBD), each with its own advantages and disadvantages. We describe here an experimental colitis model that is initiated by adoptive transfer of syngeneic splenic CD4+CD45RBhigh T cells into T and B cell deficient recipient mice. The CD4+CD45RBhigh T cell population that largely consists of na&#239;ve effector cells is capable of inducing chronic intestinal inflammation, closely resembling key aspects of human IBD. This method can be manipulated to study aspects of disease onset and progression. Additionally it can be used to study the function of innate, adaptive, and regulatory immune cell populations, and the role of environmental exposures, i.e., the microbiota, in intestinal inflammation. In this article we illustrate the methodology for inducing colitis with a step-by-step protocol. This includes a video demonstration of key technical aspects required to successfully develop this murine model of experimental colitis for research purposes.

Induction of Murine Intestinal Inflammation by Adoptive Transfer of Effector CD4+CD45RBhigh T Cells into Immunodeficient Mice

Here, we present a protocol to induce colonic inflammation in mice by adoptive transfer of syngeneic CD4+CD45RBhigh T cells into T and B cell deficient recipients. Clinical and histopathological features mimic human inflammatory bowel diseases. This method allows the study of the initiation of colonic inflammation and progression of disease.

There are many different animal models available for studying the pathogenesis of human inflammatory bowel diseases (IBD), each with its own advantages ...

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 ...

An IL-8 Transiently Transgenized Mouse Model for the In Vivo Long-term Monitoring of Inflammatory Responses

Airway inflammation is often associated with bacterial infections and represents a major determinant of lung disease. The in vivo determination of the pro-inflammatory capabilities of various factors is challenging and requires terminal procedures, such as bronchoalveolar lavage and the removal of lungs for in situ analysis, precluding longitudinal visualization in the same mouse. Here, lung inflammation is induced through the intratracheal instillation of Pseudomonas aeruginosa culture supernatant (SN) in transiently transgenized mice expressing the luciferase reporter gene under the control of a heterologous IL-8 bovine promoter. Luciferase expression in the lung is monitored by in vivo bioluminescent image (BLI) analysis over a 2.5- to 48-h timeframe following the instillation. The procedure can be repeated multiple times within 2 - 3 months, thus permitting the evaluation of the inflammatory response in the same mice without the need to terminate the animals. This approach permits the monitoring of pro- and anti-inflammatory factors acting in the lung in real time and appears suitable for functional and pharmacological studies.

An IL-8 Transiently Transgenized Mouse Model for the In Vivo Long-term Monitoring of Inflammatory Responses

The method described here allows for the visualization of IL-8 promoter-dependent inflammation activation in the lungs of mice through non-invasive bioluminescence imaging (BLI). The same animal can be subjected to BLI multiple times for up to two months from the time of delivery of the luciferase reporter construct.

Airway inflammation is often associated with bacterial infections and represents a major determinant of lung disease. The in vivo determination of the ...

A Chronic Autoimmune Dry Eye Rat Model with Increase in Effector Memory T Cells in Eyeball Tissue

Dry eye disease is a very common condition that causes morbidity and healthcare burden and decreases the quality of life. There is a need for a suitable dry eye animal model to test novel therapeutics to treat autoimmune dry eye conditions. This protocol describes a chronic autoimmune dry eye rat model. Lewis rats were immunized with an emulsion containing lacrimal gland extract, ovalbumin, and complete Freund&#39;s adjuvant. A second immunization with the same antigens in incomplete Freund&#39;s adjuvant was administered two weeks later. These immunizations were administered subcutaneously at the base of the tail. To boost the immune response at the ocular surface and lacrimal glands, lacrimal gland extract and ovalbumin were injected into the forniceal subconjunctiva and lacrimal glands 6 weeks after the first immunization. The rats developed dry eye features, including reduced tear production, decreased tear stability, and increased corneal damage. Immune profiling by flow cytometry showed a preponderance of CD3+ effector memory T cells in the eyeball.

This report describes a method to induce chronic experimental autoimmune dry eye in Lewis rats through immunization with an emulsion of rat lacrimal gland extract, ovalbumin, and complete Freund's adjuvant, followed by the injection of lacrimal gland extract and ovalbumin into the forniceal subconjunctiva and lacrimal glands six weeks later.

Dry eye disease is a very common condition that causes morbidity and healthcare burden and decreases the quality of life. There is a need for a suitable ...

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.

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

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 ...