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All procedures involving human participants have been performed in compliance with the institutional, national, and international guidelines for human welfare and have been reviewed by the local institutional review board.
Human peripheral blood must be obtained from healthy donors under informed consent, and the use of blood samples must conform to the approved guidelines established by review boards. In this study, the protocol to use human blood in a demonstration of the results of flow cytometry (Figure 1) and ELISpot assays (Figure 2).
1. Isolation and Purification of Human Peripheral Blood B Cells
<ol>
	<li>Draw ~ 10 mL of blood from the median cubital vein (in the cubital fossa anterior to the elbow) into a 15-mL tube containing K2EDTA (1.5 to 2.0 mg/mL blood) and immediately invert the tube several times to prevent clot formation.</li>
	<li>Add 35 mL of autoclaved (121 &#176;C, 15 min) red blood cell (RBC) lysis buffer (150 mM NH4Cl, 10 mM KHCO3, and 1 mM EDTA; pH 7.4) to the tube containing the fresh blood sample (&#8805; 3:1 vol/vol) and incubate at room temperature (RT) for no longer than 5 min.&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160; 
	NOTE: The appearance of light transmission through the tube indicates the completion of RBC lysis.</li>
	<li>Centrifuge at 600 x g at RT for 5 min. Ensure that the pellet is white in color.</li>
	<li>Resuspend the pellet with 10 mL of autoclaved phosphate-buffered saline (PBS, 137 mM NaCl, 2.7 mM KCl, 4.3 mM Na2HPO4, and 1.47 mM KH2PO4; pH 7.4) and centrifuge as in step 1.3.</li>
	<li>Discard the supernatant, resuspend the pellet with 10 mL of RPMI 1640 medium (supplements: 10% fetal calf serum, 100 U/mL penicillin/streptomycin, 0.25 &#181;g/mL amphotericin B, and 2 mM L-glutamine), and then plate the cells into a 10-cm culture dish (10 mL of blood per dish). Place the dish in a 37 &#176;C incubator with 5% CO2&#160;for 30 min.</li>
	<li>Gently swirl the culture dish a few times and place the culture medium (suspension cells) into 15-mL plastic conical tubes. Discard the adherent cells (mostly macrophages) on the culture dishes.</li>
	<li>Centrifuge at 600 x g at RT for 5 min. Discard the supernatant.</li>
	<li>Resuspend the pellet with 1 mL of RPMI 1640 medium. Count the cell number with a hemocytometer or an automated cell counter.&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160; 
	NOTE: The viability of isolated leukocytes is normally greater than 90% by trypan blue exclusion.</li>
	<li>Centrifuge as in step 1.7 and resuspend the cells in ~ 200 &#181;L of cold PBS buffer (0.5% bovine serum albumin (BSA) and 2 mM EDTA) at the concentration of 5 - 10 x 106&#160;cells/mL.</li>
	<li>Add 5 &#181;L of biotinylated anti-human Ab cocktail specific to blood cells (for the negative selection of B cells) per 106&#160;cells and incubate on ice for 30 min. 
	NOTE: The anti-human Ab cocktail should at least include Abs specific to CD2 (or CD3), CD14, and CD16.</li>
	<li>Add a 10-fold excess volume of sterile PBS to the cells, centrifuge at 600 x g for 5 min, and discard the supernatant.</li>
	<li>Add equal amounts of streptavidin-conjugated microbeads (5 &#181;L per 106&#160;cells) to the pellet and mix thoroughly.</li>
	<li>Incubate on ice for 30 min in a 15-mL plastic conical tube.</li>
	<li>Add 2 mL of PBS buffer into the tube.</li>
	<li>Place the tube into a magnetic stand and incubate at RT for 8 min. The brown microbeads will gradually attach to the tube wall next to the magnet.</li>
	<li>With the tube remaining in the magnetic stand, carefully transfer the supernatant into a new sterile 15-mL tube.</li>
	<li>Repeat steps 1.14 to 1.16 and combine the two supernatants that contain the untouched B cells. Discard the microbeads.</li>
	<li>Centrifuge at 600 x g at RT for 5 min. Discard the supernatant.</li>
	<li>Resuspend the pellet in RPMI 1640 medium for downstream experiments. 
	NOTE: Typically, 1 - 5 x 105&#160;B cells with a purity greater than 95% from 10 mL of peripheral blood can be isolated.</li>
</ol>
2. Purification and Separation of Memory and Na&#239;ve B Cells from Isolated B Cells
<ol>
	<li>Use cells purified from Step 1 and determine the cell number using a hemocytometer or an automatic cell counter.</li>
	<li>Resuspend the cells in 100 &#181;L of cold PBS buffer after centrifugation at 600 x g and RT for 5 min.</li>
	<li>Add 1 - 2 &#181;g of biotinylated CD27 mAb per 106&#160;cells and incubate on ice for 30 min.</li>
	<li>Add 10 mL of PBS buffer to the tube and centrifuge at 600 x g for 5 min.</li>
	<li>Discard the supernatant and resuspend the cells in 50 &#181;L of PBS buffer.</li>
	<li>Add equal amounts of streptavidin magnetic microbeads (1 - 2 &#181;g/106&#160;cells) to cells in a 15-mL plastic conical tube.</li>
	<li>Gently mix well and incubate on ice for 30 min.</li>
	<li>Add 2 - 3 mL of PBS buffer to the tube.</li>
	<li>Place the tube into a magnetic stand and incubate at RT for 8 min to allow the brown microbeads to attach to the side closest to the magnet.</li>
	<li>With the tube in the magnetic stand, carefully transfer the supernatant fraction into a new sterile tube. This fraction contains the enriched CD27-&#160;na&#239;ve B cells.</li>
	<li>Add 5 mL to the tube containing the microbeads and gently resuspend the microbeads (i.e.,&#160;the enriched fraction of CD27+&#160;memory B cells).</li>
	<li>Centrifuge at 600 x g at RT for 5 min. Resuspend the pellets with RPMI 1640 medium for the downstream experiments. 
	NOTE: Typically, ~ 30 - 60% of B cells can be purified as CD27+&#160;memory cells from the PBMCs of a healthy donor.</li>
</ol>
3. Cell Sorting for the Collection of Na&#239;ve B Cells, Memory B Cells, and PBs/PCs
<ol>
	<li>Using the cells purified from Step 1, determine the cell number using a hemocytometer or an automatic cell counter.</li>
	<li>Resuspend the cells in cold PBS buffer at the concentration of 107&#160;per mL in a 5-mL polystyrene tube.</li>
	<li>Add 1 - 2 &#181;g of human IgG per 106&#160;cells and incubate on ice for 10 min for the Fc block.</li>
	<li>Add 1 &#181;g each of anti-CD19-APC (clone: HIB19), anti-CD27-eFluor450 (clone: O323), and anti-CD38-PE (clone: HIT2) per 106&#160;cells; mix well and incubate on ice for 30 min.</li>
	<li>In the last 5 min in step 3.4, add 5 &#181;L of the commercial 7-amino actinomycin D (7-AAD).</li>
	<li>Add 2 mL of PBS to the tube, vortex, and centrifuge at 600 x g for 5 min.</li>
	<li>Resuspend the cells in sorting buffer (sterile PBS with 2% BSA and 2 mM EDTA) at a concentration of 1 - 5 x 107&#160;cells per mL in a 15-mL tube.</li>
	<li>Filter the cells through a nylon mesh cell strainer (40 &#181;m pore size) to eliminate cell clumps.</li>
	<li>Separate the cells with a flow cytometric sorter equipped with three lasers: violet (405 nm), blue (488 nm), and red (640 nm).&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160; 
	NOTE: The blue laser alone is sufficient for 3-color flow cytometry.</li>
	<li>Sort the cells into three 15-mL tubes (containing 5 mL of RPMI medium) for the simultaneous collection of na&#239;ve B cells (CD19+CD27-), memory B cells (CD19+CD27+), and PBs/PCs (CD19+CD27+/hiCD38+).&#160;&#160;&#160;&#160; 
	NOTE: Sorted na&#239;ve and memory B cells can be cultured as described in step 4.</li>
</ol>
4. In Vitro&#160;Differentiation of Isolated Human CD19+&#160;B Cells, CD19+CD27+&#160;Memory B Cells, and CD19+CD27-&#160;Na&#239;ve B Cells
<ol>
	<li>Using the cells purified in steps 1.19, 2.10, 2.11, and 3.10, determine the cell number with a hemocytometer or an automatic cell counter.</li>
	<li>Resuspend the cells with RPMI 1640 medium at a concentration of 1 - 10 x 105&#160;per mL and aliquot them into the wells of a 12-well plate.</li>
	<li>Add CpG (ODN 2006) at 5 &#181;g/106&#160;cells/mL.</li>
	<li>Culture the cells in a 37 &#176;C incubator with 5% CO2&#160;for 5 days.</li>
	<li>Harvest the cells from each well, place them separately into 15-mL tubes, add 5 mL of PBS to each tube, and centrifuge them at 600 x g and RT for 5 min.</li>
	<li>Count the cells using a hemocytometer or an automatic cell counter. Resuspend the cells at a concentration of 1 - 10 x 105&#160;per mL with RPMI 1640 medium.</li>
</ol>
5. ELISpot Assay
<ol>
	<li>Add 30 &#181;L of 35% ethanol in distilled water to each well of the ELISpot plates for 30 s.&#160;&#160;&#160;&#160; 
	NOTE: When pipetting, avoid touching the membrane in the wells at all times.</li>
	<li>Invert the ELISpot plates to remove the ethanol.</li>
	<li>Put 150 &#181;L of autoclaved ddH2O into each well and incubate them at RT for 5 min to flush off the residual ethanol; follow with a wash of sterile PBS at RT for 3 min. 
	NOTE: Steps 5.1 to 5.3 may be optional, depending on the manufacture of the plates.</li>
	<li>Put 50 &#181;L of 5 &#181;g/mL polyclonal F(ab&#39;)2&#160;fragment of anti-human Ig (IgG + IgM + IgA) (in PBS) into each well of the ELISpot plates and incubate them at 4 &#176;C overnight (preferred) or 37 &#176;C for 2 h.&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160; 
	NOTE: Seal the edges of plates with Parafilm until use.</li>
	<li>Invert the plates to remove unbound Abs, add 200 &#181;L of PBS to each well, and incubate them twice at RT for 3 min each time.</li>
	<li>Add 200 &#181;L of PBS with 5% BSA (or RPMI 1640 medium) to each well for blocking, and incubate them at RT for 2 h.</li>
	<li>Invert the plates to remove the blocking buffer. Wash each well twice with 200 &#181;L of PBS, as in step 5.5.</li>
	<li>Add 100 &#181;L of RPMI 1640 medium to each well and incubate them at 37 &#176;C.</li>
	<li>When ready to seed the cells, invert the plates to remove the RPMI 1640 medium.</li>
	<li>Seed 100 &#181;L (5 x 104), 50 &#181;L (2.5 x 104), and 25 &#181;L (1.25 x 104) of the cells (from steps 1.19, 2.10, 2.11, 3.10, and 4.6) into the wells of an ELISpot plate. With RPMI 1640 medium, bring the volume to 150 &#181;L/well.&#160;&#160;&#160;&#160;&#160;&#160; 
	NOTE: A minimum of one or two 2-fold serial dilutions for plating the cells is recommended.</li>
	<li>Incubate the plates in a 37 &#176;C incubator with 5% CO2&#160;for 8 - 14 h. Avoid moving the plates during incubation.</li>
	<li>Invert the plates to remove the cells and RPMI 1640 medium.</li>
	<li>Add 200 &#181;L/well of PBS-T (PBS with 0.05% Tween 20) and incubate them 5 times at RT, each time for 3 min. Invert the plate to remove the wash buffer between each of the 5 washes.</li>
	<li>Add either goat anti-human IgG-alkaline phosphatase (AP), Fc&#947;-specific Abs (for IgG detection, 1:5,000 in PBS-T) or goat anti-human IgM-AP, Fc&#181; fragment-specific Abs (for IgM detection, 1:5,000 in PBS-T) into the designated wells and incubate at RT for 2 h in the dark.</li>
	<li>Wash each well twice with 200 &#181;L of PBS, as in step 5.5.</li>
	<li>Add 50 &#181;L/well of bromochloroindolyl phosphate-nitro blue tetrazolium (BCIP/NBT) substrate solution. Purple-colored spots normally appear in 5 - 15 min.</li>
	<li>Add 100 &#181;L/well of ddH2O to prevent the over-development of spots.</li>
	<li>Rinse the plates with running tap water after the complete development of all spots.</li>
	<li>Remove the underdrain of the plates and allow them to air dry in the dark.</li>
	<li>Count the spots using an automated plate reader with an image acquisition/analysis unit (e.g.,&#160;an automatic scanner or manually via a dissecting microscope). 
	NOTE: The plates can be stored at RT in the dark and analyzed later.</li>
</ol>

<table><tbody><tr><td>BD Vacutainer K2E</td><td>BD Biosciences</td><td>367525</td><td>10 ml tube</td></tr><tr><td>Ficoll-Paque Plus</td><td>GE Healthcare</td><td>17-1440-02</td><td>Endotoxin-free</td></tr><tr><td>Trypan blue 0.5% solution</td><td>Biological Industries</td><td>03-102-1B</td><td></td></tr><tr><td>IMag Human B lymphocyte enrichment set</td><td>BD Biosciences</td><td>558007</td><td></td></tr><tr><td>Biotinylated CD27 mAb</td><td>Biolegend</td><td>302804</td><td>Clone O323</td></tr><tr><td>Streptavidin magnetic microbeads</td><td>BD Biosciences</td><td>9000810</td><td></td></tr><tr><td>15 ml Falcon tubes</td><td>BD Falcon</td><td>352196</td><td></td></tr><tr><td>Blue nylon mesh cell strainer, 40 &amp;mu;m</td><td>BD Falcon</td><td>352340</td><td></td></tr><tr><td>Anti-human CD19-APC</td><td>Biolegend</td><td>302212</td><td>Clone HIB19</td></tr><tr><td>Anti-human CD27-eFluor 450</td><td>eBioscience</td><td>48-0279-42</td><td>Clone O323</td></tr><tr><td>Anti-human CD38-PE-Cy7</td><td>Biolegend</td><td>303516</td><td>Clone HIT2&amp;nbsp;</td></tr><tr><td>Anti-human CD38-PE-Cy7</td><td>BD Biosciences</td><td>560677</td><td>Clone HIT2&amp;nbsp;</td></tr><tr><td>Anti-human CD45-FITC</td><td>Biolegend</td><td>304006</td><td>Clone HI30</td></tr><tr><td>Anti-human CD45-FITC</td><td>BD Biosciences</td><td>555482</td><td>Clone HI30</td></tr><tr><td>Anti-mouse/rat/human CD27-PerCP Cy5.5</td><td>Biolegend</td><td>124213</td><td>Clone LG.3A10</td></tr><tr><td>Anti-human CD27-PerCP Cy5.5</td><td>BD Biosciences</td><td>65429</td><td>Clone L128</td></tr><tr><td>Anti-human CD19-FITC</td><td>Miltenyi Biotec</td><td>130-098-064</td><td>Clone LT19</td></tr><tr><td>Anti-human CD19-FITC</td><td>GeneTex</td><td>GTX75599</td><td>Clone LT19</td></tr><tr><td>Anti-human CD20-FITC</td><td>BD Biosciences</td><td>555622</td><td>Clone 2H7</td></tr><tr><td>Biotinylated anti-human CD27</td><td>Biolegend</td><td>302804</td><td>Clone O323</td></tr><tr><td>Biotinylated anti-human CD27</td><td>eBioscience</td><td>13-0279-80</td><td>Clone O323</td></tr><tr><td>7-aminoactinomycin D (7-AAD)</td><td>BD Biosciences</td><td>559925</td><td></td></tr><tr><td>CpG (ODN 2006)&amp;nbsp;</td><td>InvivoGen</td><td>tlrl-2006</td><td>Type B CpG</td></tr><tr><td>Recombinant human IL-2</td><td>PeproTech</td><td>200-02</td><td></td></tr><tr><td>Recombinant human IL-10</td><td>PeproTech</td><td>200-10</td><td></td></tr><tr><td>Recombinant human IL-21</td><td>PeproTech</td><td>200-21</td><td></td></tr><tr><td>Recombinant human sCD40L</td><td>PeproTech</td><td>310-02</td><td></td></tr><tr><td>Protein A of&amp;nbsp;&lt;em&gt;S. aureus&lt;/em&gt;&amp;nbsp;Cowan (SAC)</td><td>Sigma-Aldrich</td><td>82526</td><td></td></tr><tr><td>Pokeweed mitogen (PWM)</td><td>Sigma-Aldrich</td><td>L9379</td><td></td></tr><tr><td>MultiScreen filter plates, 0.45 &amp;micro;m pore size</td><td>Merck Millipore</td><td>MSIPS4510</td><td>Sterile, clear 96-well filter plate with hydrophobic PVDF membrane</td></tr><tr><td>BCIP/NBT solution</td><td>Sigma-Aldrich</td><td>B6404</td><td></td></tr><tr><td>BCIP/NBT single reagent, alkaline phosphatase substrate</td><td>Merck Millipore</td><td>ES006</td><td></td></tr><tr><td>Human IgG</td><td>Jackson ImmunoResearch</td><td>009-000-003</td><td></td></tr><tr><td>Human IgG, Fc fragment</td><td>Jackson ImmunoResearch</td><td>009-000-008</td><td></td></tr><tr><td>F(ab&#039;)2&amp;nbsp;fragment of goat anti-human Ig (IgG+IgM+IgA)</td><td>Jackson ImmunoResearch</td><td>109-006-127</td><td></td></tr><tr><td>Goat anti-human IgG-alkaline phosphatase, Fc&amp;gamma; fragment specific</td><td>Jackson ImmunoResearch</td><td>109-055-008</td><td></td></tr><tr><td>Goat anti-human IgM-alkaline phosphatase, Fc&amp;micro; fragment specific</td><td>Jackson ImmunoResearch</td><td>109-055-095</td><td></td></tr><tr><td>Goat anti-human IgG-peroxidase, Fc&amp;gamma; fragment specific</td><td>Jackson ImmunoResearch</td><td>109-035-008</td><td></td></tr><tr><td>Goat anti-human IgM-peroxidase, Fc&amp;micro; fragment specific</td><td>Jackson ImmunoResearch</td><td>109-035-095</td><td></td></tr><tr><td>BD ELISPOT AEC substrate kit</td><td>BD Biosciences</td><td>551951</td><td></td></tr><tr><td>C.T.L. ImmunoSpot analyzer</td><td>C.T.L.</td><td></td><td></td></tr></tbody></table>

functional assessment of antibody-secreting b cells using elispot assay

Source: Tzeng, S. J. <a href="https://app.jove.com/v/54582">The Isolation, Differentiation, and Quantification of Human Antibody-secreting B Cells from Blood: ELISpot as a Functional Readout of Humoral Immunity.</a> J. Vis. Exp. (2016)
This video demonstrates the use of the ELISpot assay to assess the function of antibody-secreting B cells, ASCs. The antibodies secreted by the ASCs interact with the membrane-bound polyclonal antibody fragments, which are detected by the enzyme-conjugated IgG or IgM antibodies. The colored spot produced in each well indicates the presence of the specific antibody&#39;s isotype, which correlates with the B cells&#39; secretory activity.

<img alt="Figure 1" src="/files/ftp_upload/21525/21525_Figure1.jpg" /> 
Figure 1.&#160;Illustration of the Gating Strategy to Separate the Na&#239;ve B cells, Memory B cells, and PBs in the PBMCs.&#160;(A)&#160;Cells (2 x 106) were stained with 2 &#181;g each of CD19-APC, CD27-eFluor450, and CD38-PE mAbs (step 3.4). The lymphocyte compartment was gated (G1) on the dot plot for forward scattering (FSC) versus side scattering (SSC).&#160;(B)...

Functional Assessment of Antibody-Secreting B Cells Using ELISpot Assay

Source: Tzeng, S. J. The Isolation, Differentiation, and Quantification of Human Antibody-secreting B Cells from Blood: ELISpot as a Functional Readout of ...

Antibody-secreting B cells, or ASCs are differentiated B cells that secrete antibody isotypes, including IgG and IgM, to ensure protection against pathogens.
To perform a functional assessment of ASCs in vitro, begin with the enzyme-linked immunosorbent spot assay. Take an ELISpot plate containing an activated polyvinylidene fluoride membrane at the base.
Incubate the wells with polyclonal antibody fragments exclusively containing Fab regions without the antibody&#39;s Fc region. Each Fab region has a binding site for specific antibody isotypes &#8212; IgG or IgM, enabling the identification of both isotypes.
During incubation, the antibody fragments attach to the membrane, leading to immobilization.
Overlay the wells with blocking solutions to prevent non-specific binding. Seed the plate with activated antibody-secreting B cells, and incubate. The ASCs secrete different isotypes, including IgG and IgM, which bind to the membrane-bound antibody fragments.
Wash the plate with a buffer to remove unbound secreted antibodies. Treat some wells with enzyme-conjugated detection antibodies for IgM and others with detection antibodies for IgG, and incubate.
The detection antibodies bind to their respective isotypes, forming immunocomplexes.
Add colorimetric substrates to all the wells, which interact with the enzymes and produce purple-colored spots on the membrane. Add a stopping solution to stop the reaction.
The colored spot produced in each well indicates the presence of the specific antibody&#39;s isotype, correlating with the secretory activity of B cells.

functional-assessment-antibody-secreting-b-cells-using-elispot

Research

JoVE Encyclopedia of Experiments

Immunology

Immune Systems and Components

Assays and Functional Profiling

367 ビュー. 出典: Tzeng, S. J. 血液からのヒト抗体分泌B細胞の単離、分化、定量化:体液性免疫の機能的読み出しとしてのELISpot. J. Vis. Exp. (2016) このビデオでは、ELISpotアッセイを使用して抗体分泌B細胞(ASC)の機能を評価する方法を示しています。ASCから分泌された抗体は、膜結合ポリクローナル抗体フラグメントと相互作用し、酵素標識IgGまたはIgM抗体によって検出されます。各ウェル?...

ビデオ: ELISpotアッセイを用いた抗体分泌B細胞の機能評価 - 概念

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.

牛にエフェクターおよびメモリーT細胞応答を評価するための長期培養インターフェロンγ酵素結合免疫アッセイの応用

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

JoVE Journal

免疫・感染学

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

Qa-1 (HLA-E in human) belongs to a group of non-classical major histocompatibility complex 1b (MHC-Ib) molecules. Recent data suggest that Qa-1 molecules play important roles in surveying cells for structural and functional integrity, inducing immune regulation, and limiting immune responses to viral infections. Additionally, functional augmentation of Qa-1-restricted CD8+ T cells through epitope immunization has shown therapeutic effects in several autoimmune disease animal models, e.g. experimental allergic encephalomyelitis, collagen-induced arthritis, and non-obese diabetes. Therefore, there is an urgent need for a method that can efficiently and quickly identify functional Qa-1 epitopes in a protein. Here, we describe a protocol that utilizes Qa-1-restricted CD8+ T cell lines specific for an overlapping peptide (OLP) library for determining Qa-1 epitopes in a protein. This OLP library contains 15-mer overlapping peptides that cover the whole length of a protein, and adjacent peptides overlap by 11 amino acids. Using this protocol, we recently identified a 9-mer Qa-1 epitope in myelin oligodendrocyte glycoprotein (MOG). This newly mapped MOG Qa-1 epitope was shown to induce epitope-specific, Qa-1-restricted CD8+ T cells that enhanced myelin-specific immune regulation. Therefore, this protocol is useful for future investigation of novel targets and functions of Qa-1-restricted CD8+ T cells.

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

蛋白質の Qa 1 エピトープをマップするペプチド ライブラリを重複

Qa-1 (HLA-E in human) belongs to a group of non-classical major histocompatibility complex 1b (MHC-Ib) molecules. Recent data suggest that Qa-1 molecules ...

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.

ワクチン接種後のモルモット モデル メジャー T 細胞反応に最適化されたインターフェロン-γ しなさいアッセイ

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, ...

Functional Assessment of Antibody-Secreting B Cells Using ELISpot Assay

記録

Functional Assessment of Antibody-Secreting B Cells Using ELISpot Assay