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使用ELISpot和FluoroSpot檢測抗原特異性免疫反應(yīng)，通常需要在體外刺激細胞。如何選擇用于刺激的化合物很大程度上取決于細胞類型和實驗的性質(zhì)。刺激物一般可以是抗體、肽、合成化合物或微生物或植物中純化的物質(zhì)等。Mabtech提供許多不同的抗原特異性刺激肽庫（peptide pool），所有這些刺激物都經(jīng)過驗證和優(yōu)化，非常適用于作為ELISpot和FluoroSpot檢測對照或用于鑒定抗原特異性免疫反應(yīng)。
Mabtech提供的肽庫有2種：defined和scanning 形式。Scanning 形式的肽庫一般適用于不需要提前知道相關(guān)表位信息的檢測，該形式的肽庫被設(shè)計用于覆蓋整個蛋白質(zhì)或結(jié)構(gòu)域。通常以11個氨基酸重疊的15個氨基酸短肽形式提供，可以跨越整個感興趣的蛋白質(zhì)的序列。15個氨基酸短肽能夠刺激CD4+ T細胞，當加入到培養(yǎng)的細胞中時，它們可以被蛋白酶修剪成更短的肽，從而刺激CD8+ T細胞。因此，scanning 形式的肽庫可以用于刺激任何T細胞和樣品。一般來說，如果您不確定樣本中HLA/MHC的組成，scanning 形式的肽庫是一個比較好的選擇。
Defined 形式的肽庫是針對特定HLA/MHC分子的肽。這些肽已經(jīng)被鑒定為免疫顯性的，會產(chǎn)生強烈的免疫反應(yīng)。如果您知道研究樣本中的HLA組成，那么Defined 形式的肽池是首-選。
Mabtech可提供一系列針對特定研究的肽庫產(chǎn)品：
刺激物名稱 | 刺激細胞類型 | 產(chǎn)品描述 | 刺激后分泌物 | Mabtech產(chǎn)品貨號 |
PepPool: TB (EspC), scanning | T cells (scanning pool) | T cell epitopes from the Mycobacterium tuberculosis ESX-1 secretion-associated protein EspC | IFN-γ, IL-2, | 3623-1 |
PepPool: TB (ESAT-6), scanning | T cells (scanning pool) | T cell epitopes from the Mycobacterium tuberculosis early secretory antigenic target (ESAT-6) protein | IFN-γ, IL-2, | 3624-1 |
PepPool: TB (CFP-10), scanning | T cells (scanning pool) | T cell epitopes from the Mycobacterium tuberculosis culture filtrate protein (CFP-10) | IFN-γ, IL-2, | 3625-1 |
PepPool: CMV (CD4 and CD8), human | CD4+ and CD8+ T cells | T cell epitopes from Cytomegalovirus (CMV) proteins | IFN-γ, IL-2, IL-5, IL-13 | 3619-1 |
PepPool: CyCMV+RhCMV (TP-UL83+Rh112), scanning | T cells (Macaque) | T cell epitopes from cynomolgus cytomegalovirus (CyCMV) Tegument phosphoprotein UL83, and from rhesus cytomegalovirus (rhCMV) Rh112 | IFN-γ, IL-2 | 3692-1 |
PepPool: EBV (CD4 and CD8), human | CD4+ and CD8+ T cells | T cell epitopes from latent and lytic Epstein-Barr virus (EBV) proteins. | IFN-γ, IL-2 | 3641-1 |
PepPool: AdV5 (hexon), human | T cells | T cell epitopes from the human adenovirus 5 (hAdV5) hexon protein | IFN-γ, IL-2 | 3634-1 |
PepPool: Vaccinia (CD4), human | CD4 T cells | T cell epitopes from several Vaccinia proteins. Of the included peptides, 91% share >90% homology with mpox virus | IFN-γ | 3635-1 |
PepPool: Vaccinia (CD8), human | CD8 T cells | T cell epitopes from several Vaccinia proteins. Of the included peptides, 79% share >90% homology with mpox virus | IFN-γ | 3670-1 |
PepPool: SARS-CoV-2 (SNMO), human | T cells | T cell epitopes from the SARS-CoV-2 spike protein (S), nucleoprotein (N), membrane protein (M), and the open reading frame (ORF)-3a and ORF-7a proteins (O) | IFN-γ, IL-2, Granzyme B | 3622-1 |
PepPool: SARS-CoV-2 (S), human | T cells | T cell epitopes from the SARS-CoV-2 spike protein (S) | IFN-γ, IL-2, Granzyme B | 3630-1 |
PepPool: SARS-CoV-2 (NMO), human | T cells | T cell epitopes from the SARS-CoV-2 nucleoprotein (N), membrane protein (M), and the open reading frame (ORF)-3a and ORF-7a proteins (O) | IFN-γ, IL-2, Granzyme B | 3632-1 |
PepPool: SARS-CoV-2 (S1), scanning | T cells (scanning pool) | T cell epitopes from the S1 domain of the SARS-CoV-2 spike protein | IFN-γ, IL-2, Granzyme B | 3629-1 |
PepPool: SARS-CoV-2 Omicron BA.1 (S1), scanning | T cells (scanning pool) | Similar to the (S1) scanning pool, but including Omicron BA.1 mutations | IFN-γ, IL-2, Granzyme B | 3636-1 |
PepPool: BKV (CD4 and CD8), human | T cells | T cell epitopes from the large T protein, small T protein, and VP1 protein. Included peptides share 80% homology with JC virus. | IFN-γ | 3675-1 |
PepPool: TBEV (C+E), scanning | T cells (scanning pool) | T cell epitopes from the capsid (C) protein and the envelope (E) protein | IFN-γ, IL-4, IL-5 | 3672-1 |
PepPool: DENV (CD8), human | CD8 T cells | T cell epitopes from the capsid protein (C), envelope protein (E), and the non structural (NS) proteins. Coverage of all major HLA class I supertypes for Dengue virus serotypes 1, 2, 3, and 4 | IFN-γ | 3695-1 |
PepPool: DENV (CD4), human | CD4 T cells | T cell capsid protein (C), precursor membrane protein (prM), envelope protein (E), and the non structural (NS) proteins. Good HLA class coverage for Dengue virus serotypes 1, 2, 3, and 4 | IFN-γ | 3698-1 |
T細胞激活的常用試劑是植物血凝素(PHA)、PMA、離子霉素和CD3抗體。誘導(dǎo)特異性反應(yīng)的抗原也是一個比較好的選擇，因為抗原的序列明確并能更好確認特異免疫反應(yīng)的激活。例如ELISpot和FluoroSpot可以檢測抗原特異性人CD8 T細胞產(chǎn)生的IFN-γ。對于這個應(yīng)用，Mabtech可以提供陽性抗原特異性對照——PepPool: CEF (CD8), human（貨號：3616-1）。還有用于刺激CD4 T細胞的肽庫。
圖1.PepPool: CEF (CD8), human明顯刺激CD8 T細胞
記憶B細胞在分泌抗體前通常需要使用合成化合物R848 (TLR 7/8激動劑)和重組IL-2蛋白的混合物進行刺激。Mabtech可提供針對人和小鼠的B細胞刺激的R848和IL-2套裝——StimPack: Memory B cells, human（貨號：3660-1）和StimPack: Memory B cells, mouse（貨號：3661-1）。
Toll樣受體(TLRs)的配體可用于刺激單核細胞和樹突細胞。脂多糖(LPS)通過TLR4發(fā)揮作用，能有效刺激IL-1β、IL-6和TNF-α等因子的分泌?；衔颮848是TLR7/8的配體，刺激IL-1β等物質(zhì)的分泌。
適用于作為實驗對照的不同免疫細胞的刺激物：
刺激物名稱 | 刺激細胞類型 | 產(chǎn)品描述 | 刺激后分泌物 | Mabtech產(chǎn)品貨號 |
PepPool: CEF (CD8), human | CD8+ T cells (human) | Ag-specific stimulation by T cell epitopes derived from CMV, EBV and influenza. | IFN-γ, IL-2, Granzyme B, Perforin | 3616-1 |
PepPool: CEF extended (CD8), human | CD8+ T cells (human) | Ag-specific stimulation by T cell epitopes derived from CMV, EBV, and influenza | IFN-γ, IL-2, Granzyme B, Perforin | 3618-1 |
PepPool: CEFTA (CD4), human | CD4+ T cells (human) | Ag-specific stimulation by T cell epitopes derived from CMV, EBV, TT and influenza. | IFN-γ, IL-2, IL-5, IL-13 | 3617-1 |
Anti-human CD3 mAb (CD3-2), azide free | T cells (human) | Polyclonal T cell activation | IFN-γ, IL-2, IL-4, IL-5, IL-10, IL-13 and IL-17A | 3605-1 |
Anti-monkey CD3 mAb (CD3-1), azide free | T cells (non-human primate) | Polyclonal T cell activation | IFN-γ, IL-2, IL-4, IL-5, IL-10, IL-13 and IL-17A | 3610-1 |
Phytohemagglutinin (PHA) | T cells | Polyclonal T cell activation | IFN-γ, IL-2, IL-4, IL-5, IL-6, IL-10, IL-13, IL-17A, IL-21, GM-CSF, Granzyme B, Perforin | / |
StimPack: Memory B cells, human | B cells (human) | Polyclonal B-cell activation (R848 + recombinant hIL-2) | IgG, IgA, IgM | 3660-1 |
StimPack: Memory B cells, mouse | B cells (mouse) | Polyclonal B-cell activation (R848 + recombinant mIL-2) | IgG, IgA, IgM | 3661-1 |
Anti-human CD40 mAb (S2C6), azide free + recIL-4 | B cells | Polyclonal B-cell activation | IgE | 3601-1 |
Lipopolysaccharide (LPS) | Monocytes/DC | TLR4 agonist | IL-1β, IL-6, IL-8, IL-10, IL-12, IL-23, GM-CSF, TNF-α | / |
R848 | Monocytes/DC | TLR7/8 agonist | IFN-α, IL-1β, IL-6, IL-23, GM-CSF, TNF-α | / |
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