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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(xì)胞。如何選擇用于刺激的化合物很大程度上取決于細(xì)胞類型和實(shí)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(xì)胞，當(dāng)加入到培養(yǎng)的細(xì)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(xì)胞。因此，scanning 形式的肽庫可以用于刺激任何T細(xì)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(qiáng)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(xì)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(xì)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(rèn)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(xì)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(xì)胞的肽庫。
圖1.PepPool: CEF (CD8), human明顯刺激CD8 T細(xì)胞
記憶B細(xì)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(jìn)行刺激。Mabtech可提供針對人和小鼠的B細(xì)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(xì)胞和樹突細(xì)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。化合物R848是TLR7/8的配體，刺激IL-1β等物質(zhì)的分泌。
適用于作為實(shí)驗對照的不同免疫細(xì)胞的刺激物：
刺激物名稱 | 刺激細(xì)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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