Mabtech带您了解ELISpot和FluoroSpot中如何选择细胞刺激物
Mabtech提供的肽库有2种：defined和scanning 形式。Scanning 形式的肽库一般适用于不需要提前知道相关表位信息的检测，该形式的肽库被设计用于覆盖整个蛋白质或结构域。通常以11个氨基酸重叠的15个氨基酸短肽形式提供，可以跨越整个感兴趣的蛋白质的序列。15个氨基酸短肽能够刺激CD4+ T细胞，当加入到培养的细胞中时，它们可以被蛋白酶修剪成更短的肽，从而刺激CD8+ T细胞。因此，scanning 形式的肽库可以用于刺激任何T细胞和样品。一般来说，如果您不确定样本中HLA/MHC的组成，scanning 形式的肽库是一个比较好的选择。
Defined 形式的肽库是针对特定HLA/MHC分子的肽。这些肽已经被鉴定为免疫显性的，会产生强烈的免疫反应。如果您知道研究样本中的HLA组成，那么Defined 形式的肽池是首选。
Mabtech可提供一系列针对特定研究的肽库产品：
刺激物名称 | 刺激细胞类型 | 产品描述 | 刺激后分泌物 | Mabtech产品货号 |
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细胞激活的常用试剂是植物血凝素(PHA)、PMA、离子霉素和CD3抗体。诱导特异性反应的抗原也是一个比较好的选择，因为抗原的序列明确并能更好确认特异免疫反应的激活。例如ELISpot和FluoroSpot可以检测抗原特异性人CD8 T细胞产生的IFN-γ。对于这个应用，Mabtech可以提供阳性抗原特异性对照——PepPool: CEF (CD8), human（货号：3616-1）。还有用于刺激CD4 T细胞的肽库。
图1.PepPool: CEF (CD8), human明显刺激CD8 T细胞
记忆B细胞在分泌抗体前通常需要使用合成化合物R848 (TLR 7/8激动剂)和重组IL-2蛋白的混合物进行刺激。Mabtech可提供针对人和小鼠的B细胞刺激的R848和IL-2套装——StimPack: Memory B cells, human（货号：3660-1）和StimPack: Memory B cells, mouse（货号：3661-1）。
Toll样受体(TLRs)的配体可用于刺激单核细胞和树突细胞。脂多糖(LPS)通过TLR4发挥作用，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的配体，刺激IL-1β等物质的分泌。
适用于作为实验对照的不同免疫细胞的刺激物：
刺激物名称 | 刺激细胞类型 | 产品描述 | 刺激后分泌物 | Mabtech产品货号 |
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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