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      技術文章您現(xiàn)在的位置：首頁 > 技術文章 > Mabtech帶您了解ELISpot和FluoroSpot中如何選擇細胞刺激物
       
      
      
        
      Mabtech帶您了解ELISpot和FluoroSpot中如何選擇細胞刺激物
      
        
      更新時間：2024-11-29      點擊次數(shù)：334次
      
        
      使用ELISpot和FluoroSpot檢測抗原特異性免疫反應，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。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。

      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表位信息的檢測，該形式的肽庫被設計用于覆蓋整個蛋白質或結構域。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。這些肽已經被鑒定為免疫顯性的，會產生強烈的免疫反應。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品：
      刺激物名稱
      刺激細胞類型
      產品描述
      刺激后分泌物
      Mabtech產品貨號
      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。誘導特異性反應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的激活。例如ELISpot和FluoroSpot可以檢測抗原特異性人CD8 T細胞產生的IFN-γ。對于這個應用，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。
      Mabtech帶您了解ELISpot和FluoroSpot中如何選擇細胞刺激物
      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。化合物R848是TLR7/8的配體，刺激IL-1β等物質的分泌。

      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
      產品描述
      刺激后分泌物
      Mabtech產品貨號
      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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