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Mabtech帶您了解ELISpot和FluoroSpot中如何選擇細(xì)胞刺激物

更新時(shí)間：2024-11-29 點(diǎn)擊次數(shù)：857次

使用ELISpot和FluoroSpot檢測(cè)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(lèi)型和實(shí)驗(yàn)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(kù)（peptide pool），所有這些刺激物都經(jīng)過(guò)驗(yàn)證和優(yōu)化，非常適用于作為ELISpot和FluoroSpot檢測(cè)對(duì)照或用于鑒定抗原特異性免疫反應(yīng)。

Mabtech提供的肽庫(kù)有2種：defined和scanning 形式。Scanning 形式的肽庫(kù)一般適用于不需要提前知道相關(guān)表位信息的檢測(cè)，該形式的肽庫(kù)被設(shè)計(jì)用于覆蓋整個(gè)蛋白質(zhì)或結(jié)構(gòu)域。通常以11個(gè)氨基酸重疊的15個(gè)氨基酸短肽形式提供，可以跨越整個(gè)感興趣的蛋白質(zhì)的序列。15個(gè)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(shí)，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(kù)可以用于刺激任何T細(xì)胞和樣品。一般來(lái)說(shuō)，如果您不確定樣本中HLA/MHC的組成，scanning 形式的肽庫(kù)是一個(gè)比較好的選擇。

Defined 形式的肽庫(kù)是針對(duì)特定HLA/MHC分子的肽。這些肽已經(jīng)被鑒定為免疫顯性的，會(huì)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(duì)特定研究的肽庫(kù)產(chǎn)品：

刺激物名稱(chēng)	刺激細(xì)胞類(lèi)型	產(chǎn)品描述	刺激后分泌物	Mabtech產(chǎn)品貨號(hào)
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(gè)比較好的選擇，因?yàn)榭乖男蛄忻鞔_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(cè)抗原特異性人CD8 T細(xì)胞產(chǎn)生的IFN-γ。對(duì)于這個(gè)應(yīng)用，Mabtech可以提供陽(yáng)性抗原特異性對(duì)照——PepPool: CEF (CD8), human（貨號(hào)：3616-1）。還有用于刺激CD4 T細(xì)胞的肽庫(kù)。

Mabtech帶您了解ELISpot和FluoroSpot中如何選擇細(xì)胞刺激物

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(dòng)劑)和重組IL-2蛋白的混合物進(jìn)行刺激。Mabtech可提供針對(duì)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(hào)：3660-1）和StimPack: Memory B cells, mouse（貨號(hào)：3661-1）。

Toll樣受體(TLRs)的配體可用于刺激單核細(xì)胞和樹(shù)突細(xì)胞。脂多糖(LPS)通過(guò)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(shí)驗(yàn)對(duì)照的不同免疫細(xì)胞的刺激物：

刺激物名稱(chēng)	刺激細(xì)胞類(lèi)型	產(chǎn)品描述	刺激后分泌物	Mabtech產(chǎn)品貨號(hào)
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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