Mabtech ELISpot Kit 酶聯免疫斑點套組

Mabtech所研發出的ELISPOT檢測試劑是在披覆PVDF膜的培養96微孔盤上,吸附高純度,高專一性的單株抗體;當放入細胞,經過刺激細胞釋放 出細胞激素,會立即被抗體捕獲。當細胞移除後,再進一步使用生物素(Biotin)標記的二次抗體標示,結合酵素作用呈色,有反應的細胞會留下有染色的斑點,單顆細胞分泌越多,斑點越大。此法靈敏度極高,是ELISA靈敏度的20~200倍,105顆細胞中有一顆細胞分泌都可偵測出。

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ELISpot Logo

 

ELISPOT(Enzyme-linked immunospot)與ELISA原理相似,現在已經被廣泛用來偵測細胞分泌的特定反應因子,包括細胞激素(Cytokine)、趨化激素(Chemokine)或是顆粒酵素(Granzyme)。這種技術靈敏度高,可以用在細胞因子定性或免疫球蛋白分泌細胞。廣泛應用到研究感染後的免疫反應,癌症,過敏,自體免疫疾病。



ELISPOT優勢

  • 操作簡單 : 只需放入細胞刺激後,再呈色反應
  • 靈敏度高 : 可達單顆細胞層級,有利於偵測少量細胞分泌激素
  • 可大量篩選 : 96well 高通量快速進行實驗
  • 細胞需求量小 : 節省細胞樣品,刺激過後的細胞還可再利用
  • 接近體內真實狀況 : 細胞激素一分泌立即被捕獲
  • 樣品範圍廣泛 : 新鮮或是解凍後的細胞都可進行分析


應用範圍

  • 移植研究
  • 疫苗效率測試
  • 疫苗輔劑開發
  • 自體免疫研究
  • Th0/Th1/Th2細胞轉換分析
  • 癌症研究
  • 過敏機制探討
  • 傳染疾病研究
  • 體液免疫力研究



實驗原理

Mabtech所研發出的ELISPOT檢測試劑是在披覆PVDF膜的培養96微孔盤上,吸附高純度,高專一性的單株抗體;當放入細胞,經過刺激細胞釋放出細胞激素,會立即被抗體捕獲。當細胞移除後,再進一步使用生物素(Biotin)標記的二次抗體標示,結合酵素作用呈色,有反應的細胞會留下有染色的斑點,單顆細胞分泌越多,斑點越大。使用儀器對斑點進行計數和判讀,利用斑點數目多寡及大小,反應出細胞反應作用強弱。此法靈敏度極高,是ELISA靈敏度的20~200倍,105顆細胞中有一顆細胞分泌都可偵測出,靈敏度接近RT-PCR,只差在RT-PCR為mRNA層面,ELISPOT為Protein層面。

 

Coating1

Coating

Monoclonal capture antibodies are added to an ethanol-treated PVDF membrane plate.

Cell incubation2

Cell incubation

Cells are added in the presence or absence of stimuli and the plate is in-cubated to allow cytokine secretion.

Cytokine capture3

Cytokine capture

Secreted cytokines bind to capture antibodies surrounding the activated cells.

Detection antibodies4

Detection antibodies

Cells are removed and the plate is washed before biotinylated detection antibodies are added.

Streptavidin–enzyme conjugate5

Streptavidin–enzyme conjugate

Addition of a streptavidin–conjugate enables the formation of spots on the membrane.

Addition of substrate6

Addition of substrate

A colorimetric substrate forms an insoluble precipitate when catalyzed by the enzyme.



ELISPOT 分析

Mabtech已經發展 ELISPOT試劑超過20年,優化過試劑和實驗步驟,有各種套件和試劑適合不同物種與實驗應用。

Schematic illustration ELISpot assay

Schematic illustration of the ELISpot assay

Antigen specific B cell ELISpot

Antigen-specific B-cell ELISpot

 Mabtech ELISpot Basic KitMabtech ELISpot Pro Kit
ContentsELISpot
BASIC
ELISpot
PLUS
ELISpot
PRO
ELISpot platesXPre-coated platesPre-coated plates
Capture mAbOX 已經COATINGX 已經COATING
Detection mAbOOO
SA-ALP (or HRP)OOO
SubstrateXOO
Positive controlXO(certain analytes)O(certain analytes)

 

ELISpot analysis No stimuli

ELISpot analysis Peptide stimulation

ELISpot assay principle


T-cell ELISpot

T細胞被廣泛應用在研究特別的免疫反應,例如 : 特異性免疫反應、癌症、過敏和自身免疫性疾病。T-cell ELISpot也被認為是用來開發與監控新疫苗和候選疫苗。

Human IFN-γ ELISpot No stimuli

Human IFN-γ ELISpot Anti-CD3

Human IFN-γ ELISpot PHA

Human IFN-γ ELISpot PPD

Human IFN-γ ELISpot

IFN-γ secretion by PBMCs incubated overnight without stimuli or with anti-CD3, phytohaem-agglutinin (PHA) or purifi ed protein derivative (PPD).

 

B-cell ELISpot

B-cell ELISPot 是用來研究抗體免疫反應的強大工具。主要的應用包括偵測B細胞對感染和接種引起的反應。

Human IgG ELISpot

Human IgA ELISpot

Human IgG and IgA ELISpot

PBMCs were collected before and after vaccination with Pandemrix ® and secretion of influenza A (H1N1)-specifi c IgG (left) and IgA (right) was analyzed by ELISpot.



文獻選讀

疫苗效率測試-COVID19
2020 Cell Press

A Thermostable mRNA Vaccine against COVID-19.

N. Zhang et al. 分析小鼠或非人類靈長類施打 COVID-19 RNA 疫苗後 T 細胞免疫反應。

病毒效力測試 SFTSV
2019 Nature Communications

Development of a SFTSV DNA vaccine that confers complete protection against lethal infection in ferrets.

J.E. Kwak et al. 開發疫苗過程中,利用 ELISpot 分析小鼠和雪貂 (Ferrets)經由 SFTSV 疫苗施打後,疫苗誘導中和抗體和特異性T細胞的反應性。

癌症研究
2014 Nature

Checkpoint blockade cancer immunotherapy targets tumour-specific mutant antigens.

Prof. James P. Allison 致力於利用免疫檢查點抑制劑來治療癌症,在 2018 年榮獲諾貝爾生醫獎。
M Gubin et al. 在研究中, 多次使用 EliSpot 分析免疫檢查點抑制劑細胞治療後特異性T細胞的反應。

樹突細胞疫苗之研究
2018 Nature Protocol

Promoting the accumulation of tumor-specific T cells in tumor tissues by dendritic cell vaccines and chemokine-modulating agents.

N. Obermajer et al. 利用 ELISpot 協助評估樹突細胞疫苗治療過程中腫瘤內特異性 T 細胞數量變化,可偵測、定量腫瘤特異性免疫反應。

巨噬細胞之研究
2013 PLOS ONE

ApoE Production in Human Monocytes and Its Regulation by Inflammatory Cytokines

B. A.Sten et al. 利用apoE ELISpot/FluoroSpot從單顆細胞層面分析,患者在發炎過程中,細胞因子調節載脂蛋白的分泌。

Human

ANALYTEAVAILABLE ELISPOT KIT FORMATS
BasicPlusPro
ApoE
GM-CSF
Granzyme B
IFN-α2
IFN-α pan
IFN-γ * *
IgA
IgE
IgG
IgM
IL-1β
IL-2
IL-4
IL-5
IL-6
IL-8 (CXCL8)
IL-10
IL-12 (p70)
IL-12/-23 (p40)
IL-13
IL-17A
IL-21
IL-22
IL-23
IL-31
Perforin
TNF-α

Monkey

ANALYTEAVAILABLE ELISPOT KIT FORMATS
BasicPlusPro
GM-CSF
IFN-α pan
IFN-γ * *
IgA
IgG
IgM
IL-2
IL-4
IL-5
IL-6
IL-8 (CXCL8)
IL-12/-23 (p40)
IL-13
IL-17A
IL-21
IL-23
Perforin
TNF-α

Mouse

ANALYTEAVAILABLE ELISPOT KIT FORMATS
BasicPlusPro
IFN-γ
IgA
IgG
IgM
IL-2
IL-4
IL-5
IL-6
IL-10
IL-12 (p70)
IL-12/-23 (p40)
IL-17A
TNF-α

Rat

ANALYTEAVAILABLE ELISPOT KIT FORMATS
BasicPlusPro
IFN-γ

Rabbit

ANALYTEAVAILABLE ELISPOT KIT FORMATS
BasicPlusPro
IFN-γ

Cow

ANALYTEAVAILABLE ELISPOT KIT FORMATS
BasicPlusPro
IFN-γ
IL-2
IL-4
IL-8 (CXCL8)

Horse

ANALYTEAVAILABLE ELISPOT KIT FORMATS
BasicPlusPro
IFN-γ

Sheep

ANALYTEAVAILABLE ELISPOT KIT FORMATS
BasicPlusPro
IFN-γ
IL-4

Pig

ANALYTEAVAILABLE ELISPOT KIT FORMATS
BasicPlusPro
IFN-γ

Dog

ANALYTEAVAILABLE ELISPOT KIT FORMATS
BasicPlusPro
IFN-γ
IL-8 (CXCL8)

Ferret

ANALYTEAVAILABLE ELISPOT KIT FORMATS
BasicPlusPro
IFN-γ
原廠文獻查詢

  • Zhang et al. (2020). A Thermostable mRNA Vaccine against COVID-19. Cell Press 182, 1271–1283.
  • Gubin, M. (2014) 'Checkpoint blockade cancer immunotherapy targets tumour-specific mutant antigens'. Nature. 515(7528):577-581.​
  • Obermajer,N et al. (2018). Promoting the accumulation of tumor-specific T cells in tumor tissues by dendritic cell vaccines and chemokine-modulating agents. Nature Protocols volume 13, 335–357.
  • J. E. Kwak et al.(2019) Development of a SFTSV DNA vaccine that confers complete protection against lethal infection in ferrets. Nature Communtions. 10:3836.
  • Braesch-Andersen S. et al. (2013) ApoE Production in Human Monocytes and Its Regulation by Inflammatory Cytokines. PLoS ONE 8(11): e79908.