Thursday, September 15, 2005

C.T.L ELISPOT細胞激素檢測系統


Cell_Bio Blog
C.T.L ELISPOT細胞激素檢測系統

C.T.L ELISPOT Assays for Cells That Secrete Cytokines



ELISPOT(Enzyme-linked immunospot)最早是被發展來偵測分泌特定抗體的細胞,目前ELISPOT已經被廣泛用來偵測某些製造並分泌特定反應因子的細胞,這些反應因子包含細胞激素(Cytokine)、趨化激素(Chemokine)或是顆粒酵素(Granzyme)。這種技術測量細胞在被刺激後産生細胞激素的頻率,而此頻率的變化常常預示著治療和(或)病理的狀態,現已廣泛運用於歐美各大實驗室的疾病診斷和科學研究活動中,在國際上現在最熱門的細胞因子檢測方法。ELISPOT方法操作簡單,而且能提供非常類似於體內實驗的環境(ex vivo),對於測量週邊血或其中淋巴球的免疫功能而言,是一項功能強大的工具。



實驗原理

ELISPOT原理與ELISA相似,其全文為Enzyme-linked Immunospot Assay。本公司生物科學部研發出ELISPOT檢測試劑是在披覆PVDF膜的培養96微孔盤上,吸附上經純化、且無毒性(不含sodium azide、內毒素endotoxin)的抗體。當培養在微孔盤中的細胞被活化後,這些被固定且具特異性的抗體,可以捕獲細胞局部産生的細胞激素。微孔盤中的細胞被移除並清洗後,被捕獲的細胞激素可進一步使用生物素(Biotin)標記的二次抗體來標誌,其後再以結合酵素的Avidin與之作用,並加入酵素受質使其呈色,有反應作用的細胞留下約10-20mm大小染色的斑點。最後可以使用傳統方法以顯微鏡進行人工判讀,或使用高效能之影像分析系統(ELISPOT Plate Reader)進行判讀。斑點數目多寡及大小與細胞反應作用力有關,例如反應出免疫系統作用強弱。此法靈敏度極高,可用以檢測作用細胞數目,例如Th0/1/2/3、DC1/2、T1/2等。



實驗方法的改進

目前,由於96微孔盤使用材質及判讀機器的技術之長足改進,使得ELISPOT技術在使用及實用上更為方便,尤其是對於激素分泌細胞的快速定量,及針對每個細胞所分泌激素量相對比較的應用上更為顯著。PVDF膜材質的運用使得ELISPOT在分析斑點時,可以大幅增加訊號相對於雜訊的比例。PVDF膜相較於傳統的吸收膜有著更優越的捕捉能力;同時,以電腦輔助的影像收集及使用ELISPOT的專用分析軟體,可以大幅地增強ELISPOT技術,使其可以在短時間內客觀並準確地處理大量的實驗數據。

如何詮釋數據?

免疫學研究人員可以使用傳統的顯微鏡計算斑點數,或是使用ImmunoSpot® Analyzer自動分析儀。在分析ELISPOT實驗數據的過程中,ImmunoSpot® Analyzer先以高解析度鏡頭捕捉微小孔中膜上呈色影像,而後儲存成TIF檔或是壓縮成JPEG檔;這些影像可以進一步使用手動、或是自動方式計算斑點數目,如果使用ImmuneSpot分析軟體,可以先設定條件,然後同時分析斑點的大小,以推估激素分泌的多寡。










ImmunoSpot® Analyzer經由美國俄亥俄卅Case Western Reserve大學Dr. Paul Lehmann教授研究開發,該研究團隊改善增強影像儀之分析能力,開發出全自動掃描分析平台,可以精確地評估細胞激素分泌細胞生產頻率、每個細胞分泌量、及辨別細胞激素為同源或是異源分泌。新型的分析儀及其軟體可以提供各種不同的數據格式,包括未處理與處理過的膜表面影像、每個小孔中的斑點數目、每個小孔中的平均斑點數目、每個小孔中斑點大小的直方統計圖。此一全自動掃描分析平台,克服傳統顯微鏡判讀方法耗費人力、及人為客觀性等缺點,解除了ELISPOT分析技術的瓶頸問題。本公司生物科學部與美國俄亥俄卅CTL Analyzers公司合作,將在近期推出一系列自動分析儀,本公司同時提供完整、全新的ELISPOT試劑組套,希望促使ELISPOT技術得以全面推廣。



單一細胞等級高通量快速篩選

目前為止,以目視法判讀少量細胞族群的特性,如激活T細胞之激素分泌,仍被視為是免疫學技術上的一大挑戰;由於缺乏直接測量T細胞分泌激素的方法,阻礙了免疫疾病研究及治療的進展,舉例如下之免疫疾病:過敏、自動免疫疾病、癌症、移植及傳染性疾病或其疫苗在臨床試驗之研究,ELISPOT技術輔以影像自動分析,被認為是提供上述研究訊息靈敏度高且快速大量的解決方法,ELISPOT除了高靈敏度及快速偵測的優點,這項技術同時具有大量分析、細胞需求量小以及可以分析冷凍保存的淋巴球等優點。



ELISPOT應用範圍:

- 移植研究 – allograft rejection

- 疫苗開發 -亦可評估疫苗效力,或是疫苗注射路徑影響

- Th0/Th1/Th2細胞轉換分析

- 自體免疫研究 – 免疫調節及免疫治療研究

- 癌症研究 -腫瘤反應T細胞作用

- 過敏機制探討 – IL-4誘導免疫球蛋白亞型轉換,其他參與過敏機制之細胞激素研究

- 傳染疾病研究 – Lyme disease, Chlamydia infections, Helicobacter pylori infections, HIV infections, multiple sclerosis, …等

- 抗原決定位定圖

- 體液免疫力研究 – B細胞免疫球蛋白及特定亞型反應的進展研究



ELISPOT免疫斑點技術

實驗步驟

首日

1. Coating Antibody:


a. Dilute the Capture Antibody to the recommended concentration with coating buffer*. Add 100 µl of diluted antibody solution to each well of an ELISPOT plate. When using the ELISPOT Kit format, the plates are pre-coated, therefore, omit steps 1 and 2 for Kits.

b. Replace the ELISPOT plate lid and store plates at 4°C overnight.


提示:前兩天的實驗步驟中,必須在無菌操作櫃中使用 ELISPOT 組套與試劑,並用無菌技術處理細胞。以星號(*) 註記的試劑,請參照「自備試劑」配製。



次日



2. Blocking:


a. Discard the coating antibody. Wash the wells 1X with 200 µl/well of complete tissue culture medium* that contains 10% fetal bovine serum.

b. Add 200 µl/well of complete tissue culture medium*, replace the ELISPOT plate lid and allow blocking for 2 hours at room temperature.

3. Cell Activation:


提示:Kit Protocol begins here. Specific activation protocols including cell concentrations and incubation times will vary depending on the cell type, choice of stimulus, and target analyte of interest. Please note that protein transport inhibitors should not be used for ELISPOT cultures.


a. Discard the complete tissue culture media. (Not necessary for the ELISPOT Kits.)

b. Prepare mitogen or antigen which is diluted in complete tissue culture medium. Add 100 µl/well to ELISPOT plate.

c. Prepare cell suspensions at different densities, (eg, ranging from 1 x 105 cells/ml to 2 x 106 cells/ml). Note that appropriate negative controls should be prepared by adding cells to wells without the particular stimulus and by establishing background wells without cells. Cell titrations can be performed either in another cell culture plate or in tubes and then transferred to the ELISPOT plate microwells. Care should be taken not to touch or damage the coated microwell surface. Cells should be added in 100 µl volumes to ELISPOT plate microwells.

d. After adding the cells, replace the ELISPOT plate lid and incubate the plate at 37°C, 5% CO2 and 99% humidity. The duration of the incubation time will vary depending on the analyte of interest (eg, cultures are usually established for 2 – 48 hr).



第三日

4. Detection Antibody:


a. Aspirate the cultured cell suspensions from the ELISPOT plate microwells. Wash and soak the wells 2 X with 200 µl/well of distilled water (dH2O). Allow wells to soak for 3 – 4 min at each wash step.

b. Wash wells 3 X with 200 µl of PBS-Tween* per well. Discard Wash Buffer.

c. Dilute Detection Antibody in Dilution Buffer*. Add 100 µl per well.

d. Replace the ELISPOT plate lid and incubate for 2 hr at room temperature.

5. Streptavidin-Horseradish Peroxidase (SAv-HRP): (BD Pharmingen Cat. No. 557630)


a. Discard Detection Antibody solution. Wash wells 3 X with 200 µl/well of PBS-Tween. Allow wells to soak for 1 – 2 min at each wash step.

b. Dilute SAv-HRP in Dilution Buffer. Add 100 µl of diluted SAv-HRP per well.

c. Replace the ELISPOT plate lid; incubate for 1 hr at room temperature.



6. Substrate:


a. Discard SAv-HRP solution. Wash wells 4 X with 200 µl of PBS-Tween per well. Allow wells to soak for 1–2 min at each wash step.

b. Wash and soak wells 2 X with 200 µl of PBS per well.

c. Add 100 µl of AEC Substrate Solution* to each well. Monitor spot development at room temperature from 5 – 60 min. Do not let color overdevelop. This will lead to high background.

d. Stop the substrate reaction by rinsing wells thoroughly with dH2O.

e. Air-dry plate for 2 hr or overnight in the dark until the plate is completely dried. Removal of plastic tray under 96-well plate facilitates drying. Store the plate in a sealed plastic bag, in the dark, prior to analysis.


f. Enumerate spots manually by inspection under a dissecting microscope (or stationary magnifying glass) or automatically using the ImmunoSpot® Analyzer. With the ImmunoSpot® Analyzer and Software it is possible to generate data in several formats including, unprocessed and processed well membrane images, spot counts per well, mean spot size per well, and spot size histograms for each well.




* ELISPOT Assays 自備試劑


a. Coating Buffer. Dulbecco’s Phosphate Buffered Saline (PBS): 8 g NaCl; 0.2 g KCl; 1.44 g Na2HPO4•7H2O; 0.24 g KH2PO4; add dH2O to 1 liter. Adjust pH to 7.2, autoclave or sterile filter (0.2 µm-pore) and store at 4°C.

b. Complete Tissue Culture Medium: A medium consisting of RPMI 1640 (Bio-Whittaker, Cat. No. 12-167Q) or other suitable medium containing 10% FBS, 1% Penicillin-Streptomycin-L-Glutamine (Gibco-BRL Cat. No. 10378-016), and 5 X 10-5 M 2-mercaptoethanol is often used for culturing human, non-human primate, and rodent cells.

c. PBS-Tween: PBS containing 0.05% Tween-20 (Sigma, P-1379; 0.5 ml Tween-20 per 1 L PBS).

d. Dilution Buffer: PBS containing 10% FBS.

e. Substrate Solution can be prepared or can be purchased (Cat. No. 551951) for convenience from BD Biosciences.























配製 AEC 酵素受質:


1. Prepare AEC (3-amino-9-ethyl-carbazole; Sigma A-5754) stock solution: 100 mg AEC in 10 ml DMF (N,N-Dimethylformamide; Sigma D-4551). Caution: dispense DMF in fume hood. Store solution in glassware.

2. Prepare 0.1 M Acetate Solution: add 148 ml of 0.2 M acetic acid to 352 ml of 0.2 M sodium acetate. Adjust volume to 1 L with distilled water; adjust pH to 5.0.

3. For Final Substrate Solution, add 333.3 µl of AEC stock solution to 10 ml 0.1 M Acetate Solution. Filter through 0.45 µm-pore filter. Add 5 µl of H2O2 (30%) and use immediately.







 
ELISPOT應用文獻:

如需文獻全文,請洽本公司產品專員 (02)2722-5660 ext 242

Our Publications Establishing lmmunoSpot?br> as a 7/p> Validated Technology

for Direct Ex Vivo T Cell Monitoring

Single Cell Resolution/Exact Frequency Measurements of Antigen-Specific T cells Ex Vivo.

• Helms, T., B.O. Boehm, R. J. Assad, R.T. Trezza, P.V. Lehmann, and M. Tary-Lehmann. 2000. Direct visualization of cytokine-producing, recall antigen-specific CD4 memory T cells in healthy individuals and HIV patients. J. Immunol. 164: 3723-3732 (Fig. 1). 7

• Karulin, A.Y., M.D. Hesse, M. Tary-Lehmann, and P.V. Lehmann. 2000. Single-cytokine-producing CD4 memory cells prevail in vivo, in type I/type 2 immunity. /Immunol. 164:1862-1872 (Fig. 1). 7

• Hesse, M.D., A.Y. Karulin, B.O. Boehm, P.V. Lehmann, and M. Tary-Lehmann. 2001. A T cell clone's avidity is a function of its activation state./ Immunol. 167: 1353-1361 (Fig. 1). 7

• Targoni, O.S., J. Baus, H.H. Hoffstetter, M.D. Hesse, A.Y. Karulin, B.O. Boehm, T. Forsthuber, and P.V. Lehmann. 2001. Frequencies of neuroantigen-specific T cells in the CNS versus the immune periphery during the course of EAE. / Immunol. 166: 4757-4764 (Fig. 1).

Measuring Type I/Type 2 Immunity
• Helms, T., B.O. Boehm, R. J. Assad, R.T. Trezza, P.V. Lehmann, and M. Tary-Lehmann. 2000. Direct visualization of cytokine-producing, recall antigen-specific CD4 memory T cells in healthy individuals and HIV patients. / Immunol. 164: 3723-3732 (Tab. I/Fig. 3). 7

• Forsthuber, T., H.C. Yip, and P.V. Lehmann. 1996. Induction of TH1 and TH2 immunity in neonatal mice. Science. 271: 1728-1730 (Tab. 1). 7

• Chu, R.S., O.S. Targoni, A.M. Krieg, P.V. Lehmann, and C.V. Harding. 1997. CpG oligodeoxynucleotides act as adjuvants that switch on T helper 1 (Thi) immunity. /Exp. Med. 186: 1623-1631 (Fig. 2 & 3). 7

• Yip, H.C., A.Y. Karulin, M. Tary-Lehmann, M.D. Hesse, H. Radeke, P. Heeger, R.T. Trezza, F.P. Heinzel, T. Forsthuber, and P.V. Lehmann. 1999. Adjuvant guided type 1 and type 2 immunity: Infectious/non-infectious dichotomy defines the class of response. J. Immunol.162: 3942-3949

(Tab. 1). 7

• Karulin, A.Y., M.D. Hesse, H.C. Yip, and P.V. Lehmann. 2002. Indirect IL-4 pathway in Thi immunity. / Immunol. 167: in press January/February (All figures).

Cognate Cytokine Production by T Cells vs Bystander Cytokine Production





• Helms, T., B.O. Boehm, R. J. Assad, R.T. Trezza, P.V. Lehmann, and M. Tary-Lehmann. 2000. Direct visualization of cytokine-producing, recall antigen-specific CD4 memory T cells in healthy individuals and HIV patients. J. Immunol. 164: 3723-3732 (Fig. 3). 7

• Karulin, A.Y., M.D. Hesse, M. Tary-Lehmann, and P.V. Lehmann. 2000. Single-cytokine-producing CD4 memory cells prevail in vivo, in type I/type 2 immunity. J. Immunol. 164:1862-1872 (Fig 4). 7

• Karulin, A.Y., M. Hesse, H.C. Yip., and P.V. Lehmann. 2002. Indirect IL-4 pathway in Thi immunity. / Immunol. 167: in press (Tab. 1).

Measurements of Functional T Cell Avidity
• Hesse, M.D., A.Y. Karulin, B.O. Boehm, P.V. Lehmann, and M. Tary-Lehmann. 2001. A T cell clone's avidity is a function of its activation state. J. Immunol. 167: 1353-1361 (Fig. 2A). 7

• Targoni, O.S. and P.V. Lehmann. 1998. Endogenous myelin basic protein inactivates the high avidity T cell repertoire. / Exp. Med. 187: 2055-2063 (Fig. 2A). 7

• Yip, H.C., A.Y. Karulin, M. Tary-Lehmann, M.D. Hesse, H. Radeke, P. Heeger, R.T. Trezza, P.P. Heinzel, T. Forsthuber, and P.V. Lehmann. 1999. Adjuvant guided type 1 and type 2 immunity: Infectious/non-infectious dichotomy defines the class of response. J. Immunol. 162: 3942-3949 (Fig. 5C & D). 7

• Karulin, A.Y., M.D. Hesse, M. Tary-Lehmann, and P.V. Lehmann. 2000. Single-cytokine-producing CD4 memory cells prevail in vivo, in type I/type 2 immunity. J.Immunol. 164:1862-1872 (Fig. 6). 7

Cytokine Co-Expression/Two Color ELISPOT

• Karulin, A., M.Hesse, M. Tary-Lehmann, and P.V. Lehmann. 2000. Single-cytokine-producing CD4 memory cells prevail in vivo, in type I/type 2 immunity. J. Immunol. 164:1862-1872 (Figs. 2, 4, 5, 7). 7

• Heeger, P.S., T. Forsthuber, C. Shive, E. Beikert, C.P. Genain, H.H. Hofstetter, A.Y Karulin, and P.V. Lehmann. 2000. Revisiting tolerance induced by autoantigen in incomplete Freund's adjuvant. / Immunol.164:5771-5781 (Fig. 2). 7

• Karulin, A.Y., M.D. Hesse, H.C. Yip, and P.V. Lehmann. 2002. Indirect IL-4 pathway in Thi immunity. / Immunol. 167: in press January/February (Fig. 1).

Spot Size Distribution
• Karulin, A.Y., M.D. Hesse, M. Tary-Lehmann, and P.V. Lehmann. 2000.

Single-cytokine-producing CD4 memory cells prevail in vivo, in type I/type 2 immunity. /Immunol. 164:1862-1872 (Fig. 3). 7

• Hesse, M.D., A.Y. Karulin, B.O. Boehm, P.V. Lehmann, and M. Tary-Lehmann. 2001. A T cell clone's avidity is a function of its activation state. / Immunol. 167: 1353-1361 (Figs. 3 & 4).Determinant Mapping/High Throughput Screening

• Targoni, O.S. and P.V. Lehmann. 1998. Endogenous myelin basic protein inactivates

the high avidity T cell repertoire. / Exp. Med. 187: 2055-2063 (Fig. 2A). 7



• Yip, H.C, A.Y. Karulin, M. Tary-Lehmann, M.D. Hesse, H. Radeke, P. Heeger, R.T. Trezza, F.P. Heinzel, T. Forsthuber, and P.V. Lehmann. 1999. Adjuvant guided type 1 and type 2 immunity: Infectious/non-infectiousdichotomy defines the class of response. / Immunol. 162: 3942-3949 (Fig. 5A & B). 7

• Heeger, P.S., A. Valujskikh, and P.V. Lehmann. 2000. Comprehensive assessment of determinant specificity, frequency, and cytokine signature of the primed CDS cell repertoire induced by a minor transplantation antigen./ Immunol. 165: 1278-1284 (Fig. 2). 7

• Pelfrey, C.M., R.A. Rudick, A.C. Cotleur, J.-C. Lee, M. Tary-Lehmann, and P.V. Lehmann. 2000. Quantification of self-recognition in multiple sclerosis by single-cell analysis of cytokine production. / Immunol. 165:1641-1651 (Figs. 2 & 3).Assay to Assay Reproducibility of Ex Vivo Measurements on PBMC

• Helms, T., B.O. Boehm, R. J. Assad, R.T. Trezza, P.V. Lehmann, and M. Tary-Lehmann. 2000. Direct visualization of cytokine-producing, recall antigen-specific CD4 memory T cells in healthy individuals and HIV patients. / Immunol, 164: 3723-3732 (Tab. 2).

Page 2, CTL Establishing ImmunoSpotPas a Validated Technology for Ex Vivo T Cell Monitoring

TB Immunization Function Test

Ewer K, Deeks J, Alvarez L, Bryant G, Waller S, Andersen P, Monk P, Lalvani A. 2003 Apr 5. Comparison of T-cell-based assay with tuberculin skin test for diagnosis of Mycobacterium tuberculosis infection in a school tuberculosis outbreak. Lancet 361:p1168-73

Lyme Disease

Gross DM, Forsthuber T, Tary-Lehmann M et al. 1998: Identification of LFA-1 as a candidate autoantigen in treatment-resistant Lyme-Arthritis. Science 281: 703-706

Chlamydia Infection

Daugharty H, Messmer TO, Fields BS 1997: Elispot assay for Chlamydia-specific, antibody-producing cells correlated with conventional complement fixation and microimmunofluorescence. J Clin Lab Anal 11: 45-52.

Helicobacter pylori Infection

Karttunen R, Karttunen T, Ekre HP & MacDonald TT 1995: Interferon gamma and interleukin 4 secreting cells in the gastric antrum in Helicobacter pylori positive and negative gastritis. Gut 36(3): 341-345.

Mwau, et al. Design and validation of an enzyme-linked immunospot assay for use in clinical trials of candidate HIV vaccines. AIDS Research and Human Retroviruses 18 (9): 611-618 JUN 10 2002

Keir ME, Rosenberg MG, et al. Generation of CD3+CD8low thymocytes in the HIV type 1-infected thymus. J Immunol. 2002 Sep 1;169(5):2788-96.

Shacklett BL, Shaw KE, et al. Attenuated Simian Immunodeficiency Virus SIVmac-M4, with Point Mutations in the Env Transmembrane Protein Intracytoplasmic Domain, Provides Partial Protection from Mucosal Challenge with Pathogenic SIVmac251. J Virol. 2002 Nov 15;76(22):11365-11378.

Shacklett BL, Means RE, et al. Dendritic cell amplification of HIV type 1-specific CD8+ T cell responses in exposed, seronegative heterosexual women. AIDS Res Hum Retroviruses. 2002 Jul 20;18(11):805-15.

Skurnick JH, Palumbo P, et al. Correlates of nontransmission in US women at high risk of human immunodeficiency virus type 1 infection through sexual exposure. J Infect Dis. 2002 Feb 15;185(4):428-38.

Multiple Sclerosis:

McCutcheon M, Wehner N, Wensky A et al. 1997: A sensitive Elispot assay to detect low-frequency human T-lymphocytes. J Immunol Methods 210(2): 149-166.

Calabresi PA, Fields NS, Farnon EC, et al. 1998: ELI-spot of Th-1 cytokine secreting PBMCs in multiple sclerosis: correlation with MRI lesions. J. Neuroimmunol 15;85(2):212-219.

Allergy

Gabrielsson S, Paulie S, Rak S et al. 1997: Specific induction of interleukin-4-producing cells in response to in vitro allergen stimulation in atopic individuals. Clin Exp Allergy 27: 808-815.

Maggi E 1998: The Th1/Th2 paradigm in allergy. Immunotechnology 3(4):233-244.

Allograft Rejection

Tary-Lehmann M, Hricik D, Justice AC et al. 1998: Enzyme-linked immunosorbent assay spot detection of Interferon-gamma and interleukin 5-producing cells as a predictive marker for renal allograft failure. Transplantation 66: 219-224.

C.T.L EliSpot 細胞激素檢測系統

C.T.L ELISPOT細胞激素檢測系統
C.T.L ELISPOT Assays for Cells That Secrete Cytokines

ELISPOT(Enzyme-linked immunospot)最早是被發展來偵測分泌特定抗體的細胞,目前ELISPOT已經被廣泛用來偵測某些製造並分泌特定反應因子的細胞,這些反應因子包含細胞激素(Cytokine)、趨化激素(Chemokine)或是顆粒酵素(Granzyme)。這種技術測量細胞在被刺激後産生細胞激素的頻率,而此頻率的變化常常預示著治療和(或)病理的狀態,現已廣泛運用於歐美各大實驗室的疾病診斷和科學研究活動中,在國際上現在最熱門的細胞因子檢測方法。ELISPOT方法操作簡單,而且能提供非常類似於體內實驗的環境(ex vivo),對於測量週邊血或其中淋巴球的免疫功能而言,是一項功能強大的工具。

實驗原理
ELISPOT原理與ELISA相似,其全文為Enzyme-linked Immunospot Assay。本公司生物科學部研發出ELISPOT檢測試劑是在披覆PVDF膜的培養96微孔盤上,吸附上經純化、且無毒性(不含sodium azide、內毒素endotoxin)的抗體。當培養在微孔盤中的細胞被活化後,這些被固定且具特異性的抗體,可以捕獲細胞局部産生的細胞激素。微孔盤中的細胞被移除並清洗後,被捕獲的細胞激素可進一步使用生物素(Biotin)標記的二次抗體來標誌,其後再以結合酵素的Avidin與之作用,並加入酵素受質使其呈色,有反應作用的細胞留下約10-20mm大小染色的斑點。最後可以使用傳統方法以顯微鏡進行人工判讀,或使用高效能之影像分析系統(ELISPOT Plate Reader)進行判讀。斑點數目多寡及大小與細胞反應作用力有關,例如反應出免疫系統作用強弱。此法靈敏度極高,可用以檢測作用細胞數目,例如Th0/1/2/3、DC1/2、T1/2等。

實驗方法的改進
目前,由於96微孔盤使用材質及判讀機器的技術之長足改進,使得ELISPOT技術在使用及實用上更為方便,尤其是對於激素分泌細胞的快速定量,及針對每個細胞所分泌激素量相對比較的應用上更為顯著。PVDF膜材質的運用使得ELISPOT在分析斑點時,可以大幅增加訊號相對於雜訊的比例。PVDF膜相較於傳統的吸收膜有著更優越的捕捉能力;同時,以電腦輔助的影像收集及使用ELISPOT的專用分析軟體,可以大幅地增強ELISPOT技術,使其可以在短時間內客觀並準確地處理大量的實驗數據。
如何詮釋數據?
免疫學研究人員可以使用傳統的顯微鏡計算斑點數,或是使用ImmunoSpot® Analyzer自動分析儀。在分析ELISPOT實驗數據的過程中,ImmunoSpot® Analyzer先以高解析度鏡頭捕捉微小孔中膜上呈色影像,而後儲存成TIF檔或是壓縮成JPEG檔;這些影像可以進一步使用手動、或是自動方式計算斑點數目,如果使用ImmuneSpot分析軟體,可以先設定條件,然後同時分析斑點的大小,以推估激素分泌的多寡。

ImmunoSpot® Analyzer經由美國俄亥俄卅Case Western Reserve大學Dr. Paul Lehmann教授研究開發,該研究團隊改善增強影像儀之分析能力,開發出全自動掃描分析平台,可以精確地評估細胞激素分泌細胞生產頻率、每個細胞分泌量、及辨別細胞激素為同源或是異源分泌。新型的分析儀及其軟體可以提供各種不同的數據格式,包括未處理與處理過的膜表面影像、每個小孔中的斑點數目、每個小孔中的平均斑點數目、每個小孔中斑點大小的直方統計圖。此一全自動掃描分析平台,克服傳統顯微鏡判讀方法耗費人力、及人為客觀性等缺點,解除了ELISPOT分析技術的瓶頸問題。本公司生物科學部與美國俄亥俄卅CTL Analyzers公司合作,將在近期推出一系列自動分析儀,本公司同時提供完整、全新的ELISPOT試劑組套,希望促使ELISPOT技術得以全面推廣。

單一細胞等級高通量快速篩選
目前為止,以目視法判讀少量細胞族群的特性,如激活T細胞之激素分泌,仍被視為是免疫學技術上的一大挑戰;由於缺乏直接測量T細胞分泌激素的方法,阻礙了免疫疾病研究及治療的進展,舉例如下之免疫疾病:過敏、自動免疫疾病、癌症、移植及傳染性疾病或其疫苗在臨床試驗之研究,ELISPOT技術輔以影像自動分析,被認為是提供上述研究訊息靈敏度高且快速大量的解決方法,ELISPOT除了高靈敏度及快速偵測的優點,這項技術同時具有大量分析、細胞需求量小以及可以分析冷凍保存的淋巴球等優點。

ELISPOT應用範圍:
- 移植研究 – allograft rejection
- 疫苗開發 -亦可評估疫苗效力,或是疫苗注射路徑影響
- Th0/Th1/Th2細胞轉換分析
- 自體免疫研究 – 免疫調節及免疫治療研究
- 癌症研究 -腫瘤反應T細胞作用
- 過敏機制探討 – IL-4誘導免疫球蛋白亞型轉換,其他參與過敏機制之細胞激素研究
- 傳染疾病研究 – Lyme disease, Chlamydia infections, Helicobacter pylori infections, HIV infections, multiple sclerosis, …等
- 抗原決定位定圖
- 體液免疫力研究 – B細胞免疫球蛋白及特定亞型反應的進展研究

ELISPOT免疫斑點技術
實驗步驟
首日
1. Coating Antibody:
a. Dilute the Capture Antibody to the recommended concentration with coating buffer*. Add 100 µl of diluted antibody solution to each well of an ELISPOT plate. When using the ELISPOT Kit format, the plates are pre-coated, therefore, omit steps 1 and 2 for Kits.
b. Replace the ELISPOT plate lid and store plates at 4°C overnight.

提示:前兩天的實驗步驟中,必須在無菌操作櫃中使用 ELISPOT 組套與試劑,並用無菌技術處理細胞。以星號(*) 註記的試劑,請參照「自備試劑」配製。
次日

2. Blocking:
a. Discard the coating antibody. Wash the wells 1X with 200 µl/well of complete tissue culture medium* that contains 10% fetal bovine serum.
b. Add 200 µl/well of complete tissue culture medium*, replace the ELISPOT plate lid and allow blocking for 2 hours at room temperature.
3. Cell Activation:

提示:Kit Protocol begins here. Specific activation protocols including cell concentrations and incubation times will vary depending on the cell type, choice of stimulus, and target analyte of interest. Please note that protein transport inhibitors should not be used for ELISPOT cultures.

a. Discard the complete tissue culture media. (Not necessary for the ELISPOT Kits.)
b. Prepare mitogen or antigen which is diluted in complete tissue culture medium. Add 100 µl/well to ELISPOT plate.
c. Prepare cell suspensions at different densities, (eg, ranging from 1 x 105 cells/ml to 2 x 106 cells/ml). Note that appropriate negative controls should be prepared by adding cells to wells without the particular stimulus and by establishing background wells without cells. Cell titrations can be performed either in another cell culture plate or in tubes and then transferred to the ELISPOT plate microwells. Care should be taken not to touch or damage the coated microwell surface. Cells should be added in 100 µl volumes to ELISPOT plate microwells.
d. After adding the cells, replace the ELISPOT plate lid and incubate the plate at 37°C, 5% CO2 and 99% humidity. The duration of the incubation time will vary depending on the analyte of interest (eg, cultures are usually established for 2 – 48 hr).

第三日
4. Detection Antibody:
a. Aspirate the cultured cell suspensions from the ELISPOT plate microwells. Wash and soak the wells 2 X with 200 µl/well of distilled water (dH2O). Allow wells to soak for 3 – 4 min at each wash step.
b. Wash wells 3 X with 200 µl of PBS-Tween* per well. Discard Wash Buffer.
c. Dilute Detection Antibody in Dilution Buffer*. Add 100 µl per well.
d. Replace the ELISPOT plate lid and incubate for 2 hr at room temperature.
5. Streptavidin-Horseradish Peroxidase (SAv-HRP): (BD Pharmingen Cat. No. 557630)

a. Discard Detection Antibody solution. Wash wells 3 X with 200 µl/well of PBS-Tween. Allow wells to soak for 1 – 2 min at each wash step.
b. Dilute SAv-HRP in Dilution Buffer. Add 100 µl of diluted SAv-HRP per well.
c. Replace the ELISPOT plate lid; incubate for 1 hr at room temperature.

6. Substrate:
a. Discard SAv-HRP solution. Wash wells 4 X with 200 µl of PBS-Tween per well. Allow wells to soak for 1–2 min at each wash step.
b. Wash and soak wells 2 X with 200 µl of PBS per well.
c. Add 100 µl of AEC Substrate Solution* to each well. Monitor spot development at room temperature from 5 – 60 min. Do not let color overdevelop. This will lead to high background.
d. Stop the substrate reaction by rinsing wells thoroughly with dH2O.
e. Air-dry plate for 2 hr or overnight in the dark until the plate is completely dried. Removal of plastic tray under 96-well plate facilitates drying. Store the plate in a sealed plastic bag, in the dark, prior to analysis.

f. Enumerate spots manually by inspection under a dissecting microscope (or stationary magnifying glass) or automatically using the ImmunoSpot® Analyzer. With the ImmunoSpot® Analyzer and Software it is possible to generate data in several formats including, unprocessed and processed well membrane images, spot counts per well, mean spot size per well, and spot size histograms for each well.

* ELISPOT Assays 自備試劑
a. Coating Buffer. Dulbecco’s Phosphate Buffered Saline (PBS): 8 g NaCl; 0.2 g KCl; 1.44 g Na2HPO4•7H2O; 0.24 g KH2PO4; add dH2O to 1 liter. Adjust pH to 7.2, autoclave or sterile filter (0.2 µm-pore) and store at 4°C.
b. Complete Tissue Culture Medium: A medium consisting of RPMI 1640 (Bio-Whittaker, Cat. No. 12-167Q) or other suitable medium containing 10% FBS, 1% Penicillin-Streptomycin-L-Glutamine (Gibco-BRL Cat. No. 10378-016), and 5 X 10-5 M 2-mercaptoethanol is often used for culturing human, non-human primate, and rodent cells.
c. PBS-Tween: PBS containing 0.05% Tween-20 (Sigma, P-1379; 0.5 ml Tween-20 per 1 L PBS).
d. Dilution Buffer: PBS containing 10% FBS.
e. Substrate Solution can be prepared or can be purchased (Cat. No. 551951) for convenience from BD Biosciences.

配製 AEC 酵素受質:

1. Prepare AEC (3-amino-9-ethyl-carbazole; Sigma A-5754) stock solution: 100 mg AEC in 10 ml DMF (N,N-Dimethylformamide; Sigma D-4551). Caution: dispense DMF in fume hood. Store solution in glassware.
2. Prepare 0.1 M Acetate Solution: add 148 ml of 0.2 M acetic acid to 352 ml of 0.2 M sodium acetate. Adjust volume to 1 L with distilled water; adjust pH to 5.0.
3. For Final Substrate Solution, add 333.3 µl of AEC stock solution to 10 ml 0.1 M Acetate Solution. Filter through 0.45 µm-pore filter. Add 5 µl of H2O2 (30%) and use immediately.

ELISPOT應用文獻:
如需文獻全文,請洽本公司產品專員 (02)2722-5660 ext 242
Our Publications Establishing lmmunoSpot?br> as a 7/p> Validated Technology
for Direct Ex Vivo T Cell Monitoring
Single Cell Resolution/Exact Frequency Measurements of Antigen-Specific T cells Ex Vivo.
• Helms, T., B.O. Boehm, R. J. Assad, R.T. Trezza, P.V. Lehmann, and M. Tary-Lehmann. 2000. Direct visualization of cytokine-producing, recall antigen-specific CD4 memory T cells in healthy individuals and HIV patients. J. Immunol. 164: 3723-3732 (Fig. 1). 7
• Karulin, A.Y., M.D. Hesse, M. Tary-Lehmann, and P.V. Lehmann. 2000. Single-cytokine-producing CD4 memory cells prevail in vivo, in type I/type 2 immunity. /Immunol. 164:1862-1872 (Fig. 1). 7
• Hesse, M.D., A.Y. Karulin, B.O. Boehm, P.V. Lehmann, and M. Tary-Lehmann. 2001. A T cell clone's avidity is a function of its activation state./ Immunol. 167: 1353-1361 (Fig. 1). 7
• Targoni, O.S., J. Baus, H.H. Hoffstetter, M.D. Hesse, A.Y. Karulin, B.O. Boehm, T. Forsthuber, and P.V. Lehmann. 2001. Frequencies of neuroantigen-specific T cells in the CNS versus the immune periphery during the course of EAE. / Immunol. 166: 4757-4764 (Fig. 1).
Measuring Type I/Type 2 Immunity
• Helms, T., B.O. Boehm, R. J. Assad, R.T. Trezza, P.V. Lehmann, and M. Tary-Lehmann. 2000. Direct visualization of cytokine-producing, recall antigen-specific CD4 memory T cells in healthy individuals and HIV patients. / Immunol. 164: 3723-3732 (Tab. I/Fig. 3). 7
• Forsthuber, T., H.C. Yip, and P.V. Lehmann. 1996. Induction of TH1 and TH2 immunity in neonatal mice. Science. 271: 1728-1730 (Tab. 1). 7
• Chu, R.S., O.S. Targoni, A.M. Krieg, P.V. Lehmann, and C.V. Harding. 1997. CpG oligodeoxynucleotides act as adjuvants that switch on T helper 1 (Thi) immunity. /Exp. Med. 186: 1623-1631 (Fig. 2 & 3). 7
• Yip, H.C., A.Y. Karulin, M. Tary-Lehmann, M.D. Hesse, H. Radeke, P. Heeger, R.T. Trezza, F.P. Heinzel, T. Forsthuber, and P.V. Lehmann. 1999. Adjuvant guided type 1 and type 2 immunity: Infectious/non-infectious dichotomy defines the class of response. J. Immunol.162: 3942-3949
(Tab. 1). 7
• Karulin, A.Y., M.D. Hesse, H.C. Yip, and P.V. Lehmann. 2002. Indirect IL-4 pathway in Thi immunity. / Immunol. 167: in press January/February (All figures).
Cognate Cytokine Production by T Cells vs Bystander Cytokine Production


• Helms, T., B.O. Boehm, R. J. Assad, R.T. Trezza, P.V. Lehmann, and M. Tary-Lehmann. 2000. Direct visualization of cytokine-producing, recall antigen-specific CD4 memory T cells in healthy individuals and HIV patients. J. Immunol. 164: 3723-3732 (Fig. 3). 7
• Karulin, A.Y., M.D. Hesse, M. Tary-Lehmann, and P.V. Lehmann. 2000. Single-cytokine-producing CD4 memory cells prevail in vivo, in type I/type 2 immunity. J. Immunol. 164:1862-1872 (Fig 4). 7
• Karulin, A.Y., M. Hesse, H.C. Yip., and P.V. Lehmann. 2002. Indirect IL-4 pathway in Thi immunity. / Immunol. 167: in press (Tab. 1).
Measurements of Functional T Cell Avidity
• Hesse, M.D., A.Y. Karulin, B.O. Boehm, P.V. Lehmann, and M. Tary-Lehmann. 2001. A T cell clone's avidity is a function of its activation state. J. Immunol. 167: 1353-1361 (Fig. 2A). 7
• Targoni, O.S. and P.V. Lehmann. 1998. Endogenous myelin basic protein inactivates the high avidity T cell repertoire. / Exp. Med. 187: 2055-2063 (Fig. 2A). 7
• Yip, H.C., A.Y. Karulin, M. Tary-Lehmann, M.D. Hesse, H. Radeke, P. Heeger, R.T. Trezza, P.P. Heinzel, T. Forsthuber, and P.V. Lehmann. 1999. Adjuvant guided type 1 and type 2 immunity: Infectious/non-infectious dichotomy defines the class of response. J. Immunol. 162: 3942-3949 (Fig. 5C & D). 7
• Karulin, A.Y., M.D. Hesse, M. Tary-Lehmann, and P.V. Lehmann. 2000. Single-cytokine-producing CD4 memory cells prevail in vivo, in type I/type 2 immunity. J.Immunol. 164:1862-1872 (Fig. 6). 7
Cytokine Co-Expression/Two Color ELISPOT
• Karulin, A., M.Hesse, M. Tary-Lehmann, and P.V. Lehmann. 2000. Single-cytokine-producing CD4 memory cells prevail in vivo, in type I/type 2 immunity. J. Immunol. 164:1862-1872 (Figs. 2, 4, 5, 7). 7
• Heeger, P.S., T. Forsthuber, C. Shive, E. Beikert, C.P. Genain, H.H. Hofstetter, A.Y Karulin, and P.V. Lehmann. 2000. Revisiting tolerance induced by autoantigen in incomplete Freund's adjuvant. / Immunol.164:5771-5781 (Fig. 2). 7
• Karulin, A.Y., M.D. Hesse, H.C. Yip, and P.V. Lehmann. 2002. Indirect IL-4 pathway in Thi immunity. / Immunol. 167: in press January/February (Fig. 1).
Spot Size Distribution
• Karulin, A.Y., M.D. Hesse, M. Tary-Lehmann, and P.V. Lehmann. 2000.
Single-cytokine-producing CD4 memory cells prevail in vivo, in type I/type 2 immunity. /Immunol. 164:1862-1872 (Fig. 3). 7
• Hesse, M.D., A.Y. Karulin, B.O. Boehm, P.V. Lehmann, and M. Tary-Lehmann. 2001. A T cell clone's avidity is a function of its activation state. / Immunol. 167: 1353-1361 (Figs. 3 & 4).Determinant Mapping/High Throughput Screening
• Targoni, O.S. and P.V. Lehmann. 1998. Endogenous myelin basic protein inactivates
the high avidity T cell repertoire. / Exp. Med. 187: 2055-2063 (Fig. 2A). 7

• Yip, H.C, A.Y. Karulin, M. Tary-Lehmann, M.D. Hesse, H. Radeke, P. Heeger, R.T. Trezza, F.P. Heinzel, T. Forsthuber, and P.V. Lehmann. 1999. Adjuvant guided type 1 and type 2 immunity: Infectious/non-infectiousdichotomy defines the class of response. / Immunol. 162: 3942-3949 (Fig. 5A & B). 7
• Heeger, P.S., A. Valujskikh, and P.V. Lehmann. 2000. Comprehensive assessment of determinant specificity, frequency, and cytokine signature of the primed CDS cell repertoire induced by a minor transplantation antigen./ Immunol. 165: 1278-1284 (Fig. 2). 7
• Pelfrey, C.M., R.A. Rudick, A.C. Cotleur, J.-C. Lee, M. Tary-Lehmann, and P.V. Lehmann. 2000. Quantification of self-recognition in multiple sclerosis by single-cell analysis of cytokine production. / Immunol. 165:1641-1651 (Figs. 2 & 3).Assay to Assay Reproducibility of Ex Vivo Measurements on PBMC
• Helms, T., B.O. Boehm, R. J. Assad, R.T. Trezza, P.V. Lehmann, and M. Tary-Lehmann. 2000. Direct visualization of cytokine-producing, recall antigen-specific CD4 memory T cells in healthy individuals and HIV patients. / Immunol, 164: 3723-3732 (Tab. 2).
Page 2, CTL Establishing ImmunoSpotPas a Validated Technology for Ex Vivo T Cell Monitoring
TB Immunization Function Test
Ewer K, Deeks J, Alvarez L, Bryant G, Waller S, Andersen P, Monk P, Lalvani A. 2003 Apr 5. Comparison of T-cell-based assay with tuberculin skin test for diagnosis of Mycobacterium tuberculosis infection in a school tuberculosis outbreak. Lancet 361:p1168-73
Lyme Disease
Gross DM, Forsthuber T, Tary-Lehmann M et al. 1998: Identification of LFA-1 as a candidate autoantigen in treatment-resistant Lyme-Arthritis. Science 281: 703-706
Chlamydia Infection
Daugharty H, Messmer TO, Fields BS 1997: Elispot assay for Chlamydia-specific, antibody-producing cells correlated with conventional complement fixation and microimmunofluorescence. J Clin Lab Anal 11: 45-52.
Helicobacter pylori Infection
Karttunen R, Karttunen T, Ekre HP & MacDonald TT 1995: Interferon gamma and interleukin 4 secreting cells in the gastric antrum in Helicobacter pylori positive and negative gastritis. Gut 36(3): 341-345.
Mwau, et al. Design and validation of an enzyme-linked immunospot assay for use in clinical trials of candidate HIV vaccines. AIDS Research and Human Retroviruses 18 (9): 611-618 JUN 10 2002
Keir ME, Rosenberg MG, et al. Generation of CD3+CD8low thymocytes in the HIV type 1-infected thymus. J Immunol. 2002 Sep 1;169(5):2788-96.
Shacklett BL, Shaw KE, et al. Attenuated Simian Immunodeficiency Virus SIVmac-M4, with Point Mutations in the Env Transmembrane Protein Intracytoplasmic Domain, Provides Partial Protection from Mucosal Challenge with Pathogenic SIVmac251. J Virol. 2002 Nov 15;76(22):11365-11378.
Shacklett BL, Means RE, et al. Dendritic cell amplification of HIV type 1-specific CD8+ T cell responses in exposed, seronegative heterosexual women. AIDS Res Hum Retroviruses. 2002 Jul 20;18(11):805-15.
Skurnick JH, Palumbo P, et al. Correlates of nontransmission in US women at high risk of human immunodeficiency virus type 1 infection through sexual exposure. J Infect Dis. 2002 Feb 15;185(4):428-38.
Multiple Sclerosis:
McCutcheon M, Wehner N, Wensky A et al. 1997: A sensitive Elispot assay to detect low-frequency human T-lymphocytes. J Immunol Methods 210(2): 149-166.
Calabresi PA, Fields NS, Farnon EC, et al. 1998: ELI-spot of Th-1 cytokine secreting PBMCs in multiple sclerosis: correlation with MRI lesions. J. Neuroimmunol 15;85(2):212-219.
Allergy
Gabrielsson S, Paulie S, Rak S et al. 1997: Specific induction of interleukin-4-producing cells in response to in vitro allergen stimulation in atopic individuals. Clin Exp Allergy 27: 808-815.
Maggi E 1998: The Th1/Th2 paradigm in allergy. Immunotechnology 3(4):233-244.
Allograft Rejection
Tary-Lehmann M, Hricik D, Justice AC et al. 1998: Enzyme-linked immunosorbent assay spot detection of Interferon-gamma and interleukin 5-producing cells as a predictive marker for renal allograft failure. Transplantation 66: 219-224.