ワクチンと新規治療が提起する複雑な問題に対応する ELISPOT

新しいワクチンや免疫療法の登場と臨床現場への投入が加速するのに伴い、ますます高度化する試験プラットフォームを中心とした個別化医療、免疫プロファイリング、免疫監視などの分野が次々と誕生しています。なかでも、ELISpot(Enzyme-Linked ImmunoSpot、酵素免疫スポット)群の免疫アッセイは、単細胞解析の機能的アッセイとして最も広く使用されています。1

ELISPOT チャートTrialtrove(Citeline.com)の2017年のデータによると、ELISpot アッセイは 160 件を超えるオープン試験で使用されました(図 1)。このように臨床での利用が増えた主な理由は次の通りです:

  • 先進国で進む高齢化に伴う感染症や慢性疾患の有病率の増加
  • オンコロジーの新しいワクチンや免疫療法における免疫アッセイの広範な利用
  • 試験の自動化や解析時間の短縮といった技術的進歩
  • バイオテクノロジー分野の成長

Looking for the Right Spot

In the ELISpot assay, cell-secreted analytes (cytokine, immunoglobulin, etc.) are captured by an antibody-coated membrane at the bottom of ELISA-like plates. After removal of cells, the captured analyte is visualized by deposition of a colored substrate, which results in the formation of spots on the membrane. Spots are a representation of cells that secrete the analyte of interest, the amount of analyte secreted and its kinetics.2 The spots are then counted and quantitative reports are generated like the frequency of antigen-specific T-cells3 following immunization or treatment.4 The ELISpot assay where human interferon gamma is detected is the IFN-γ ELISpot assay.

IFN-γ
 ELISpot 

The induction of a potent lymphocyte effector function is central to the activity of novel biological therapeutics. Due to its known role, IFN-γ  has become a predominant marker for the induction of cellular immune responses.5-7 Correspondingly, for the past two decades, the IFN-γ ELISpot assay is a highly sensitive yet “simple” platform to detect and quantify antigen-specific cellular responses. It has also become the benchmark for analysis of T-cell responses according to Good Clinical Laboratory Practice guidance.8 It is used in vaccines, transplantation, HIV, cancer and allergy, 4,5,6,7  and more. The IFN-γ ELISpot assay carries nearly 55% of all ELISpot testing needs in ongoing clinical trial evaluation. Because of that, and the ongoing industry expansion into polyfunctional cell analysis,9,10 much attention has been given to the quality of the IFN-γ ELISpot assay.

However, the simplicity of the IFN-γ ELISpot assay only seems apparent. Results from proficiency panels initiated by several organizations revealed drastic differences in the reported results by participant labs.11,12,13 There are several strictly controlled variables that influence the assay outcome. Those include the test medium, the antigen (stimulus), cell storage conditions, cell viability, staff training levels and the actual counting and quantification approach of the ELISpot plates.

A Holistic Approach to ELISpot Development, Qualification and Validation

Successful implementation of any ELISpot testing solution during clinical trials does not happen in a vacuum if assay variables and sample integrity are to be kept in check. Part of the Covance IFN-γ ELISpot integrated solution is a dedicated Targeted Cells Isolation team, who is responsible for processing/producing the cellular component that goes into the assay (peripheral blood mononuclear cells), T-cells, B-cells, etc.), avoiding costly sample processing, minimizing risk and additional fees incurred when shipping live frozen cells to different specialty laboratories.

Likewise, regulatory proficiency, global reach, proven logistics and a dedicated, scalable ELISpot solution with increasing testing volumes – according to the specific study needs – are all critical to the success of a clinical trial program using ELISpot. Covance, at its Central Laboratories (CLS), offers a wide array of tests in the areas of genomics, flow cytometry and histology – all under one roof as these tests generate mutually complementary data to that of ELISpot assays. This enables sponsors to create functional immunoprofiles that capture the overall cellular immunity status of clinical trial subjects.

Covance has offered IFN-γ ELISpot testing for several years in our Translational Biomarker Solutions laboratories. However, and more importantly, Covance just added IFN-γ ELISpot capabilities at our Vaccine and Novel Immunotherapeutic Laboratory (within the CLS) in Indianapolis, offering sponsors a one-stop solution for any IFN-γ ELISpot assay in their clinical testing programs.

Positioned for the Future

In addition to the traditional colorimetric quantification of secreted cytokines, the Covance ELISpot platform allows for the development and execution of multiplexed FluoroSpot assays (Figure 2), the next-generation of ELISpot, which enables simultaneous measurement of different analytes.14,15  The capability to validate novel FluoroSpot assays using a modular approach will allow for a “plug-and-play” flexible offering that will decrease development time for sponsor-specific antigens and targets. The ELISpot platform at Covance’s Vaccine and Novel Immunotherapeutic Laboratory is ideally positioned to support upcoming developments in the field.


参照
1-Czerkinsky CC, et al J Immunol Methods. 1983;65:109-121.
2- Karulin A.Y, Lehmann, PV. Methods Mol Biol. 2012;792:125-143.
3- Nanan R, et al  J Gen Virol. 2000;81:1313-1319.
4- Carvalho L.H, et al. J Immunol Methods. 2001;252:207-218.
5- Flynn K., et al. Immunity. 1998;8:683-691.
6- Bercovici N, et al. Clin Diag Lab Immunol. 2000;7:859-864.
7- Carter LL, Swain SL. Curr Opin Immunol. 1997;9:177-182.
8- Ezzelle J, et al. J Pharm Biomed Anal. 2008;46(1):18-29.
9- Dillenbeck T, et al. Cells. 2014;3(4):1116-1130.
10- Gazagne A, et al. J Immunol Methods. 2003;283:91-98.
11- Janetzki S, et al. Cancer Immunol. Immunother. 2008;57:303-315.
12- Britten C.M, et al. Cancer Immunol Immunother. 2008;57:289-302.
13- Cox J.H, et al. AIDS Res Hum Retroviruses. 2005;21:68-81.
14- Janetzki S, et al. Cells. 2014;3(4):1102-1115.
15- Gazagne A, et al. J Immunol Methods. 2003;283(1-2):91-98.