Towards a Full Automation of the ELISpot Assay for Safe and Parallelized Immunomonitoring.

The ELISpot assay, as a sensitive and specific method, enables the detection of cytokines for immunological purposes and in vaccine development. Here we describe the successful transfer of the manual procedure to a commercially available automated liquid handling platform, based on the work described in Neubauer et al. (Cytotechnology 69:57-73, 2017). Different kinds of technical issues (dead volume reduction, instrumental handling limitations, liquid class improvement) have been solved and biological effects (reagents concentration, selectivity tests, dispensing way, etc.) have been controlled during the implementation process. At the end a maximum of 6% mean delta difference and a lower mean dispersion than the manual assay were reached as well as a turnaround time of four to six times higher than the manual process.

[1]  S Janetzki,et al.  Evaluation of Elispot assays: influence of method and operator on variability of results. , 2004, Journal of immunological methods.

[2]  Sylvia Janetzki,et al.  Results and harmonization guidelines from two large-scale international Elispot proficiency panels conducted by the Cancer Vaccine Consortium (CVC/SVI) , 2007, Cancer Immunology, Immunotherapy.

[3]  M. John,et al.  Automation of the ELISpot assay for high-throughput detection of antigen-specific T-cell responses. , 2009, Journal of immunological methods.

[4]  Holden T Maecker,et al.  Precision and linearity targets for validation of an IFNγ ELISPOT, cytokine flow cytometry, and tetramer assay using CMV peptides , 2008, BMC Immunology.

[5]  Jon Wakefield,et al.  The automated counting of spots for the ELISpot assay. , 2006, Journal of immunological methods.

[6]  Sylvia Janetzki,et al.  Guidelines for the automated evaluation of Elispot assays , 2015, Nature Protocols.

[7]  Sylvia Janetzki,et al.  Measurement of cytokine release at the single cell level using the ELISPOT assay. , 2006, Methods.

[8]  A. Meyerhans,et al.  Towards standardized automated immunomonitoring: an automated ELISpot assay for safe and parallelized functionality analysis of immune cells , 2016, Cytotechnology.

[9]  D. Ankerst,et al.  Impact of cryopreservation on tetramer, cytokine flow cytometry, and ELISPOT , 2005, BMC Immunology.

[10]  Sylvia Janetzki,et al.  Response determination criteria for ELISPOT: toward a standard that can be applied across laboratories. , 2012, Methods in molecular biology.

[11]  Ursula Graf-Hausner,et al.  Development of a Novel Automated Cell Isolation, Expansion, and Characterization Platform , 2011, Journal of laboratory automation.

[12]  Sergey S Zadorozhny,et al.  Mathematical algorithms for automatic search, recognition, and detection of spots in ELISPOT assay. , 2012, Methods in molecular biology.