Abstract
The ELISpot assay is used for the detection of T cell responses in clinical trials and vaccine evaluations. Standardization and reproducibility are necessary to compare the results worldwide, inter- and intra-assay variability being critical factors. To assure operator safety as well as high-quality experiment performance, the ELISpot assay was implemented on an automated liquid handling platform, a Tecan Freedom EVO. After validation of the liquid handling, automated loading of plates with cells and reagents was investigated. With step by step implementation of the manual procedure and liquid dispensing optimization on the robot platform, a fully automated ELISpot assay was accomplished with plates remaining in the system from the plate blocking step to spot development. The mean delta difference amounted to a maximum of 6%, and the mean dispersion was smaller than in the manual assay. Taken together, we achieved with this system not only a lower personnel attendance but also higher throughput and a more precise and parallelized analysis. This platform has the potential to guarantee validated, safe, fast, reproducible and cost-efficient immunological and toxicological assays in the future.
Highlights
Developed first in 1983 to detect antibody secreting cells (Czerkinsky et al 1983), the enzyme linked Immunospot (ELISpot) assay improved continually and gained more and more attention over the years (Czerkinsky et al 1988)
In this paper we focus on the automation of the ELISpot assay due to the infectious potential of the assay and the necessity to reduce the risk for the personnel
After measurement of cell concentration and viability on the automated cell analyzer Vi-Cell XR (Beckman Coulter, Krefeld, Germany), peripheral blood mononuclear cells (PBMC) were resuspended in 10 ml R10 medium with a concentration of 1 9 106 cells/ml and cultured overnight (37 °C, 5% CO2) for direct use in the ELISpot assay
Summary
Developed first in 1983 to detect antibody secreting cells (Czerkinsky et al 1983), the enzyme linked Immunospot (ELISpot) assay improved continually and gained more and more attention over the years (Czerkinsky et al 1988). Measurement and characterization of immune cell activities in clinical and cancer trials (Cox et al 2006), it helps to evaluate new vaccines in order to control and prevent infectious diseases such as those caused by mycobacterium tuberculosis (Bathoorn et al 2011; Kobashi et al 2010), hepatitis-C-virus and HIV (Lee et al 1989) Despite this potential, inter-operator and interassay inconsistency of the measurement is one of the most critical limitations of the method (Janetzki 2004). The CVC described in a guideline the recommendations for successful assay outcome such as established laboratory standard operation procedures (SOP), counting method, cell preparation, serum quality and spot evaluation These conditions result from two ELISpot proficiency panels initiated in 2005 which were aimed to identify deficient practices and common sources of variability between laboratories to increase standardization (Janetzki et al 2008). Despite this progress of automation, the ELISpot assay is still a very error-
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