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Interferon-gamma (IFNγ) ELISpot and FluoroSpot are widely used assays to detect functional cell responses in immunotherapy clinical studies. Recognized for their importance in vaccine development studies to quantitate immune responses, these assays have more recently risen to the forefront in cell and gene therapy as well as cancer immunotherapy fields where responses against cancer neoantigens are not easily detectable above assay background. Here, we test a new class of fetal bovine serum (FBS), CultraPure FBS, in ex vivo ELISpot and FluoroSpot assays and cultured FluoroSpot assays following in vitro expansion. Several CultraPure FBS lots that have been specially formulated through the process of lyophilization (lyo-FBS) were compared to liquid CultraPure FBS. We stimulated human PBMCs with antigen-specific peptide pools diluted in media supplemented with liquid CultraPure FBS or lyo-FBS and found equivalent cytokine production with negligible to no assay background with both liquid and lyo-FBS formats. Moreover, the lyo-FBS showed lot-to-lot consistency and 90-day refrigerated (4 °C) stability in both ex vivo direct and in vitro cultured assays. In addition, we present here a method using lyo-FBS for the expansion of low-frequency antigen-specific T cells, mimicking the low frequency seen with cancer neoantigens by utilizing a cultured FluoroSpot assay. Our results demonstrate the presence of Granzyme B, interferon-gamma (IFNγ), and tumor necrosis factor (TNF) production by antigen-specific polyfunctional T cells following a 9-day culture using media supplemented with lyo-FBS., (© 2024. The Author(s), under exclusive license to Springer Science+Business Media, LLC, part of Springer Nature.)

The ELISpot assay has a solid place in the immune monitoring field for over 40 years. It is an assay that can assess the function of single immune cells in a straightforward and easy-to-learn approach. Its use in basic research, translational, and clinical work has been documented in countless publications. Harmonization guidelines and invaluable tools for optimal assay performance and evaluation exist. However, the validation of an established ELISpot protocol has been left to diverse opinions about how to interpret and tackle typical validation parameters. This chapter addresses important considerations for ELISpot validation, including the interpretations of validation parameters for a meaningful description of assay performance., (© 2024. The Author(s), under exclusive license to Springer Science+Business Media, LLC, part of Springer Nature.)

The number of approved or investigational late phase viral vector gene therapies (GTx) has been rapidly growing. The adeno-associated virus vector (AAV) technology continues to be the most used GTx platform of choice. The presence of pre-existing anti-AAV immunity has been firmly established and is broadly viewed as a potential deterrent for successful AAV transduction with a possibility of negative impact on clinical efficacy and a connection to adverse events. Recommendations for the evaluation of humoral, including neutralizing and total antibody based, anti-AAV immune response have been presented elsewhere. This manuscript aims to cover considerations related to the assessment of anti-AAV cellular immune response, including review of correlations between humoral and cellular responses, potential value of cellular immunogenicity assessment, and commonly used analytical methodologies and parameters critical for monitoring assay performance. This manuscript was authored by a group of scientists involved in GTx development who represent several pharma and contract research organizations. It is our intent to provide recommendations and guidance to the industry sponsors, academic laboratories, and regulatory agencies working on AAV-based GTx viral vector modalities with the goal of achieving a more consistent approach to anti-AAV cellular immune response assessment., (© 2023. The Author(s), under exclusive licence to American Association of Pharmaceutical Scientists.)

The use of conventional serum for supplementation of media in cell-based and single-cell functional assays has been a major challenge for assay performance, standardization, optimization, and reproducibility. It has been identified as the leading cause of variability and suboptimal performance in large, international Elispot proficiency panels (Janetzki et al., 2008; Rountree et al., 2016). Extensive pretesting and optimization activities are one approach to overcome these challenges, but they are time-consuming and resource-intensive because suitable lots of serum are difficult to identify and secure in sufficient quantities to provide stability in long-term studies. Advancements in manufacturing methods have resulted in a new class of serum with the potential to solve these challenges. An IFNɣ Elispot study was designed by the External Quality Assurance Program Oversight Laboratory (EQAPOL) at Duke Human Vaccine Institute's (DHVI) Immunology and Virology Quality Assessment Center (IVQAC) to test this new class of serum against their in-house, validated control serum, which is regarded as a global standard in performance for high functionality, recovery, and viability. Commonly used serum-free media were also included in the study. The results of this study compellingly demonstrate that this new class of serum produces high responses and low background reactivity comparable to the included serum standard, with excellent recovery and viability of cells. A protocol for ongoing testing has been developed to continuously validate new batches of this serum with the goal to make available to the field a pretested and validated serum that can be used with confidence in functional cell-based assays., Competing Interests: Declaration of Competing Interest Guido Ferrari reports financial support and equipment, drugs, or supplies were provided by Seraworx, LLC. Devin Davis reports a relationship with Seraworx, LLC that includes: employment and equity or stocks. Sylvia Janetzki reports a relationship with Seraworx, LLC that includes: consulting or advisory., (Copyright © 2023 The Authors. Published by Elsevier B.V. All rights reserved.)

Adeno-associated virus (AAV)-based gene therapies have recently shown promise as a novel treatment for hereditary diseases. Due to the viral origin of the vector capsid, however, cellular immune response may be elicited that could eliminate transduced target cells. To monitor cellular immune responses in clinical trials, we optimized and bioanalytically validated a sensitive, robust, and reliable interferon-γ (IFN-γ) enzyme-linked immunospot (ELISpot) assay. For method performance validation, human peripheral blood mononuclear cells (PBMCs) were stimulated with peptides derived from AAV5 capsid proteins and the encoded transgene product, human blood clotting factor VIII (FVIII), in addition to positive controls, such as peptides from the 65-kDa phosphoprotein of cytomegalovirus. We statistically assessed the limit of detection and confirmatory cutpoint, evaluated precision and linearity, and confirmed specificity using HIV peptides. Robustness parameter ranges and sample stability periods were established. The validated IFN-γ ELISpot assay was then implemented in an AAV5-FVIII gene therapy clinical trial. Cellular immune responses against the AAV5 capsid were observed in most participants as soon as 2 weeks following dose administration; only limited responses against the transgene product were detected. These data underscore the value of using validated methods for monitoring cellular immunity in AAV gene therapy trials., Competing Interests: K.S.P., J.H., T.K., B.R.L., K.L., S.J.Z., and C.V. are current employees and/or shareholders of BioMarin Pharmaceutical. M.T.H. and R.O. are both current employees of Precision for Medicine. S.J. provided consultancy on the project and is currently employed by Zellnet Consulting., (© 2021 The Author(s).)

Background and Aims: Photochemical internalization (PCI) is a technology for inducing release of endocytosed antigens into the cell cytosol via a light-induced process. Preclinical experiments have shown that PCI improves MHC class I antigen presentation, resulting in strongly enhanced CD8+ T-cell responses to polypeptide antigens. In PCI vaccination a mixture of the photosensitizing compound fimaporfin, vaccine antigens, and an adjuvant is administered intradermally followed by illumination of the vaccination site. This work describes an open label, phase I study in healthy volunteers, to assess the safety, tolerability, and immune response to PCI vaccination in combination with the adjuvant poly-ICLC (Hiltonol) (ClinicalTrials.gov Identifier: NCT02947854)., Methods: The primary objective of the study was to assess the safety and local tolerance of PCI mediated vaccination, and to identify a safe fimaporfin dose for later clinical studies. A secondary objective was to analyze the immunological responses to the vaccination. Each subject received 3 doses of HPV16 E7 peptide antigens and two doses of Keyhole Limpet Hemocyanin (KLH) protein. A control group received Hiltonol and vaccine antigens only, whereas the PCI groups in addition received fimaporfin + light. Local and systemic adverse effects were assessed by standard criteria, and cellular and humoral immune responses were analyzed by ELISpot, flow cytometry, and ELISA assays., Results: 96 healthy volunteers were vaccinated with fimaporfin doses of 0.75-50 µg. Doses below 17.5 µg were safe and tolerable, higher doses exhibited local tolerability issues in some study subjects, mainly erythema, and pain during illumination. There were few, and only mild and expected systemic adverse events. The employment of PCI increased the number of subjects exhibiting a T-cell response to the HPV peptide vaccine about 10-fold over what was achieved with the antigen/Hiltonol combination without PCI. Moreover, the use of PCI seemed to result in a more consistent and multifunctional CD8+ T-cell response. An enhancement of the humoral immune response to KLH vaccination was also observed., Conclusions: Using PCI in combination with Hiltonol for intradermal vaccination is safe at fimaporfin doses below 17.5 µg, and gives encouraging immune responses to peptide and protein based vaccination., Competing Interests: TO and AH are employees of PCI Biotech AS and own shares and share options in the Company. AH and PS are inventors on several patents and patent Applications on the PCI Technology. HO and SJ work as consultants for PCI Biotech, and SJ is working for ZellNet Consulting, Inc. VE is an employee of PCI Biotech. The remaining authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2021 Otterhaug, Janetzki, Welters, Håkerud, Nedberg, Edwards, Boekestijn, Loof, Selbo, Olivecrona, van der Burg and Høgset.)

Results from the first gating proficiency panel of intracellular cytokine staining (ICS) highlighted the value of using a consensus gating approach to reduce the variability across laboratories in reported %CD8 + or %CD4 + cytokine-positive cells. Based on the data analysis from the first proficiency panel, harmonization guidelines for a consensus gating protocol were proposed. To validate the recommendations from the first panel and to examine factors that were not included in the first panel, a second ICS gating proficiency panel was organized. All participants analyzed the same set of Flow Cytometry Standard (FCS) files using their own gating protocol. An optional learning module was provided to demonstrate how to apply the previously established gating recommendations and harmonization guidelines to actual ICS data files. Eighty-three participants took part in this proficiency panel. The results from this proficiency panel confirmed the harmonization guidelines from the first panel. These recommendations addressed the (1) placement of the cytokine-positive gate, (2) identification of CD4 + CD8 + double-positive T cells, (3) placement of lymphocyte gate, (4) inclusion of dim cells, (5) gate uniformity, and (6) proper adjustment of the biexponential scaling. In addition, based on the results of this proficiency gating panel, two new recommendations were added to expand the harmonization guidelines: (1) inclusion of dump channel marker to gate all live and dump negative cells and (2) backgating to confirm the correct placement of gates across all populations. © 2020 International Society for Advancement of Cytometry., (© 2020 International Society for Advancement of Cytometry.)

Since the publication of the Society for Immunotherapy of Cancer's (SITC) original cancer immunotherapy biomarkers resource document, there have been remarkable breakthroughs in cancer immunotherapy, in particular the development and approval of immune checkpoint inhibitors, engineered cellular therapies, and tumor vaccines to unleash antitumor immune activity. The most notable feature of these breakthroughs is the achievement of durable clinical responses in some patients, enabling long-term survival. These durable responses have been noted in tumor types that were not previously considered immunotherapy-sensitive, suggesting that all patients with cancer may have the potential to benefit from immunotherapy. However, a persistent challenge in the field is the fact that only a minority of patients respond to immunotherapy, especially those therapies that rely on endogenous immune activation such as checkpoint inhibitors and vaccination due to the complex and heterogeneous immune escape mechanisms which can develop in each patient. Therefore, the development of robust biomarkers for each immunotherapy strategy, enabling rational patient selection and the design of precise combination therapies, is key for the continued success and improvement of immunotherapy. In this document, we summarize and update established biomarkers, guidelines, and regulatory considerations for clinical immune biomarker development, discuss well-known and novel technologies for biomarker discovery and validation, and provide tools and resources that can be used by the biomarker research community to facilitate the continued development of immuno-oncology and aid in the goal of durable responses in all patients., Competing Interests: Competing interests: SHL – Consulting: Amgen, Merck, Genmab, Xencor, Bristol-Myers Squibb, Regeneron; Funding support: Bristol-Myers Squibb, Merck, Vaccinex; Contracted research: Astellas, Merck, Xencor, Boehringer Ingelheim, Kite Pharma, Vedanta. SB – Employee: Roche Tissue Diagnostics. SG – Funding: Bristol-Myers Squibb, Rexahn, Incyte, Novartis, LSK BioPharma, Five Prime, Mirati, QED Bioscience, Debiopharm, Merck, Pfizer, AstraZeneca, MedImmune, Clovis, and Immunocore; Spouse stock ownership: Salarius Pharmaceuticals. BAH – Research funding: Merck, GlaxoSmithKline, Tempest Therapeutics, Leap Therapeutics, A*STAR Singapore, Sanofi; Consulting fees: Novartis, Merck, G1 Therapeutics. SJ – President and owner: ZellNet Consulting, Inc. TOK – Owner: Biolab Services; Employee: Immodulon Therapeutics. YK – Travel funds: AgonOx, Beckman Coulter; Consulting: Curiox; Research and travel funding: Shimadzu Corporation; Research funding from Bristol-Myers Squibb. YDM – President and owner: FlowKnowHow LLC; Business relationship ZellNet Consulting, Inc. RDN – Shareholder: Eli Lilly, Bristol-Myers Squibb. TS – Employee: Regeneron; IP rights: Regeneron; Sockholder: Regeneron. HTM – Scientific advisory board: Cytek, Inc., Caris Life Sciences; Stockholder: BD Biosciences; Spouse employed by: AbbVie Inc. KD, JCJBG, AWL, CM, SRS, YZ – Nothing to disclose. SITC Staff: AK, BL, LL, RL, SMW – Nothing to disclose., (© Author(s) (or their employer(s)) 2020. Re-use permitted under CC BY. Published by BMJ.)

The ELISpot assay prevails as one of the most sensitive and meaningful assays for the detection of antigen-specific, effector immune responses. Acquisition of cellular analyte for ELISpot analysis is typically not problematic when derived from tissues enriched in lymphocytes (e.g., lymphoid organs and blood); however, cell processing becomes more difficult when lymphocytes represent only a very minor population relative to the source tissue, especially when the source tissue is in limited supply (e.g., small mouse tumors). Traditional enzymatic-based methods for dissociating tumors often result in poor yields, inconsistent lymphocyte enrichment, and can have deleterious effects on lymphocyte phenotype and function. To address these limitations, we have developed an enzyme-free protocol for processing tumor infiltrating lymphocytes (TILs) from small mouse tumors, which enables the enumeration of antigen-specific effector lymphocytes using ELISpot analysis. This procedure is predicated on the dissociation of tumor tissue using gentle agitation with a paddle blender followed by a brief in vitro culture period to remove adherent cells, as well as to revive lymphocytes from a non-responsive state. Although this method is demonstrated with mouse intracerebral tumors, we have found that this protocol is applicable to peripheral tumors and may likely extend to alternative tissue sources wherein lymphocytes exist in low numbers., (Copyright © 2018 The Authors. Published by Elsevier B.V. All rights reserved.)

Much has been written about Elispot and how to optimally run the assay for a wide variety of applications. But only a limited number of articles exist addressing the analysis step, the plate evaluation. Comparing that fact with the vast amount of analysis advise available for other single cell immune assay, for example, intracellular cytokine staining, the overall impression may be that Elispot evaluation is just simple enough to not require extensive elaboration and guidance. At first thought this appears reasonable because how difficult can it be counting colored spots on a white background. In addition, automated Elispot readers were already introduced more than 20 years ago (Herr et al., J Immunol Methods 203, 141-152, 1997), easing the strenuous load of manual counting and providing means to decrease the subjectivity in Elispot analysis. Just shortly thereafter however, the first report was published about the subjectivity and operator-dependency of plate evaluation even when using automated reader systems (Janetzki et al., J Immunol Methods 291, 175-183, 2004). Later, the plate evaluation was identified as a main factor causing variability in Elispot results, triggering the inclusion of recommendations on handling of artifacts and the audits of plate reading results in the Initial Elispot Harmonization guidelines (Janetzki et al., Cancer Immunol Immunother 57, 303-315, 2008; Britten et al., Cancer Immunol Immunother 57, 289-302, 2008). In follow-up, a large international study with 75 laboratories was conducted to address the current approaches taken to evaluate Elispot plates and to establish consensus guidelines for plate evaluation (Janetzki et al., Nat Protoc 10, 1098-1115, 2015). This article addresses the special challenges of plate evaluation, gives explanations for unusual observation, and provides overall recommendations on how to work through the labyrinth of available algorithms and reader settings to obtain reliable Elispot data.

This report summarizes the symposium, 'Immunotherapy Biomarkers 2016: Overcoming the Barriers', which was held on April 1, 2016 at the National Institutes of Health in Bethesda, Maryland. The symposium, cosponsored by the Society for Immunotherapy of Cancer (SITC) and the National Cancer Institute (NCI), focused on emerging immunotherapy biomarkers, new technologies, current hurdles to further progress, and recommendations for advancing the field of biomarker development.

Immunotherapies have emerged as one of the most promising approaches to treat patients with cancer. Recently, there have been many clinical successes using checkpoint receptor blockade, including T cell inhibitory receptors such as cytotoxic T-lymphocyte-associated antigen 4 (CTLA-4) and programmed cell death-1 (PD-1). Despite demonstrated successes in a variety of malignancies, responses only typically occur in a minority of patients in any given histology. Additionally, treatment is associated with inflammatory toxicity and high cost. Therefore, determining which patients would derive clinical benefit from immunotherapy is a compelling clinical question. Although numerous candidate biomarkers have been described, there are currently three FDA-approved assays based on PD-1 ligand expression (PD-L1) that have been clinically validated to identify patients who are more likely to benefit from a single-agent anti-PD-1/PD-L1 therapy. Because of the complexity of the immune response and tumor biology, it is unlikely that a single biomarker will be sufficient to predict clinical outcomes in response to immune-targeted therapy. Rather, the integration of multiple tumor and immune response parameters, such as protein expression, genomics, and transcriptomics, may be necessary for accurate prediction of clinical benefit. Before a candidate biomarker and/or new technology can be used in a clinical setting, several steps are necessary to demonstrate its clinical validity. Although regulatory guidelines provide general roadmaps for the validation process, their applicability to biomarkers in the cancer immunotherapy field is somewhat limited. Thus, Working Group 1 (WG1) of the Society for Immunotherapy of Cancer (SITC) Immune Biomarkers Task Force convened to address this need. In this two volume series, we discuss pre-analytical and analytical (Volume I) as well as clinical and regulatory (Volume II) aspects of the validation process as applied to predictive biomarkers for cancer immunotherapy. To illustrate the requirements for validation, we discuss examples of biomarker assays that have shown preliminary evidence of an association with clinical benefit from immunotherapeutic interventions. The scope includes only those assays and technologies that have established a certain level of validation for clinical use (fit-for-purpose). Recommendations to meet challenges and strategies to guide the choice of analytical and clinical validation design for specific assays are also provided.

There is growing recognition that immunotherapy is likely to significantly improve health outcomes for cancer patients in the coming years. Currently, while a subset of patients experience substantial clinical benefit in response to different immunotherapeutic approaches, the majority of patients do not but are still exposed to the significant drug toxicities. Therefore, a growing need for the development and clinical use of predictive biomarkers exists in the field of cancer immunotherapy. Predictive cancer biomarkers can be used to identify the patients who are or who are not likely to derive benefit from specific therapeutic approaches. In order to be applicable in a clinical setting, predictive biomarkers must be carefully shepherded through a step-wise, highly regulated developmental process. Volume I of this two-volume document focused on the pre-analytical and analytical phases of the biomarker development process, by providing background, examples and "good practice" recommendations. In the current Volume II, the focus is on the clinical validation, validation of clinical utility and regulatory considerations for biomarker development. Together, this two volume series is meant to provide guidance on the entire biomarker development process, with a particular focus on the unique aspects of developing immune-based biomarkers. Specifically, knowledge about the challenges to clinical validation of predictive biomarkers, which has been gained from numerous successes and failures in other contexts, will be reviewed together with statistical methodological issues related to bias and overfitting. The different trial designs used for the clinical validation of biomarkers will also be discussed, as the selection of clinical metrics and endpoints becomes critical to establish the clinical utility of the biomarker during the clinical validation phase of the biomarker development. Finally, the regulatory aspects of submission of biomarker assays to the U.S. Food and Drug Administration as well as regulatory considerations in the European Union will be covered.

The presented protocol for Elispot plate evaluation summarizes how to implement the recommendations developed following the establishment of a large-scale international Elispot plate-reading panel and subsequent multistep consensus-finding process. The panel involved >100 scientists from various immunological backgrounds. The protocol includes the description and justification of steps for setting reading parameters to obtain accurate, reliable and precise automated analysis results of Elispot plates. Further, necessary adjustments for out-of-specification situations are described and examples are provided. The plate analysis, including parameter adjustments, auditing of results and necessary annotations, should be achievable within a time range of 10-30 min per plate. Adoption of these guidelines should enable a further reduction in assay variability and an increase in the reliability and comparability of results obtained by Elispot. These guidelines conclude the ongoing harmonization efforts for the enzymatic Elispot assay.

The ELISpot, a heterogeneous immunoassay, is widely used for detection of low abundant analytes. It is a reliable and robust assay to monitor responses of the immune system at the single-cell level by capturing secreted molecules of interest with specific, membrane-bound antibodies. Those molecules are then made visible by a cascade of ELISA-related development steps. The final results are distinct spots on the membrane as an imprint of the cell secreting the captured molecules, not only allowing their quantification but also providing insight on the kinetics and strength of secretion. This chapter describes the optimized protocol steps of the ELISpot technique, important improvements and tools available for the community, and the current expansion of the technique into polyfunctional cell analysis.

Immune monitoring of functional responses is a fundamental parameter to establish correlates of protection in clinical trials evaluating vaccines and therapies to boost antigen-specific responses. The IFNg ELISPOT assay is a well-standardized and validated method for the determination of functional IFNg-producing T-cells in peripheral blood mononuclear cells (PBMC); however, its performance greatly depends on the quality and integrity of the cryopreserved PBMC. Here, we investigate the effect of overnight (ON) resting of the PBMC on the detection of CD8-restricted peptide-specific responses by IFNg ELISPOT. The study used PBMC from healthy donors to evaluate the CD8 T-cell response to five pooled or individual HLA-A2 viral peptides. The results were analyzed using a modification of the existing distribution free resampling (DFR) recommended for the analysis of ELISPOT data to ensure the most rigorous possible standard of significance. The results of the study demonstrate that ON resting of PBMC samples prior to IFNg ELISPOT increases both the magnitude and the statistical significance of the responses. In addition, a comparison of the results with a 13-day preculture of PBMC with the peptides before testing demonstrates that ON resting is sufficient for the efficient evaluation of immune functioning.

ELISpot is one of the most commonly used immune monitoring assays, which allows the functional assessment of the immune system at the single cell level. With its outstanding sensitivity and ease of performance, the assay has recently advanced from the mere single function cell analysis to multifunctional analysis by implementing detection reagents that are labeled with fluorophores (FluoroSpot), allowing the detection of secretion patterns of two or more analytes in a single well. However, the automated evaluation of such assays presents various challenges for image analysis. Here we dissect the technical and methodological requirements for a reliable analysis of FluoroSpot assays, introduce important quality control measures and provide advice for proper interpretation of results obtained by automated imaging systems.

During more than 25 years of application in immunological sciences, ELISPOT has been established as a routine, robust, versatile, and reliable assay. From basic research to clinical immune monitoring, ELISPOT is being used to address the quantification and (to a lesser extent) functional characterization of immune cells secreting different molecules in the context of health and disease, immune intervention, and therapy in humans and other species [Kalyuzhny (Ed.) (2005) Handbook of Elispot: methods and protocols, Vol. 302, Humana Press Inc., Totowa, NJ]. Over the last decade, ELISPOT assays have been increasingly implemented as an immune-monitoring tool in clinical trials [Schmittel et al. J Immunother 23:289-295, 2000; Whiteside Immunol Invest 29:149-162, 2000; Nagata et al. Ann N Y Acad Sci 1037:10-15, 2004; Cox et al. (2005) Cellular immune assays for evaluation of vaccine efficacy in developing countries., In Manual of Clinical Immunology Laboratory (Rose, N. R., Hamilton, R. G., and Detrick, B., Eds.), p 301, ASM Press, Washington, DC; Cox et al. Methods 38:274-282, 2006]. While the principles of the original protocol have changed little since its first introduction [Czerkinsky J Immunol Methods 110:29-36, 1988], individual laboratories have adapted assay procedures based on experimental needs, availability of reagents and equipment, obtained recommendations, and gained experience, leading to a wide disparity of applied ELISPOT protocols with inevitable consequences. This chapter addresses the resulting challenges for ELISPOT use in clinical trial settings, and discusses the influence of harmonization strategies as a tool for overcoming these challenges. Furthermore, harmonization is discussed in the context of assay standardization and validation strategies.

The Cancer Vaccine Consortium of the Sabin Vaccine Institute (CVC/SVI) is conducting an ongoing large-scale immune monitoring harmonization program through its members and affiliated associations. This effort was brought to life as an external validation program by conducting an international Elispot proficiency panel with 36 laboratories in 2005, and was followed by a second panel with 29 participating laboratories in 2006 allowing for application of learnings from the first panel. Critical protocol choices, as well as standardization and validation practices among laboratories were assessed through detailed surveys. Although panel participants had to follow general guidelines in order to allow comparison of results, each laboratory was able to use its own protocols, materials and reagents. The second panel recorded an overall significantly improved performance, as measured by the ability to detect all predefined responses correctly. Protocol choices and laboratory practices, which can have a dramatic effect on the overall assay outcome, were identified and lead to the following recommendations: (A) Establish a laboratory SOP for Elispot testing procedures including (A1) a counting method for apoptotic cells for determining adequate cell dilution for plating, and (A2) overnight rest of cells prior to plating and incubation, (B) Use only pre-tested serum optimized for low background: high signal ratio, (C) Establish a laboratory SOP for plate reading including (C1) human auditing during the reading process and (C2) adequate adjustments for technical artifacts, and (D) Only allow trained personnel, which is certified per laboratory SOPs to conduct assays. Recommendations described under (A) were found to make a statistically significant difference in assay performance, while the remaining recommendations are based on practical experiences confirmed by the panel results, which could not be statistically tested. These results provide initial harmonization guidelines to optimize Elispot assay performance to the immunotherapy community. Further optimization is in process with ongoing panels.

During the last 20 yr, the enzyme-linked immunospot (ELISPOT) assay has emerged as one of the most important and widely used assays to monitor immune responses in humans and a variety of other species. With the ELISPOT assay, immune cell frequencies can be measured at the single cell level without elaborate expansion or manipulation of cell populations. Its usefulness has led to its application in vaccine design and development and, most importantly, in monitoring vaccination efforts. The impact of results measured with this assay can be profound. In addition to ease of performance, repeatability and reliability are major features expected of an ELISPOT assay. The focus today is on standardization of the technique, validation strategies to comply with these required features, and accommodation of the growing demand of Good Laboratory Practice (GLP) compliance. This chapter will give the experienced scientists as well as newcomers to the field an overview over the major standardization issues for each step of the protocol. Guidelines are given on how to validate the ELISPOT performance.

In this study, a comprehensive comparative analysis of different evaluation methods of Elispot plates was performed. Three investigators using three different evaluation approaches read 50 randomly selected wells at three different time points. The methods were: (1) manual evaluation using a stereomicroscope, (2) automated evaluation using an image analysis reader system with reading parameters established by each investigator, and (3) automated evaluation using a reader system with preset reading parameters using assay-specific controls. We demonstrate that manual evaluation had the highest variability both within the same method and when comparing all methods, followed by automated evaluation with investigator-dependent parameters. The variability was low only when all investigators used the same parameters established using assay-specific controls. This variability was independent of operator or spot number per well. Based on this study, recommendations for standardization and validation procedures of Elispot assay performance and evaluation procedures are presented.