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Research paper thumbnail of Guidelines for the automated evaluation of Elispot assays

Nature Protocols, 2015

The presented protocol for Elispot plate evaluation summarizes how to implement the recommendatio... more 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.

Research paper thumbnail of 48 Phase I study of HSPPC-96 (oncophage®) vaccine in patients with completely resected pancreatic adenocarcinoma

European Journal of Cancer Supplements, 2003

Research paper thumbnail of Response Determination Criteria for ELISPOT: Toward a Standard that Can Be Applied Across Laboratories

Methods in Molecular Biology, 2011

ELISPOT assay readout is often dichomized as positive or negative responses according to prespeci... more ELISPOT assay readout is often dichomized as positive or negative responses according to prespecified criteria. However, these criteria can vary widely across institutions. The adoption of a common response criterion is a key step toward cross-laboratory comparability. This chapter describes the two main approaches to response determination, identifying the strengths and limitations of each. Nonparametric statistical tests and consideration of data quality are recommended and instructions provided for their ready implementation by nonstatisticians and statisticians alike.

Research paper thumbnail of MIATA modules

Research paper thumbnail of Harmonization of Immune Biomarker Assays for Clinical Studies

Science Translational Medicine, 2011

Assays that measure a patient&amp... more Assays that measure a patient's immune response play an increasingly important role in the development of immunotherapies. The inherent complexity of these assays and independent protocol development between laboratories result in high data variability and poor reproducibility. Quality control through harmonization--based on integration of laboratory-specific protocols with standard operating procedures and assay performance benchmarks--is one way to overcome these limitations. Harmonization guidelines can be widely implemented to address assay performance variables. This process enables objective interpretation and comparison of data across clinical trial sites and also facilitates the identification of relevant immune biomarkers, guiding the development of new therapies.

Research paper thumbnail of A critical assessment for the value of markers to gate-out undesired events in HLA-peptide multimer staining protocols

Journal of Translational Medicine, 2011

Research paper thumbnail of A systematic approach to biomarker discovery; Preamble to "the iSBTc-FDA taskforce on immunotherapy biomarkers

Journal of Translational Medicine, 2008

Research paper thumbnail of Heat Shock Protein Vaccines Against Cancer

Journal of Immunotherapy, 1993

Vaccination of mice with heat shock proteins (HSPs) derived from a tumor makes the mice resistant... more Vaccination of mice with heat shock proteins (HSPs) derived from a tumor makes the mice resistant to the tumor from which the HSP was obtained. This phenomenon has been demonstrated with three HSPs--gp96, hsp90, and hsp70. Vaccination with HSPs also elicits antigen-specific cytotoxic T lymphocytes (CTLs). The specific immunogenicity of HSPs derives apparently, not from the HSPs per se, but from the peptides bound to them. These observations provide the basis for a new generation of vaccines against cancer. The HSP-based cancer vaccines circumvent two of the most intractable hurdles to cancer immunotherapy. One of them is the possibility that human cancers, like cancers of experimental animals, are antigenically distinct. The prospect of identification of immunogenic antigens of individual cancers from patients is daunting to the extent of being impractical. The observation that HSPs chaperone antigenic peptides of the cells from which they are derived circumvents this extraordinary hurdle. Second, most current approaches to cancer immunotherapy focus on determining the CTL-recognized epitopes of cancer cell lines. This approach requires the availability of cell lines and CTLs against cancers. These reagents are unavailable for an overwhelming proportion of human cancers. In contrast, the HSP-based vaccines do not depend on the availability of cell lines or CTLs nor do they require definition of the antigenic epitopes of cancer cells. These advantages, among others, make HSPs attractive and novel immunogens against cancer.

Research paper thumbnail of Evaluation of Elispot assays: influence of method and operator on variability of results

Journal of Immunological Methods, 2004

Research paper thumbnail of The role of the reporting framework MIATA within current efforts to advance immune monitoring

Journal of Immunological Methods, 2014

Research paper thumbnail of Immunization of cancer patients with autologous cancer-derived heat shock protein gp96 preparations: A pilot study

International Journal of Cancer, 2000

Research paper thumbnail of T Cell Assays and MIATA: The Essential Minimum for Maximum Impact

Research paper thumbnail of Recommendations from the iSBTc-SITC/FDA/NCI Workshop on Immunotherapy Biomarkers

Clinical Cancer Research, 2011

Research paper thumbnail of Harmonization guidelines for HLA-peptide multimer assays derived from results of a large scale international proficiency panel of the Cancer Vaccine Consortium

Cancer Immunology, Immunotherapy, 2009

Research paper thumbnail of Response definition criteria for ELISPOT assays revisited

Cancer Immunology, Immunotherapy, 2010

Research paper thumbnail of Results and harmonization guidelines from two large-scale international Elispot proficiency panels conducted by the Cancer Vaccine Consortium (CVC/SVI)

Cancer Immunology, Immunotherapy, 2008

Research paper thumbnail of Serum-free freezing media support high cell quality and excellent ELISPOT assay performance across a wide variety of different assay protocols

Cancer Immunology, Immunotherapy, 2013

Research paper thumbnail of Guidelines for the automated evaluation of Elispot assays

Nature Protocols, 2015

The presented protocol for Elispot plate evaluation summarizes how to implement the recommendatio... more 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.

Research paper thumbnail of Standardization and validation issues of the ELISPOT assay

Methods in molecular biology (Clifton, N.J.), 2005

During the last 20 yr, the enzyme-linked immunospot (ELISPOT) assay has emerged as one of the mos... more 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 standardizati...

Research paper thumbnail of Response Determination Criteria for ELISPOT: Toward a Standard that Can Be Applied Across Laboratories

Methods in Molecular Biology, 2011

ELISPOT assay readout is often dichomized as positive or negative responses according to prespeci... more ELISPOT assay readout is often dichomized as positive or negative responses according to prespecified criteria. However, these criteria can vary widely across institutions. The adoption of a common response criterion is a key step toward cross-laboratory comparability. This chapter describes the two main approaches to response determination, identifying the strengths and limitations of each. Nonparametric statistical tests and consideration of data quality are recommended and instructions provided for their ready implementation by nonstatisticians and statisticians alike.

Research paper thumbnail of Guidelines for the automated evaluation of Elispot assays

Nature Protocols, 2015

The presented protocol for Elispot plate evaluation summarizes how to implement the recommendatio... more 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.

Research paper thumbnail of 48 Phase I study of HSPPC-96 (oncophage®) vaccine in patients with completely resected pancreatic adenocarcinoma

European Journal of Cancer Supplements, 2003

Research paper thumbnail of Response Determination Criteria for ELISPOT: Toward a Standard that Can Be Applied Across Laboratories

Methods in Molecular Biology, 2011

ELISPOT assay readout is often dichomized as positive or negative responses according to prespeci... more ELISPOT assay readout is often dichomized as positive or negative responses according to prespecified criteria. However, these criteria can vary widely across institutions. The adoption of a common response criterion is a key step toward cross-laboratory comparability. This chapter describes the two main approaches to response determination, identifying the strengths and limitations of each. Nonparametric statistical tests and consideration of data quality are recommended and instructions provided for their ready implementation by nonstatisticians and statisticians alike.

Research paper thumbnail of MIATA modules

Research paper thumbnail of Harmonization of Immune Biomarker Assays for Clinical Studies

Science Translational Medicine, 2011

Assays that measure a patient&amp... more Assays that measure a patient's immune response play an increasingly important role in the development of immunotherapies. The inherent complexity of these assays and independent protocol development between laboratories result in high data variability and poor reproducibility. Quality control through harmonization--based on integration of laboratory-specific protocols with standard operating procedures and assay performance benchmarks--is one way to overcome these limitations. Harmonization guidelines can be widely implemented to address assay performance variables. This process enables objective interpretation and comparison of data across clinical trial sites and also facilitates the identification of relevant immune biomarkers, guiding the development of new therapies.

Research paper thumbnail of A critical assessment for the value of markers to gate-out undesired events in HLA-peptide multimer staining protocols

Journal of Translational Medicine, 2011

Research paper thumbnail of A systematic approach to biomarker discovery; Preamble to "the iSBTc-FDA taskforce on immunotherapy biomarkers

Journal of Translational Medicine, 2008

Research paper thumbnail of Heat Shock Protein Vaccines Against Cancer

Journal of Immunotherapy, 1993

Vaccination of mice with heat shock proteins (HSPs) derived from a tumor makes the mice resistant... more Vaccination of mice with heat shock proteins (HSPs) derived from a tumor makes the mice resistant to the tumor from which the HSP was obtained. This phenomenon has been demonstrated with three HSPs--gp96, hsp90, and hsp70. Vaccination with HSPs also elicits antigen-specific cytotoxic T lymphocytes (CTLs). The specific immunogenicity of HSPs derives apparently, not from the HSPs per se, but from the peptides bound to them. These observations provide the basis for a new generation of vaccines against cancer. The HSP-based cancer vaccines circumvent two of the most intractable hurdles to cancer immunotherapy. One of them is the possibility that human cancers, like cancers of experimental animals, are antigenically distinct. The prospect of identification of immunogenic antigens of individual cancers from patients is daunting to the extent of being impractical. The observation that HSPs chaperone antigenic peptides of the cells from which they are derived circumvents this extraordinary hurdle. Second, most current approaches to cancer immunotherapy focus on determining the CTL-recognized epitopes of cancer cell lines. This approach requires the availability of cell lines and CTLs against cancers. These reagents are unavailable for an overwhelming proportion of human cancers. In contrast, the HSP-based vaccines do not depend on the availability of cell lines or CTLs nor do they require definition of the antigenic epitopes of cancer cells. These advantages, among others, make HSPs attractive and novel immunogens against cancer.

Research paper thumbnail of Evaluation of Elispot assays: influence of method and operator on variability of results

Journal of Immunological Methods, 2004

Research paper thumbnail of The role of the reporting framework MIATA within current efforts to advance immune monitoring

Journal of Immunological Methods, 2014

Research paper thumbnail of Immunization of cancer patients with autologous cancer-derived heat shock protein gp96 preparations: A pilot study

International Journal of Cancer, 2000

Research paper thumbnail of T Cell Assays and MIATA: The Essential Minimum for Maximum Impact

Research paper thumbnail of Recommendations from the iSBTc-SITC/FDA/NCI Workshop on Immunotherapy Biomarkers

Clinical Cancer Research, 2011

Research paper thumbnail of Harmonization guidelines for HLA-peptide multimer assays derived from results of a large scale international proficiency panel of the Cancer Vaccine Consortium

Cancer Immunology, Immunotherapy, 2009

Research paper thumbnail of Response definition criteria for ELISPOT assays revisited

Cancer Immunology, Immunotherapy, 2010

Research paper thumbnail of Results and harmonization guidelines from two large-scale international Elispot proficiency panels conducted by the Cancer Vaccine Consortium (CVC/SVI)

Cancer Immunology, Immunotherapy, 2008

Research paper thumbnail of Serum-free freezing media support high cell quality and excellent ELISPOT assay performance across a wide variety of different assay protocols

Cancer Immunology, Immunotherapy, 2013

Research paper thumbnail of Guidelines for the automated evaluation of Elispot assays

Nature Protocols, 2015

The presented protocol for Elispot plate evaluation summarizes how to implement the recommendatio... more 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.

Research paper thumbnail of Standardization and validation issues of the ELISPOT assay

Methods in molecular biology (Clifton, N.J.), 2005

During the last 20 yr, the enzyme-linked immunospot (ELISPOT) assay has emerged as one of the mos... more 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 standardizati...

Research paper thumbnail of Response Determination Criteria for ELISPOT: Toward a Standard that Can Be Applied Across Laboratories

Methods in Molecular Biology, 2011

ELISPOT assay readout is often dichomized as positive or negative responses according to prespeci... more ELISPOT assay readout is often dichomized as positive or negative responses according to prespecified criteria. However, these criteria can vary widely across institutions. The adoption of a common response criterion is a key step toward cross-laboratory comparability. This chapter describes the two main approaches to response determination, identifying the strengths and limitations of each. Nonparametric statistical tests and consideration of data quality are recommended and instructions provided for their ready implementation by nonstatisticians and statisticians alike.