A high-throughput pipeline for validation of antibodies

Sikorski, K.; Mehta, Adi; Inngjerdingen, Marit; Thakor, Flourina Kumar; Kling, Simon; Kalina, Tomáš; Nyman, Tuula A.; Stensland, Maria; Zhou, Wei; Souza, Gustavo; Holden, Lars; Stuchlý, Jan; Templin, Markus F.; Lund-Johansen, Fridtjof

doi:10.1038/s41592-018-0179-8

Cited by 41 publications

(35 citation statements)

References 19 publications

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“…In the upcoming 11th HLDA workshop, this methodological framework will be used to measure and cluster antibody reactivities across subsets to help assign new CD nomenclature. This approach follows the strategy proposed by the International Working Group for Antibody Validation (IWGAV) that has documented expression patterns for 3,706 antibodies in immunoprecipitates (50,51). Including future reactivity patterns of HLDA 11 in the CD Maps resource will enhance its role as a benchmark for the research community.…”

Section: Discussionmentioning

confidence: 99%

CD Maps - Dynamic Profiling of CD1 to CD100 Surface Expression on Human Leukocyte and Lymphocyte Subsets

Fišer

Kalina

Pérez-Andrés

et al. 2019

Blood

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CD molecules are surface molecules expressed on cells of the immune system that play key roles in immune cell-cell communication and sensing the microenvironment. These molecules are essential markers for the identification and isolation of leukocytes and lymphocyte subsets and their malignant counterparts. Here, we present the results of the first phase of the CD Maps study, mapping the expression of CD1-100 (n=110) on 47 immune cell subsets from blood, thymus and tonsil using an 8-color standardized EuroFlow approach and quantification of expression. The resulting dataset included median antibody binding capacities (ABC) and percentage of positivity for all markers on all subsets and was developed into an interactive CD Maps web resource. Using the resource, we examined differentially expressed proteins between granulocyte, monocyte and dendritic cell subsets, and profiled dynamic expression of markers during thymocyte differentiation, T-cell maturation, and between functionally distinct B-cell subset clusters. The CD Maps resource will serve as a benchmark of antibody reactivities ensuring improved reproducibility of flow cytometry-based research. Moreover, it will provide a full picture of the surfaceome of human immune cells and serves as a useful platform to increase our understanding of leukocyte biology, as well as, to facilitate the identification of new biomarkers and therapeutic targets of immunological and hematological diseases. CD Maps project is supported with reagents from BD Biosciences, BioLegend and Exbio. TK is supported by Ministry of Health Czech Republic grant 15-26588A and LO1604. KF is supported by Ministry of Health Czech Republic grant NV18-08-00385. Disclosures No relevant conflicts of interest to declare.

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Section: Discussionmentioning

confidence: 99%

CD Maps - Dynamic Profiling of CD1 to CD100 Surface Expression on Human Leukocyte and Lymphocyte Subsets

Fišer

Kalina

Pérez-Andrés

et al. 2019

Blood

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“…However, another high-throughput method for antibody validation was developed by Lund-Johansen's group, who used flow cytometry to quantify the immunoprecipitation of a target protein on a microbead (54), later adding the resolution of thousands of microbeads each immunoprecipitating its target protein from cell lysates after size-exclusion chromatography and differential detergent lysis (42,43,55). The addition of the automated analysis tool (56) and the creation of a pipeline for antibody reactivity profiling coupled to the massspectrometry validation (53) of target proteins has made this approach useful for the large-scale antibody performance validation of intracellular targets.…”

Section: New Methodsmentioning

confidence: 99%

“…Immunoprecipitation combined with mass spectrometry has recently proven to be a very powerful tool to validate antibody specificity. It allows the identification of proteins that selectively bind to an antibody, including any off-target proteins (53). The role of these high content techniques with a digital, and thus, data analysis amenable output will increase.…”

Section: Biochemical Validationmentioning

confidence: 99%

Relevance of Antibody Validation for Flow Cytometry

2019

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Antibody reagents are the key components of multiparametric flow cytometry analysis. Their quality performance is an absolute requirement for reproducible flow cytometry experiments. While there is an enormous body of antibody reagents available, there is still a lack of consensus about which criteria should be evaluated to select antibody reagents with the proper performance, how to validate antibody reagents for flow cytometry, and how to interpret the validation results. The achievements of cytometry moved the field to a higher number of measured parameters, large data sets, and computational data analysis approaches. These advancements pose an increased demand for antibody reagent performance quality. This review summarizes the codevelopment of cytometry, antibody development, and validation strategies. It discusses the diverse issues of the specificity, cross-reactivity, epitope, titration, and reproducibility features of antibody reagents, and this review discusses the validation principles and methods that are currently available and those that are emerging. We argue that significant efforts should be invested by antibody users, developers, manufacturers, and publishers to increase the quality and reproducibility of published studies. More validation data should be presented by all stakeholders; however, the data should be presented in sufficient experimental detail to foster reproducibility, and community effort shall lead to the public availability of large data sets that can serve as a benchmark for antibody performance.Flow cytometry has developed into an indispensable technique in the research and clinical investigation of immune and hematologic systems, with increasing applications in other cell biology disciplines. There are three main pillars in the practical application of this technology, namely the instrumentation, the analytical methods for large data sets, and the reagents used to design biological experiments. Despite dramatic developments in instrumentation (polychromatic (1), mass (2,3), and spectral (4,5) cytometry) and the significant developments in data analysis techniques for high content analyses (6-8), the last pillar of this trio remains consistent across time in its importance to the application: the fluorescent antibody conjugate.Our and others' experiences indicate that nearly half of antibodies, sold by companies or described by academic groups, do not function for the recommended application. They present staining patterns that conflict with those reported in the literature, show unexpected cross-reactivity, or have even failed the most basic specificity tests (9-11). There is growing alarm about results that cannot be reproduced by other research groups, including data published in high-impact journals. Antibodies are believed to be, in part, responsible for inconsistent experimental results and the publication of inaccurate data in the scientific literature (12,13).During recent years, both industry and academic groups have increased their efforts to increase the quality of t...

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“…Proteomic orthogonal validation includes targeted proteomics and MS (48,49). A high-throughput "capture Western blotting" that utilizes biotinylated protein samples and microsphere-based barcoded antibody assays (PAGE-MAP) has also recently been developed to aid validation using the IWGAV pillars (50). Although these are valuable tools for confirming antibody specificity through an analysis of protein levels between samples, they have issues differentiating co-migrating proteins of a similar molecular weight (71).…”

Section: Orthogonal and Independent Approachesmentioning

confidence: 99%

“…The assay can be manual, slow, and time-intensive during most steps and difficult to automate at scale. Electroblotting to a membrane and subsequent antibody incubations can also lead to substantial antigen loss (50). Capillary electrophoresis and microfluidic Western blotting (66) represent two areas of innovation of the core assay concept.…”

Section: The Future Is Now: Multiplex Fluorescent Western Blot Targetmentioning

confidence: 99%

Antibody validation for Western blot: By the user, for the user

Pillai-Kastoori¹,

Heaton²,

Shiflett³

et al. 2019

J. Biol. Chem.

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Well-characterized antibody reagents play a key role in the reproducibility of research findings, and inconsistent antibody performance leads to variability in Western blotting and other immunoassays. The current lack of clear, accepted standards for antibody validation and reporting of experimental details contributes to this problem. Because the performance of primary antibodies is strongly influenced by assay context, recommendations for validation and usage are unique to each type of immunoassay. Practical strategies are proposed for the validation of primary antibody specificity, selectivity, and reproducibility using Western blot analysis. The antibody should produce reproducible results within and between Western blotting experiments and the observed effect confirmed with a complementary or orthogonal method. Routine implementation of standardized antibody validation and reporting in immunoassays such as Western blotting may promote improved reproducibility across the global life sciences community.

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A high-throughput pipeline for validation of antibodies

Cited by 41 publications

References 19 publications

CD Maps - Dynamic Profiling of CD1 to CD100 Surface Expression on Human Leukocyte and Lymphocyte Subsets

CD Maps - Dynamic Profiling of CD1 to CD100 Surface Expression on Human Leukocyte and Lymphocyte Subsets

Relevance of Antibody Validation for Flow Cytometry

Antibody validation for Western blot: By the user, for the user

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