Relevance of Antibody Validation for Flow Cytometry

Abstract

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...

Analytical performance of a 25‐marker spectral cytometry immune monitoring assay in peripheral blood

“…The recent Cytometry Part A Special Issue: Rigor and Reproducibility highlighted the importance of antibody validation, sample preparation and instrument, assay and post‐analysis standardization [21–23]. These practices are indispensable to successful analytical performance or complete validation of high‐dimensional assays.…”

High-specificity antibodies and detection methods for quantifying phosphorylated tau from clinical samples

“…For example, Kalina et al. [ 42 ] and Pillai-Kastoori et al. [ 43 ] express the importance of validating antibodies using their intended experiment.…”

Reproducibility of Flow Cytometry Through Standardization: Opportunities and Challenges

“…Notably, some epitopes are decreased or lost upon cryopreservation (e.g., in CD62L) (35); in other cases, cell fixation can decrease epitope availability for particular antibody clones (77,78), resulting in alteration in the measured signal intensity. Accompanying article in this Special issue is dedicated to antibody validation (79).…”

“…This might simplify sample preparation and reduce errors. Ideally, the demand for reproducibility would also motivate reagent manufacturers to develop and declare intensity based quality controls of the antibody conjugates (79). Reduction of technical errors together with the broader use of a particular immunostaining panel might motivate the development of data analysis tools, that will be more accessible also to non-programmers.…”