Antibody Validation for Flow Cytometry
Tomas Kalina, Childhood Leukemia Investigation Prague, Czech Republic
Fridtjof Lund-Johansen, Oslo University Hospital, Norway
Anis Larbi, Singapore Immunology Network, Singapore
Kelly Lundsten, BioLegend, USA
Ruud Hulspas, Cellular Technologies Bioconsulting LLC, USA
Pablo Engel, Department of Cellular Biology and Pathology, University of Barcelona Medical School, Spain
Antibody validation in flow cytometry may be a better-established practice than in other antibody applications, particularly in clinical flow cytometry for diagnostic purposes. Antibody validation for flow cytometry was first addressed in 1982 by the Human Cell Differentiation Molecules, an organization that runs the Human Leucocyte Differentiation Antigens (HLDA) workshops to study leukocyte surface molecules. Through these HLDA workshops, information on novel molecules has accumulated rapidly, and led to the characterization and formal designation of more than 400 molecules. This has allowed users to put a lot of trust in (albeit a relatively small number) vendors, who are expected to have validated and verified reactivity, specificity, selectivity, and sensitivity in relevant cell populations and cell types. However, best practice demands that the user finds and validates suitable antibodies for any flow cytometric assay, while there is no consensus on acceptable antibody validation methods. Consequently, the validity of flow cytometry results may be put in question.
This tutorial will present the current status of antibody validation from an academic perspective, as well as from the manufacturer perspective. We will discuss current irreproducibility issues and their relevance to cytometry. We will summarize the needs and challenges specific to flow cytometry, and will discuss future methods needed for antibody validation.
Intended Audience: Flow cytometrists, reagent manufacturers/vendors, lab personnel
Intended Outcome: Understanding of antibody validation needs, current practices and methods