Importance of validating antibody panels: Anti-PD-L1 clone binds AF700 fluorophore

J Immunol Methods. 2020 Aug:483:112795. doi: 10.1016/j.jim.2020.112795. Epub 2020 May 26.


Researchers routinely use antibodies to assess the expression levels of proteins on the surface or intracellularly in a variety of different cell types. In this current study we highlight the importance of careful validation of antibodies for analysis of protein expression by flow cytometry and how failure to do so can significantly impact the interpretation of the data generated leading to false-positive results. There has been increasing awareness of the role the programmed death receptor 1 (PD-1) pathway plays in health and disease and a potential that programme death ligand 1 (PD-L1) may play a role in inflammatory disease. We aimed to investigate PD-L1 expression on human neutrophils isolated from healthy individuals and patients diagnosed with chronic obstructive pulmonary disease (COPD). We observed an increase in surface expression of PD-L1 by human neutrophils when incubated with AlexaFluor™700-conjugated anti-CD16. Through careful interrogation and antibody validation, we found a novel interaction between a commercially available anti-PD-L1 antibody and the AlexaFluor™700 fluorophore, resulting in this observed increase in PD-L1 signal. Surface expression of PD-L1 was not observed on neutrophils from healthy volunteers or patients with COPD when clone 29E.2A3 of anti-PD-L1 was not used with AlexaFluor™700-conjugated anti-CD16. This highlights the importance of robust antibody validation to ensure antibody compatibility in the context of multi-parametric flow cytometry panels. We also show that, without these validation experiments, novel neutrophil phenotypes could be falsely reported - an important consideration when there is increasing interest in neutrophil heterogeneity.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Adult
  • Aged
  • Aged, 80 and over
  • Antibodies / immunology*
  • Antibody Specificity
  • B7-H1 Antigen / immunology
  • B7-H1 Antigen / metabolism*
  • Case-Control Studies
  • Cross Reactions
  • False Positive Reactions
  • Female
  • Flow Cytometry*
  • Fluorescent Dyes / chemistry
  • GPI-Linked Proteins / immunology
  • GPI-Linked Proteins / metabolism
  • Humans
  • Male
  • Neutrophils / immunology
  • Neutrophils / metabolism*
  • Phenotype
  • Pulmonary Disease, Chronic Obstructive / diagnosis
  • Pulmonary Disease, Chronic Obstructive / immunology
  • Pulmonary Disease, Chronic Obstructive / metabolism*
  • Receptors, IgG / immunology
  • Receptors, IgG / metabolism*
  • Reproducibility of Results
  • Young Adult


  • Antibodies
  • B7-H1 Antigen
  • CD274 protein, human
  • FCGR3B protein, human
  • Fluorescent Dyes
  • GPI-Linked Proteins
  • Receptors, IgG