LAUSR

Background Understanding protein expression patterns within tissue compartments is imperative to investigating a range of biological questions. Historically, low plex immunohistochemical (IHC) approaches have been employed to assess the spatial heterogeneity of protein expression in tissue slices, but these techniques are of limited utility due to the challenge of measuring multiple targets in parallel. Compounding this limitation is the necessity of validating all antibodies which is resource intensive. Antibodies with poor quality have led to wasted time and resources, including false positives and non-reproducible results.1 2 Here we review the antibody validation process for the GeoMx® Digital Spatial Profiler (DSP) which enables investigation of high-plex, validated, spatially resolved protein targets from a single slide mounted formalin-fixed paraffin-embedded (FFPE) or fresh frozen sample. The robust validation process is in line with recent suggestions for antibody validation from SITC.3 Methods Unconjugated and oligo-conjugated antibodies are screened by IHC to assess staining sensitivity, patterns, and more importantly ensure that the oligo-conjugation has not adversely affected antibody performance. Upon approval by a pathologist, the antibodies are incorporated into a core or module and further validated using the GeoMx DSP. Using FFPE cell pellet arrays (CPAs) containing positive and negative control pellets, we assess the specificity as defined as a lack of signal in negative control pellets and a robust signal in positive control pellets. Antibodies with robust signals are then screened on tissue microarrays (TMAs) composed of healthy and diseased tissues to ensure that they will perform as expected in real samples and yield sufficient signal over background. Finally, after antibodies pass functional validation, we assess the performance of antibodies within panels of antibodies that will be commercialized. Results In total, approximately 60% of off-the-shelf antibodies tested for use in GeoMx assays pass the entire validation process and are put into commercial assays. Passing requirements include exhibiting a maximum positive signal divided by the limit of detection, plus two standard deviations (SD) that is greater than or equal to 5 in both CPAs and TMAs for individual antibodies; such a threshold gives a false positive rate of less than 10%. Conclusions Unvalidated or poorly validated antibodies can result in false positives and non-reproducible results. Following the robust validation process outlined here, approximately 40% of off-the-shelf antibodies are removed from panels, underscoring the importance of antibody validation prior to incorporating new antibodies into experiments. References Taussig MJ, Fonseca C, and Trimmer JS. Antibody validation: a view from the mountains. N Biotechnol. 2018; 45:1–8. Bordeaux J, Welsh AW, Agarwal S, Killiam E, Baquero MT, Hanna JA, Anagnostou VK, Rimm DL. Antibody validation. Biotechniques 2010;48(3):197–209. Taube, et al. The Society for Immunotherapy of Cancer statement on best practices for multiplex immunohistochemistry (IHC) and immunofluorescence (IF) staining and validation. J Immunother Cancer 2020;8(1):e000155.