Using Imaging Mass Cytometry to Define Cell Identities and Interactions in Human Tissues

In the evolving landscape of highly multiplexed imaging techniques that can be applied to study complex cellular microenvironments, this review characterizes the use of imaging mass cytometry (IMC) to study the human kidney. We provide technical details for antibody validation, cell segmentation, and data analysis specifically tailored to human kidney samples, and elaborate on phenotyping of kidney cell types and novel insights that IMC can provide regarding pathophysiological processes in the injured or diseased kidney. This review will provide the reader with the necessary background to understand both the power and the limitations of IMC and thus support better perception of how IMC analysis can improve our understanding of human disease pathogenesis and can be integrated with other technologies such as single cell sequencing and proteomics to provide spatial context to cellular data.

Anti-drug Antibody Validation Testing and Reporting Harmonization

Evolving immunogenicity assay performance expectations and a lack of harmonized anti-drug antibody validation testing and reporting tools have resulted in significant time spent by health authorities and sponsors on resolving filing queries. Following debate at the American Association of Pharmaceutical Sciences National Biotechnology Conference, a group was formed to address these gaps. Over the last 3 years, 44 members from 29 organizations (including 5 members from Europe and 10 members from FDA) discussed gaps in understanding immunogenicity assay requirements and have developed harmonization tools for use by industry scientists to facilitate filings to health authorities. Herein, this team provides testing and reporting strategies and tools for the following assessments: (1) pre-study validation cut point; (2) in-study cut points, including procedures for applying cut points to mixed populations; (3) system suitability control criteria for in-study plate acceptance; (4) assay sensitivity, including the selection of an appropriate low positive control; (5) specificity, including drug and target tolerance; (6) sample stability that reflects sample storage and handling conditions; (7) assay selectivity to matrix components, including hemolytic, lipemic, and disease state matrices; (8) domain specificity for multi-domain therapeutics; (9) and minimum required dilution and extraction-based sample processing for titer reporting. Graphical Abstract

Listening to the Whispers in Neuroimmune Crosstalk: A Comprehensive Workflow to Investigate Neurotrophin Receptor p75NTR Under Endogenous, Low Abundance Conditions

Inflammatory conditions are critically influenced by neuroimmune crosstalk. Cytokines and neurotrophic factors shape the responses of both nervous and immune systems. Although much progress has been made, most findings to date are based on expression of recombinant (tagged) proteins. The examination of receptor interactions by immunoprecipitation (IP) at endogenous levels provides further insight into the more subtle regulations of immune responses. Here, we present a comprehensive workflow and an optimized IP protocol that provide step-by-step instructions to investigate neurotrophin receptor p75NTR at endogenous, low abundance levels: from lysate preparation and confirmation of receptor expression to antibody validation and successful detection of protein-protein interactions. We employ human melanoma cell line A375 to validate specific antibodies and IP conditions, and apply these methods to explore p75NTR interactions in human leukemic plasmacytoid dendritic cell line PMDC05 detecting 14-3-3ϵ:p75NTR interaction in this cell type. With p75NTR as an exemplary protein, our approach provides a strategy to detect specific interaction partners even under endogenous, low abundance expression conditions.

Antibodies validated for routinely processed tissues stain frozen sections unpredictably

Background: Antibody validation for tissue staining is required for reproducibility; criteria to ensure validity have been published recently. The majority of these recommendations imply the use of routinely processed (formalin-fixed, paraffin-embedded) tissue. Materials & methods: We applied to lightly fixed frozen sections a panel of 126 antibodies validated for formalin-fixed, paraffin-embedded tissue with extended criteria. Results: Less than 30% of the antibodies performed as expected with all fixations. 35% preferred one fixation over another, 13% gave nonspecific staining and 23% did not stain at all. Conclusion: Individual antibody variability of the paratope’s fitness for the fixed antigen may be the cause. Revalidation of established antibody panels is required when they are applied to sections whose fixation and processing are different from the tissue where they were initially validated.

Antibody validation for protein expression on tissue slides: a protocol for immunohistochemistry

Antibodies play a crucial role in basic research and clinical decision-making. However, there are no standardized algorithms or guidelines to ensure their accuracy and validity. There have been efforts to generate consensus, but, with the exception of clinical labs, antibody validation remains variable in the literature and sometimes in clinical practice. Here we focus on immunohistochemistry, an example of a scientific and clinical tool where validation of antibodies is critical. We describe a protocol that we use to validate antibodies specifically for immunohistochemistry, including some of the pillars of antibody validation from Uhlen et al. 2016, as an example of a rigorous approach to build antibody-based tests for both basic and translational science labs and for the clinic.

34 Multi-step antibody validation for the geomx® digital spatial profiler

BackgroundUnderstanding 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.3MethodsUnconjugated 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.ResultsIn 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%.ConclusionsUnvalidated 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.ReferencesTaussig 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.

Validating Immunohistochemistry Assay Specificity in Investigative Studies: Considerations for a Weight of Evidence Approach

Immunohistochemistry (IHC) is a fundamental molecular technique that provides information on protein expression in the context of spatial localization and tissue morphology. IHC is used in all facets of pathology from identifying infectious agents or characterizing tumors in diagnostics, to characterizing cellular and molecular processes in investigative and experimental studies. Confidence in an IHC assay is primarily driven by the degree to which it is validated. There are many approaches to validate an IHC assay’s specificity including bioinformatics approaches using published protein sequences, careful design of positive and negative tissue controls, use of cell pellets with known target protein expression, corroboration of IHC findings with western blots and other analytical methods, and replacement of the primary antibody with an appropriate negative control reagent. Each approach has inherent strengths and weaknesses, and the thoughtful use of these approaches provides cumulative evidence, or a weight of evidence, to support the IHC assay’s specificity and build confidence in a study’s conclusions. Although it is difficult to be 100% confident in the specificity of any IHC assay, it is important to consider how validation approaches provide evidence to support or to question the specificity of labeling, and how that evidence affects the overall interpretation of a study’s results. In this review, we discuss different approaches for IHC antibody validation, with an emphasis on the characterization of antibody specificity in investigative studies. While this review is not prescriptive, it is hoped that it will be thought provoking when considering the interpretation of IHC results.

Profiles of histidine-rich glycoprotein associate with age and risk of all-cause mortality

Despite recognizing aging as a common risk factor of many human diseases, little is known about its molecular traits. To identify age-associated proteins circulating in human blood, we screened 156 individuals aged 50–92 using exploratory and multiplexed affinity proteomics assays. Profiling eight additional study sets (N = 3,987), performing antibody validation, and conducting a meta-analysis revealed a consistent age association (P = 6.61 × 10−6) for circulating histidine-rich glycoprotein (HRG). Sequence variants of HRG influenced how the protein was recognized in the immunoassays. Indeed, only the HRG profiles affected by rs9898 were associated with age and predicted the risk of mortality (HR = 1.25 per SD; 95% CI = 1.12–1.39; P = 6.45 × 10−5) during a follow-up period of 8.5 yr after blood sampling (IQR = 7.7–9.3 yr). Our affinity proteomics analysis found associations between the particular molecular traits of circulating HRG with age and all-cause mortality. The distinct profiles of this multipurpose protein could serve as an accessible and informative indicator of the physiological processes related to biological aging.

Proximity Ligation Assay as a Tool for Antibody Validation in Human Tissues

Immunohistochemistry (IHC) is the accepted standard for spatial analysis of protein expression in tissues. IHC is widely used for cancer diagnostics and in basic research. The development of new antibodies to proteins with unknown expression patterns has created a demand for thorough validation. We have applied resources from the Human Protein Atlas project and the Antibody Portal at National Cancer Institute to generate protein expression data for 12 proteins across 39 cancer cell lines and 37 normal human tissue types. The outcome of IHC on consecutive sections from both cell and tissue microarrays using two independent antibodies for each protein was compared with in situ proximity ligation (isPLA), where binding by both antibodies is required to generate detection signals. Semi-quantitative scores from IHC and isPLA were compared with expression of the corresponding 12 transcripts across all cell lines and tissue types. Our results show a more consistent correlation between mRNA levels and isPLA as compared to IHC. The main benefits of isPLA include increased detection specificity and decreased unspecific staining compared to IHC. We conclude that implementing isPLA as a complement to IHC for analysis of protein expression and in antibody validation pipelines can lead to more accurate localization of proteins in tissue.