Antibody characterization is critical to enhance reproducibility in biomedical research.

Antibodies are used in many areas of biomedical and clinical research, but many of these antibodies have not been adequately characterized, which casts doubt on the results reported in many scientific papers. This problem is compounded by a lack of suitable control experiments in many studies. In this article we review the history of the antibody characterization crisis, and we document efforts and initiatives to address the problem, notably for antibodies that target human proteins. We also present recommendations for a range of stakeholders - researchers, universities, journals, antibody vendors and repositories, scientific societies and funders - to increase the reproducibility of studies that rely on antibodies

Interaction of erythrocytes (RBC's) with nanostructured surfaces

2022 Summer.Includes bibliographical references.Titanium and its alloys are used to make different blood-contacting medical devices such as stents, artificial heart valves, and catheters for cardiovascular diseases due to their superior biocompatibility. Thrombus formation begins on the surface of these devices as soon as they encounter blood. This leads to the formation of blood clots, which obstructs the flow of blood that leads to severe complications. Recent advancements in nanoscale fabrication and superhydrophobic surface modification techniques have demonstrated that these surfaces have antiadhesive properties and the ability to reduce thrombosis. In this study, the interaction of erythrocytes and whole blood clotting kinetics on superhydrophobic titanium nanostructured surfaces was investigated. These surfaces were characterized for their wettability (contact angle), surface morphology and topography (scanning electron microscopy (SEM)), and crystallinity (glancing angled X-Ray diffraction (GAXRD)). Erythrocyte morphology on different surfaces was characterized using SEM and overall cell viability was demonstrated through fluorescence microscopy. The hemocompatibility of these surfaces was characterized using commercially available assays: thrombin generation assay --> thrombin generation, hemolytic assay --> hemolysis, and complement convertase assay --> complement activity. The results indicate that superhydrophobic titanium nanostructured surfaces had lower erythrocyte adhesion, less morphological changes in adhered cells, lower thrombin generation, lower complement activation, and were less cytotoxic compared to control surfaces. Thus, superhydrophobic titanium nanostructured surfaces may be a promising approach to prevent thrombosis for several blood-contacting medical devices

Redox-sensitive TRP ion channels in the interaction between human lung myofibroblasts and oxidative stress

In idiopathic pulmonary fibrosis (IPF), human lung myofibroblasts (HLMFs) are observed in “fibroblastic foci” which are thought to represent abnormal persistence of myofibroblasts, in part because of failure of normal apoptosis. Oxidative stress is observed in many diseases including IPF. To date however, therapeutics directed specifically at “oxidative stress” have had no important clinical benefit for patients. Part of this failure is our lack of a nuanced understanding of the specific redox signalling and homeostasis abnormalities in disease. Some members of the transient receptor potential cation channels (TRP) are sensitive to redox status and may contribute to important cellular activities in response to oxidative stress. I describe the production and validation of new tools for the study of expression of redox-sensitive TRP cation channels family members, ankyrin 1 (TRPA1) and melastatin 2 (TRPM2). I describe the mRNA, protein, and functional ion current expression of these channels in cells derived from human lungs, including HLMFs. Importantly I highlight problems with the sensitivity and specificity of anti-TRPA1 antibodies widely used in the literature. Transforming factor β1(TGFβ1) is strongly implicated in driving IPF pathogenesis, and I show that TGFβ1 reduces TRPA1 expression in HLMFs. TRPA1 activation was found to play a role in HLMF cell death induced by H2O2 and other agonists, and TGFβ1 exposure induced resistance to this agonist-dependent HLMF death via the downregulation of TRPA1. This may go towards explaining the persistence of myofibroblasts in IPF and other fibrotic diseases. Finally, I study for the first time the H2O2 elimination dynamics in HLMFs using genetically encoded redox sensors. These novel tools have been adapted to enable the future study of specific interactions between TRP channels and other genes with cellular redox homeostasis.</p

YCharOS open antibody characterisation data: Lessons learned and progress made

YCharOS is a collaborative initiative aimed at characterising antibodies against the entire human proteome. As of August 2023, they have presented comprehensive knockout characterisation data for 812 antibodies and 78 proteins using techniques such as Western blot, immunoprecipitation, and immunofluorescence. YCharOS consolidates its data into reports (one protein per report) available on Zenodo, a public repository controlled by CERN, to ensure open access. To enhance the visibility of their work, the group is progressively converting their Zenodo reports into F1000 articles, collected on the YCharOS Gateway, and indexed via PubMed. Their data is also accessible through searches on the Antibody Registry. The provided data is a valuable resource for researchers when selecting antibodies for specific applications, although certain limitations should be considered. The data accumulated thus far has illuminated the extent of the problem when poorly performing antibodies are employed in research. While the scientific community was already aware that this was likely a widespread issue, the establishment of a collaborative open science project with industry partners introduces an innovative solution that holds the potential to yield significant returns on investment in the public interest. This potential is substantiated by the number of antibodies that have either been withdrawn or had their recommended usage altered by the vendor. However, despite the discovery of high-performing renewable antibodies for most of the studied proteins, this accounts for a tiny fraction of the human proteome and the commercial antibody market. To realise the full potential of this work, end-users must adjust their antibody procurement and usage practises in line with the provided data. This editorial offers a guide on how individual scientists can utilise the YCharOS data, in addition to sharing the insights gained from the data thus far with the wider scientific community

Tales of the unexpected

A single event can completely change the direction of a career in science; four researchers share their stories

Lack of Benefit of High Flow Nasal Oxygen Therapy as Ceiling of Treatment for Severe COVID-19 Pneumonitis in Elderly Frail Patients: A Single Centre Observational Study

Background: Severe COVID-19 pneumonitis in elderly frail patients is associated with poor outcomes, and therefore invasive mechanical ventilation is often deemed an inappropriate course of action. Some evidence suggests high-flow nasal oxygen (HFNO) may prevent the need for invasive ventilation in other groups of patients, but whether it is an appropriate ceiling of care for older frail patients is unknown. Methods: We retrospectively identified patients with severe COVID-19 pneumonitis requiring FiO2>60% who were deemed inappropriate for invasive ventilation or non-invasive continuous positive airway pressure ventilation (CPAP). Our local protocol based on national guidance suggested these patients should be considered for HFNO. We observed whether the patients received HFNO or standard oxygen therapy (SOT) and compared mortality and survival time in these groups. Results: We identified 81 patients meeting the inclusion criteria. From this group, 24 received HFNO and 57 received SOT. The HFNO group was similar in age, BMI and co-morbidities to the SOT group but less frail, as determined by the Clinical Frailty Scale (CFS). All 24 patients that received HFNO died in comparison to 46 patients (80.7%) in the SOT group. Mortality in the HFNO group was significantly higher than in the SOT group. Conclusion: Elderly frail patients with severe COVID-19 pneumonitis deemed inappropriate for invasive ventilation and did not benefit from HFNO. Further, HFNO may have been associated with harm in this group.</p

Improving the integrity and reproducibility of research that uses antibodies: a technical, data sharing, behavioral and policy challenge

Antibodies are one of the most important reagents used in biomedical and fundamental research, used to identify, and quantify proteins, contribute to knowledge of disease mechanisms, and validate drug targets. Yet many antibodies used in research do not recognize their intended target, or recognize additional molecules, compromising the integrity of research findings and leading to waste of resources, lack of reproducibility, failure of research projects, and delays in drug development. Researchers frequently use antibodies without confirming that they perform as intended in their application of interest. Here we argue that the determinants of end-user antibody choice and use are critical, and under-addressed, behavioral drivers of this problem. This interacts with the batch-to-batch variability of these biological reagents, and the paucity of available characterization data for most antibodies, making it more difficult for researchers to choose high quality reagents and perform necessary validation experiments. The open-science company YCharOS works with major antibody manufacturers and knockout cell line producers to characterize antibodies, identifying high-performing renewable antibodies for many targets in neuroscience. This shows the progress that can be made by stakeholders working together. However, their work so far applies to only a tiny fraction of available antibodies. Where characterization data exists, end-users need help to find and use it appropriately. While progress has been made in the context of technical solutions and antibody characterization, we argue that initiatives to make best practice behaviors by researchers more feasible, easy, and rewarding are needed. Global cooperation and coordination between multiple partners and stakeholders will be crucial to address the technical, policy, behavioral, and open data sharing challenges. We offer potential solutions by describing our Only Good Antibodies initiative, a community of researchers and partner organizations working toward the necessary change. We conclude with an open invitation for stakeholders, including researchers, to join our cause. </p

Improving the integrity and reproducibility of research that uses antibodies: a technical, data sharing, behavioral and policy challenge

Antibodies are one of the most important reagents used in biomedical and fundamental research, used to identify, and quantify proteins, contribute to knowledge of disease mechanisms, and validate drug targets. Yet many antibodies used in research do not recognize their intended target, or recognize additional molecules, compromising the integrity of research findings and leading to waste of resources, lack of reproducibility, failure of research projects, and delays in drug development. Researchers frequently use antibodies without confirming that they perform as intended in their application of interest. Here we argue that the determinants of end-user antibody choice and use are critical, and under-addressed, behavioral drivers of this problem. This interacts with the batch-to-batch variability of these biological reagents, and the paucity of available characterization data for most antibodies, making it more difficult for researchers to choose high quality reagents and perform necessary validation experiments. The open-science company YCharOS works with major antibody manufacturers and knockout cell line producers to characterize antibodies, identifying high-performing renewable antibodies for many targets in neuroscience. This shows the progress that can be made by stakeholders working together. However, their work so far applies to only a tiny fraction of available antibodies. Where characterization data exists, end-users need help to find and use it appropriately. While progress has been made in the context of technical solutions and antibody characterization, we argue that initiatives to make best practice behaviors by researchers more feasible, easy, and rewarding are needed. Global cooperation and coordination between multiple partners and stakeholders will be crucial to address the technical, policy, behavioral, and open data sharing challenges. We offer potential solutions by describing our Only Good Antibodies initiative, a community of researchers and partner organizations working toward the necessary change. We conclude with an open invitation for stakeholders, including researchers, to join our cause. </p

Scaling of an antibody validation procedure enables quantification of antibody performance in major research applications.

Antibodies are critical reagents to detect and characterize proteins. It is commonly understood that many commercial antibodies do not recognize their intended targets, but information on the scope of the problem remains largely anecdotal, and as such, feasibility of the goal of at least one potent and specific antibody targeting each protein in a proteome cannot be assessed. Focusing on antibodies for human proteins, we have scaled a standardized characterization approach using parental and knockout cell lines (Laflamme et al., 2019) to assess the performance of 614 commercial antibodies for 65 neuroscience-related proteins. Side-by-side comparisons of all antibodies against each target, obtained from multiple commercial partners, have demonstrated that: (i) more than 50% of all antibodies failed in one or more applications, (ii) yet, ~50-75% of the protein set was covered by at least one high-performing antibody, depending on application, suggesting that coverage of human proteins by commercial antibodies is significant; and (iii) recombinant antibodies performed better than monoclonal or polyclonal antibodies. The hundreds of underperforming antibodies identified in this study were found to have been used in a large number of published articles, which should raise alarm. Encouragingly, more than half of the underperforming commercial antibodies were reassessed by the manufacturers, and many had alterations to their recommended usage or were removed from the market. This first study helps demonstrate the scale of the antibody specificity problem but also suggests an efficient strategy toward achieving coverage of the human proteome; mine the existing commercial antibody repertoire, and use the data to focus new renewable antibody generation efforts