Screening of PCRP transcription 1 factor antibodies in biochemical assays
Ontology highlight
ABSTRACT:
PROVIDER: PRJNA638411 | ENA |
REPOSITORIES: ENA
Dataset's files
| Action | DRS | |||
|---|---|---|---|---|
| SRR11969000.fastq.gz | Fastqsanger.gz | |||
| SRR11969100.fastq.gz | Fastqsanger.gz | |||
| SRR11969001.fastq.gz | Fastqsanger.gz | |||
| SRR11969101.fastq.gz | Fastqsanger.gz | |||
| SRR11969002.fastq.gz | Fastqsanger.gz |
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Project description:Screening of PCRP transcription factor antibodies in biochemical assays
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Project description:Antibodies offer a powerful means to interrogate specific proteins in a complex milieu, and where epitope tagging is impractical. However, antibody reliability is problematic. The Protein Capture Reagents Program (PCRP) has generated over a thousand renewable monoclonal antibodies against presumptive chromatin proteins in an effort to improve reliability. However, these reagents have not been widely field tested. We therefore screened them in a variety of chromatin-based assays. 887 unique antibodies against 681 unique chromatin targets were assayed by ChIP-exo. A subset was further tested in ChIP-seq, CUT&RUN, STORM super-resolution microscopy, immunoblots, and PBM protein-DNA interaction assays. At least 5% of the tested antibodies were clearly validated by the most stringent of criteria. However, many others produced data that was distinctly different from background, but whose validity was ambiguous. We demonstrate and discuss the metrics and limitation to antibody validation in chromatin-based assays.
2020-06-09 | GSE151287 | GEO
Project description:Antibodies offer a powerful means to interrogate specific proteins in a complex milieu, and where epitope tagging is impractical. However, antibody availability and reliability are problematic. The Protein Capture Reagents Program (PCRP) generated over a thousand renewable monoclonal antibodies against human-presumptive chromatin proteins in an effort to improve reliability. However, these reagents have not been widely field-tested. We therefore screened their ability in a variety of assays. 887 unique antibodies against 681 unique chromatin proteins, of which 605 are putative sequence-specific transcription factors (TFs), were assayed by ChIP-exo. Subsets were further tested in ChIP-seq, CUT&RUN, STORM super-resolution microscopy, immunoblots, and protein binding microarray (PBM) experiments. At least 6% of the tested antibodies were validated for use in ChIP-based assays by the most stringent of our criteria. An additional 34% produced data suggesting they warranted further testing for clearer validation. We demonstrate and discuss the metrics and limitations to antibody validation in chromatin-based assays.
2020-09-01 | GSE152144 | GEO
Project description:The study of chromatin and its organization involves use of antibodies directed against thousands of distinct proteins spanning numerous isoforms and post-translationally modified variants in the human nucleus. Antibodies are essential where epitope-tagging is impractical (e.g., human tissue, cell lines from a wide variety of origins). However, antibody reliability is problematic. The Protein Capture Reagents Program (PCRP) has generated about a thousand renewable monoclonal antibodies that are expected to be more reliable, but these reagents have not been field tested. Since they were generated primarily against chromatin proteins, we tested them in a variety of chromatin-based assays. 887 unique antibodies against 681 unique chromatin targets were assayed by ChIP-exo. A subset were further tested in ChIP-seq, CUT&RUN, STORM super-resolution microscopy, immunoblots, and PBM protein-DNA interaction assays. While this represents only a small portion of the antibody validation/utility space, and noting that optimization may be needed in each assay, our findings suggest that 10-20% of the PCRP-derived antibodies have utility in a particular biochemical assay. We also describe validation strategies.
2020-06-09 | GSE151326 | GEO
Project description:The H3D virtual screening cascade contains models for Mycobacterium tuberculosis and Plasmodium falciparum IC50 predictions, as well as ADME, cytotoxicity, and solubility assays.
Model Type: Predictive machine learning model.
Model Relevance: This panel of models provides predictions for the H3D virtual screening cascade.
Model Encoded by: Miquel Duran-Frigola (Ersilia)
Metadata Submitted in BioModels by: Zainab Ashimiyu-Abdusalam
Implementation of this model code by Ersilia is available here:
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2024-05-13 | MODEL2403270001 | BioModels
Project description:The H3D virtual screening cascade contains models for Mycobacterium tuberculosis and Plasmodium falciparum IC50 predictions, as well as ADME, cytotoxicity, and solubility assays.
Model Type: Predictive machine learning model.
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Metadata Submitted in BioModels by: Zainab Ashimiyu-Abdusalam
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2024-05-13 | MODEL2403270001 | BioModels
Project description:Post-transcriptional modifications are important for transfer RNAs (tRNAs) to be efficient and accurate in translation on the ribosome. The m1G37 modification on a subset of tRNAs in bacteria are generated by a conserved methyltransferase TrmD and is essential for bacterial growth. Previous studies showed that m1G37 has an important role in preventing translational frameshifting and also that this modification is coupled with aminoacylation of tRNAs for proline. Here we performed suppressor screening to isolate a mutant E. coli cell that lacks TrmD but is viable, and the whole-genome sequencing revealed several mutations on prolyl-tRNA synthetase (ProRS) gene conferring cell viability in the absence of TrmD. Biochemical assays confirmed uncoupling of m1G37 modification and aminoacylation, and cell-based assays uncovered the critical role of m1G37 in supporting Wobble decoding.
2022-09-24 | E-MTAB-12178 | biostudies-arrayexpress
Project description:This project is aimed at characterizing the interactions of SARS-CoV-2 Spike protein and its variants with multiple full-length antibodies and monitoring the accompanying conformational dynamics. Different categories of antibodies are tested that recognize different domains of the Spike protein. The project aims at identifying the effects of weak, moderate and strong neutralizing antibodies on Spike protein and decipher their mechanisms of action. In addition to the direct binding effects, distal allosteric effects are also determined. A range of biophysical experiments, biochemical assays, and molecular dynamics simulations are used as orthogonal approaches. The rationale is to identify regions on the SARS-CoV-2 Spike protein that acts as indicators for antibody binding and use these hotspots to develop better neutralizing antibodies against SARS-CoV-2 and any future viral pandemics.
2023-10-09 | PXD043818 | Pride
Project description:The appended raw files, csv files and other documents were deposited into the public domain in support for the publication "Internalization of therapeutic antibodies into Dendritic cells as a risk factor for immunogenicity" by Michel Siegel, Anna-Lena Bolender, Axel Ducret, Johannes Fraidling, Katharina Hartman, Cary M Looney, Olivier Rohr, Timothy Hickling, Martin Lechmann, Celine Marban-Doran and Thomas Kraft.
The abstract reads as follows: "Immunogenicity, defined as the unwanted immune response triggered by the administration of therapeutic antibodies, leads to the production of anti-drug antibodies and is a significant risk factor for the development of therapeutic antibodies. Immunogenicity is a multifactorial phenomenon and risk factors influencing each and every step of the immune response may have major overall consequences. Previously, it has been observed that biophysical properties such as positive charge patches affect the biodistribution of therapeutic antibodies, pharmacokinetic properties like nonspecific clearance by altering their uptake into endothelial cells.
The internalization of therapeutic antibodies into dendritic cells plays a crucial role in immunogenicity. Cellular internalization is mainly facilitated by nonspecific endocytosis or fluid phase pinocytosis. It involves the cell engulfing surrounding fluid, leading to the internalization of its contents. The biophysical properties of therapeutic antibodies, such as positive charge patches, can influence this process as described for endothelial cells. We therefore hypothesize that internalization of therapeutic antibodies into dendritic cells is influenced by their biophysical properties. Investigating the link between charge patches and dendritic cells internalization using tool antibodies with large positive or negative charge patches, we observed that antibodies with large positive charge patches showed higher lysosomal accumulation and epitope presentation, an important risk factor for immunogenicity.
To understand the subsequent impact on T cell activation, we inserted a CD4+ T cell epitope from ovalbumin (a model antigen with well-characterized T cell epitopes) into the same tool compounds. This insertion led to no significant changes in dendritic cell internalization or T cell epitope presentation compared to wild-type antibodies, allowing for a direct comparison of the impact of internalization T cell epitope presentation on T cell activation. However, the insertion of the ovalbumin CD4+ T cell epitope into the tool antibodies increased the occurrence of a specific T cell response, coupled with enhanced internalization and epitope presentation. This increased activation is a key risk factor in immunogenicity, emphasizing the need for careful consideration during the development of biotherapeutics.
In conclusion, positive charge patches-mediated internalization and presentation significantly influence the immunogenicity of therapeutic antibodies."
doi: 10.3389/fimmu.2024.1406643
The data deposited here supports the generation of Fig. 2 and 4 of this manuscript.
2024-08-26 | MSV000095695 | MassIVE