In this report, we describe the development of a mini-array system suitable for high-throughput quantification of proteins. This mini-array is a multiplexed, sandwich-type ELISA that measures the concentration of seven different human cytokines--TNF-alpha, IFN alpha, IFN gamma, IL-1 alpha, IL-1 beta, IL-6, and IL-10--from a single sample in each well of a 96-well plate. The mini-array is produced by spotting monoclonal antibodies (mAbs) in a 3 x 3 pattern in the bottom of the wells of 96-well polystyrene plates. Cytokines that are captured by the arrayed mAbs are detected by using biotinylated mAbs, followed by the addition of a streptavidin-horseradish peroxidase (HRP) conjugate and a chemiluminescent substrate. The light produced from the HRP-catalyzed oxidation of the substrate is measured at each spot in the array by imaging the entire plate with a commercially available CCD camera. Here, we demonstrate that these 96-well-plate format mini-arrays have performance characteristics that make them suitable for the high-throughput screening of anti-inflammatory compounds.
A key component of efforts to address the reproducibility crisis in biomedical research is the development of rigorously validated and renewable protein-affinity reagents. As part of the US National Institutes of Health (NIH) Protein Capture Reagents Program (PCRP), we have generated a collection of 1,406 highly validated immunoprecipitation- and/or immunoblotting-grade mouse monoclonal antibodies (mAbs) to 737 human transcription factors, using an integrated production and validation pipeline. We used HuProt human protein microarrays as a primary validation tool to identify mAbs with high specificity for their cognate targets. We further validated PCRP mAbs by means of multiple experimental applications, including immunoprecipitation, immunoblotting, chromatin immunoprecipitation followed by sequencing (ChIP-seq), and immunohistochemistry. We also conducted a meta-analysis that identified critical variables that contribute to the generation of high-quality mAbs. All validation data, protocols, and links to PCRP mAb suppliers are available at http://proteincapture.org.
Abstract:A key component to overcoming the reproducibility crisis in biomedical research is the development of readily available, rigorously validated and renewable protein affinity reagents. As part of the NIH Protein Capture Reagents Program (PCRP), we have generated a collection of 1406 highly validated, immunoprecipitation (IP) and/or immunoblotting (IB) grade, mouse monoclonal antibodies (mAbs) to 736 human transcription factors. We used HuProt™ human protein microarrays to identify mAbs that recognize their cognate targets with exceptional specificity. Using an integrated production and validation pipeline, we validated these mAbs in multiple experimental applications, and have distributed them to the Developmental Studies Hybridoma Bank (DSHB) and several commercial suppliers. This study allowed us to perform a meta-analysis that identified critical variables that contribute to the generation of high quality mAbs. We find that using full-length antigens for immunization, in combination with HuProt™ analysis, provides the highest overall success rates. The efficiencies built into this pipeline ensure substantial cost savings compared to current standard practices.
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