RESUMEN
In the wake of the reproducibility crisis and numerous discussions on how commercially available antibodies as research tool contribute to it, The Antibody Society developed a series of 10 webinars to address the issues involved. The webinars were delivered by speakers with both academic and commercial backgrounds. This report highlights the problems, and offers solutions to help the scientific community appropriately identify the right antibodies and to validate them for their research and development projects. Despite the various solutions proposed here, they must be applied on a case-by-case basis. Each antibody must be verified based on the content of the product sheet, and subsequently through experimentation to confirm integrity, specificity and selectivity. Verification needs to focus on the precise application and tissue/cell type for which the antibody will be used, and all verification data must be reported openly. The various approaches discussed here all have caveats, so a combination of solutions must be considered.
Asunto(s)
Anticuerpos Monoclonales/química , Especificidad de Anticuerpos , Ingeniería de Proteínas , Estudios de Validación como Asunto , HumanosRESUMEN
Since their discovery, the matrix metalloproteinase (MMP) family proteases have been considered as therapeutic targets in numerous diseases and disorders. Unfortunately, clinical trials with MMP inhibitors have failed to yield any clinical benefits of these inhibitors. These failures were largely due to a lack of MMP-selective agents; accordingly, it has become important to identify a platform with which high selectivity can be achieved. To this end, we propose using MMP-targeting antibodies that can achieve high specificity in interactions with their targets. Using a scaffold of single-domain antibodies, here we raised a panel of MMP10-selective antibodies through immunization of llamas, a member of the camelid family, whose members generate conventional heavy/light-chain antibodies and also smaller antibodies lacking light-chain and CH1 domains. We report the generation of a highly selective and tightly binding MMP10 inhibitor (Ki < 2 nm). Using bio-layer interferometry-based binding assays, we found that this antibody interacts with the MMP10 active site. Activity assays demonstrated that the antibody selectively inhibits MMP10 over its closest relative, MMP3. The ability of a single-domain antibody to discriminate between the most conserved MMP pair via an active site-directed mechanism of inhibition reported here supports the potential of this antibody as a broadly applicable scaffold for the development of selective, tightly binding MMP inhibitors.
Asunto(s)
Metaloproteinasa 10 de la Matriz/metabolismo , Inhibidores de la Metaloproteinasa de la Matriz/farmacología , Anticuerpos de Dominio Único/farmacología , Animales , Camélidos del Nuevo Mundo , Humanos , Inmunización , Cinética , Biblioteca de Péptidos , Proteínas Recombinantes/metabolismo , Saccharomyces cerevisiae/metabolismo , Especificidad por Sustrato/efectos de los fármacos , alfa 1-Antitripsina/metabolismoRESUMEN
Studying TRP channel expressing nociceptors requires the identification of the respective subpopulations as well as the quantification of dynamic cellular events. However, the heterogeneity of sensory neurons and associated nonneuronal cells demands the analysis of large numbers of cells to reflect the distribution of entire populations. Here we report a detailed workflow how to apply high-content screening (HCS) microscopy to signaling events in TRPV1-positive neurons as well as an approach to use the selective elimination of TRPV1 positive cells from dissociated rat sensory ganglia as base for transcriptomic analysis of TRPV1-positive cells and/or as control for TRPV1 antibody specificity.
Asunto(s)
Células Receptoras Sensoriales/metabolismo , Canales Catiónicos TRPV/metabolismo , Animales , Células Cultivadas , Técnica del Anticuerpo Fluorescente/métodos , Masculino , Ratones Endogámicos C57BL , Microscopía/métodos , Microscopía Fluorescente , Nociceptores/metabolismo , Ratas , Ratas Sprague-Dawley , Canales Catiónicos TRPV/inmunologíaRESUMEN
With the increasing availability of collections of antibodies, their evaluation in terms of binding selectivity becomes an important but challenging task. Planar antigen microarrays are very suitable tools to address this task and provide a powerful proteomics platform for the characterization of the binding selectivity of antibodies toward thousands of antigens in parallel. In this chapter, we describe our in-house developed procedures for the generation of high-density planar antigen microarrays with over 21,000 features. We also provide the details of the assay protocol, which we routinely use for the assessment of binding selectivity of the polyclonal antibodies generated within the Human Protein Atlas.
Asunto(s)
Especificidad de Anticuerpos/inmunología , Antígenos/inmunología , Análisis por Matrices de Proteínas/métodos , Proteómica/métodos , Anticuerpos/inmunología , Antígenos/genética , Humanos , Unión Proteica/inmunologíaRESUMEN
With the increasing number of binding reagents for affinity-based investigations of the human proteome, high-throughput tools for the characterization of the used reagents become essential. For the analysis of binding selectivity, bead-based antigen arrays offer a miniaturized and parallelized assay platform to meet such needs, as they enable two-dimensional multiplexing to analyze up to 384 samples against up to 500 analytes in a single round of analysis. In this chapter, we describe our protocols for the generation of multiplex bead arrays built on immobilized protein fragments, as well as biotinylated peptides. Combined together, these two versions of antigen arrays offer a versatile approach for multiplexed characterization of antibody binding selectivity, off-target interactions, as well as mapping for the amino acids of epitopes involved in antibody binding.
Asunto(s)
Especificidad de Anticuerpos/inmunología , Antígenos/inmunología , Mapeo Epitopo/métodos , Análisis por Matrices de Proteínas/métodos , Animales , Anticuerpos/inmunología , Epítopos/genética , Epítopos/inmunología , Humanos , Proteoma/genética , Proteoma/inmunologíaRESUMEN
Antibody microarrays offer high-throughput immunoassays for multiplexed analyses of clinical samples. For such approaches, samples are either labeled in solution to enable a direct readout on the single binder assay format or detected by matched pairs of capture and detection antibodies in dual binder assay format, also known as sandwich assays. Aiming to benefit from the flexibility and capacity offered by single binder assay readout and the specificity and sensitivity of dual binder assays, we developed a multiplexed dual binder procedure that is based on a sequential, rather than combined, antigen binding. The method, entitled dual capture assay (DCA), is composed of an initial antigen capture by antibodies on beads, followed by labeling of captured protein targets on beads, combinatorial elution steps at high and low pH, and a readout using a secondary bead array. Compared to classical single binder assays, the described method demonstrated several advantages such as reduced contribution of off-target binding, lower noise levels, and improved correlation when comparing with clinical reference values. This procedure describes a novel and versatile immunoassay strategy for proteome profiling in body fluids.
Asunto(s)
Inmunoensayo/métodos , Proteoma , Proteómica/métodos , Anticuerpos Inmovilizados , Humanos , Análisis por Matrices de Proteínas , Sensibilidad y Especificidad , Coloración y Etiquetado , Flujo de TrabajoRESUMEN
Antibody selectivity represents a major issue in the development of efficient immuno-therapeutics and detection assays. Its description requires a comparison of the affinities of the antibody for a significant number of antigen variants. In the case of peptide antigens, this task can now be addressed to a significant level of details owing to improvements in spot peptide array technologies. They allow the high-throughput mutational analysis of peptides with, depending on assay design, an evaluation of binding stabilities. Here, we examine the cross-reactive capacity of an antibody fragment using the PEPperCHIP(®) technology platform (PEPperPRINT GmbH, Heidelberg, Germany; >8800 peptides per microarray) combined with the surface plasmon resonance characterization (Biacore(®) technology; GE-Healthcare Biacore, Uppsala, Sweden) of a subset of interactions. ScFv1F4 recognizes the N-terminal end of oncoprotein E6 of human papilloma virus 16. The spot permutation analysis (i.e. each position substituted by all amino acids except cysteine) of the wild type decapeptide (sequence (6)TAMFQDPQER(15)) and of 15 variants thereof defined the optimal epitope and provided a ranking for variant recognition. The SPR affinity measurements mostly validated the ranking of complex stabilities deduced from array data and defined the sensitivity of spot fluorescence intensities, bringing further insight into the conditions for cross-reactivity. Our data demonstrate the importance of throughput and quantification in the assessment of antibody selectivity.